Nothing Special   »   [go: up one dir, main page]

CN101057975A - Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application - Google Patents

Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application Download PDF

Info

Publication number
CN101057975A
CN101057975A CN 200610165104 CN200610165104A CN101057975A CN 101057975 A CN101057975 A CN 101057975A CN 200610165104 CN200610165104 CN 200610165104 CN 200610165104 A CN200610165104 A CN 200610165104A CN 101057975 A CN101057975 A CN 101057975A
Authority
CN
China
Prior art keywords
virus
sequence
gene
epitope
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN 200610165104
Other languages
Chinese (zh)
Other versions
CN101057975B (en
Inventor
田波
张玉霞
刘长梅
高福
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of CAS
Original Assignee
Institute of Microbiology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Microbiology of CAS filed Critical Institute of Microbiology of CAS
Priority to CN200610165104A priority Critical patent/CN101057975B/en
Publication of CN101057975A publication Critical patent/CN101057975A/en
Application granted granted Critical
Publication of CN101057975B publication Critical patent/CN101057975B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a cocktail vaccine that is anti-immune tolerance and anti-immunodeficiency virus and its application. Said cocktail vaccine at least comprises two or more elements and any viral antigen recombinant protein and medical shaping. Said element is recombinant DNA and/ or recombinant virus; it is chosen from (A) neutralizing antibody, receptor antagonist, membrane fusion blocking agent, cell factor coding sequence, and/ or DNA interference sequence; (B) immunoadjuvants coded sequence; (C) antigen gene, viral antigen and immunoadjuvants fusion gene, viral antigen epitope coded sequence, multi-epitope fusion gene and/or viral antigen epitope -MHC-I SCT gene; (D) two seven-peptide multiplexed sequence HR1 and HR2 of membrana capsularis virus fusion protein and/ or coded sequence of its polymer HR212. The expression carrier of all antiviral elements comprises recombinant DNA carrier, vaccinia virus carrier and/ or adenoviral carrier.

Description

The cocktail vaccine of a kind of anti immune tolerance and immunodeficiency virus and application thereof
Technical field
The invention belongs to field of immunology and vaccine development technology, be specifically related to the cocktail vaccine and the application thereof of anti immune tolerance and immunodeficiency virus.
Background technology
1. immunologic tolerance virus and amynologic basis thereof: immunologic tolerance virus is meant the virus that can cause the body immune system respond low.Though each viral pathogenesis is not quite similar, they all cause with virus continue exist, ability drop or disappearance that B cell and T cell produce antibody and cellular immunization be the disease of feature.In the numerous viruses that start an inflammation of the liver, hepatitis B virus (HBV) and hepatitis C virus (HCV) are most important two immunologic tolerance viruses.
The immunologic tolerance virus immunity of organism of can effectively escaping causes immunologic tolerance, makes the immunologic mechanism of human body no longer resist former generation immunne response, makes virus long-term existence in vivo.HBV is most typical immunologic tolerance virus, and susceptible mother produces 90% neonate becomes the virus carrier, and the HBV patient that partly is grown up also can form the chronic infection of hepatitis B.Ctl response ability drop, disappearance or clone that the variation of the persistent infection of HBV and antigenic overexpression and virus often causes the Th1 class cell relevant with immunomodulating viral special in virus replication lasting in patient's body, the thymus to be cloned and is correlated with the infection cell cracking with virus sweep in rejecting, peripheral blood and the liver reject.The persistent infection of HBV and associated liver cirrhosis and hepatocarcinoma have caused great economy and social pressure to the patient to society.
The primary structure antigen of HBV comprises surface antigen S, and (comprising glycosylated big antigen S1S2S, middle antigen S2S and little antigen S) and core protein C also have polymerase P in addition and regulate albumen e and x.S and C antigen can cause the generation of neutralizing antibody.Core protein C is conservative relatively, and the special cytotoxic T lymphocyte (CTL) that it produces is significant to virus sweep.Concerning HBV, the antigenic great expression of e can make Th1 class cell be suppressed, and the great expression of surface antigen makes immune system exhaustion (exhausted).
HCV immunologic tolerance mechanism is different with HBV, and HCV mostly is low-level infection, is difficult for producing active immunne response, and the HCV gene very easily produces variation in addition, causes immunologic escape, produces persistent infection and immunologic tolerance.
191 aminoacid of HCV polyprotein N-terminal are core protein, are the main components of virus nucleocapsid.The N-terminal of core protein is rich in basic amino acid and high conservative.Be the envelope protein E1 of virus behind the core protein, E2 is the I type transmembrane protein of high glycosylation.In all HCV albumen, the conservative of E2 is the most weak, is immunoreactive main target.Most HCV non-structural protein (NS2-NS5B) by viral genome duplicate essential.HCV is a kind of virus of height variation, and this variation makes HCV form complicated and diversified HCV quasispecies group in the infected's body, might exert an influence to body disease-resistant poison immunne response.The formation of immune evasion strain then may be relevant with persistent viral infection.Two kinds of immune evasion may mechanism: the active antagonism that (1) HCV replys IFN, can block the phosphorylation and the activity of interferon regulatory factor 3 as HCV NS3/4 serine protease; E2 and NS5 albumen can suppress the function of RNA deopendent protein kinase, thereby influence the antiviral effect of IFN.(2) passive variation takes place in bone-marrow-derived lymphocyte or T lymphocyte antigen epi-position on the HCV albumen under immunity of organisms, thereby escapes the attack of adaptive immune response.
2. immunodeficiency virus and amynologic basis thereof: typical case's representative of immunodeficiency virus is human immunodeficiency virus (HIV).HIV causes CD4 by infecting CD4 T cell +The T lymphocyte quantity constantly descends.In addition, HIV infects the cell toxicant function reduction also cause imbalance of B-cell function and NK cell, and the quantity that makes the dendritic cell (DC) with immunologic function is in blood and distribution in the tissue and quantity change.HIV infects organs such as also can causing central nervous system and gastrointestinal tract and produces pathological changes.Because immunologic function degression, the probability of concurrent various cancers of patient and infectious disease heightens, and finally causes acquired immune deficiency syndrome (AIDS) (AIDS).
The structural gene of HIV comprises gag (P55, P17, P24), Pol and env.Pol gene code polymerase precursor protein (P34) forms protease, intergrase, reverse transcriptase, ribonuclease H through cutting, and it is essential to be virus multiplication institute.About 863 the amino acid whose precursor proteins of env gene code form gp160, gp120 and gp41 through glycosylation.During gp120 contains and antigenic determinant, proved among the HIV and epitope on gp120 V3 ring, V3 ring district is the critical function district of envelope protein, plays an important role in virus and cell fusion.Gp120 links to each other with non-covalent bond with transmembrane protein gp41.Gp41 participates in target cell and merges, and impels the virus infection cell.Experiment shows that gp41 also has than strong antigen, can induce the generation antibody response.The controlling gene of HIV comprises Tat, Rev, Nef, Vif, Vpu, Vpr at least.The immunization therapy of HIV should be set up blocking virus and be continued to infect CD4 +The mechanism of T cell, that suppresses the inner HIV of infection cell continues to duplicate and produce neutralizing antibody and ctl response.
3. the progress of immunologic tolerance virus and immunodeficiency virus vaccine
(1) HBV vaccine research progress: the preventative SAV of HBV has obtained using and having reduced to a great extent widely the infection of HBV, but this kind vaccine can not be treated the chronic infection of HBV.The method that traditional treatment HBV infects comprises α-IFN and chemicals, and they have important effect aspect the duplicating of control virus, but but can not remove virus (Seeger and Mason 2000) effectively.Though the dna vaccination of pure strategy comes into one's own because bringing out cell and humoral immunization, the result of dna vaccination in the toy experiment but can not realize (Payette et al.2006 at large animal on one's body; Zhao et al.2006).Along with to HBV with and induce an illness research go deep into, there has been the clinical experiment of part to adopt chemicals and the bonded comprehensive therapy of protein vaccine, though obtained certain achievement (Yang, Lee et al.2006), but conventional medicament overcoming immunologic escape that virus variation causes, reducing virus replication and recover and also do not have optimistic report aspect the ctl response ability of body.So far still not having approved HBV therapeutic vaccine comes out.
(2) HCV vaccine research progress: the hepatitis C that HCV causes, the chronicity rate of its infection be up to 60-80%, and cause liver cirrhosis and hepatocarcinoma.HCV mainly infects by blood transfusion, the new incidence rate that infects of the close inspection may command HCV of blood, and according to above-mentioned situation, the vaccine research of HCV mainly is the research at therapeutic vaccine.Because HCV gene alterable height, lack the suitable cell culture system small animal model of unifying again, effectively the recombiant vaccine research and development are hindered.However, gene vaccine, polypeptide and protein vaccine, based on researchs such as the vaccine of DC cell and viruslike particle vaccines all by extensive concern (Manns et al.2001).When the experimental vaccine of hepatitis C is started to walk,, promptly seek special antigenic component, come immune host in the hope of obtaining neutrality or protection antibody nothing more than traditional vaccine strategy.Under study for action, can induce the anti-memebrane protein of high titre (anti-envelope) antibody to produce but can only watch for animals with reorganization E1/E2 protein immunization orangutan and avoid virus infraction (Foms et al, 1998 at a low price; Zhou et al.2000; Lee et al 1998).Gene vaccine was at first reported in 1992, recently development rapidly, this technology is expelled to host's muscle or Intradermal with the dna direct of coding for antigens, can the inducing specific humoral immunization and broad-spectrum cellullar immunologic response more, the order-checking of HCV whole genome sequence makes the research of HCV gene vaccine progressively go deep into (Jiao et al.2004), but the application of recombinant DNA vaccine is subjected to the restriction of carrier feature equally.The challenge that the HCV vaccine faces, the variation of high degeneration, especially envelope protein that remains virus protein is more main, and the LDL albumen relevant with HCV make that most of virion has can not neutrality, become the challenge of vaccine research.Because the height variability that the weak protection of HCV neutralizing antibody is active, viral and the destruction of body's immunity, the vaccine research thinking of pure strategy can not produce important therapeutical effect (Inchauspe et al.2003) in the immunization therapy of HCV.The present situation of HCV clinical research presses for people, and to establish a kind of be the comprehensive therapy strategy of feature according to virus infection process and human body immunoreation.
(3) HIV vaccine research progress: after early 1980s was found acquired immune deficiency syndrome (AIDS), people promptly began to attempt prevention and the therapeutic vaccine of HIV as far back as 1986.Main vaccine strategy comprises inactivated vaccine, the attenuated live vaccine of HIV; though sustainable generation antibody of vaccine and CTL immunoreation that these obtain by the processing to virus; and but the adult Rhesus Macacus of protection test reduces the SIV infection; but these monkeys finally still develop into acquired immune deficiency syndrome (AIDS) (Baba T W et al.; 1999), thus people to deactivation or attenuated live vaccine the application in human body hold conservative attitude.Research to the HIV recombinant subunit vaccine does not obtain applicable achievement yet, enter the HIV memebrane protein gp160 antigen vaccine of clinical experiment first as the U.S., though gp120 can be incorporated on the cd4 cell and blocks the invasion of HIV to a certain extent, but because HIV has other approach and combines with the virus receptor of cell surface, CTL can not be brought out behind this kind subunit vaccine application human body and neutralizing antibody can not be produced, the III phase clinical experiment of carrying out in the U.S. and Thailand end up in failure (Dennis R B et al., 2004).The HIV virus sample particle vaccines is a kind of vaccine that the virus protein of generation was assembled into the granular structure after applying portion HIV DNA infected.Owing to having very strong immunogenicity in primate, it comes into one's own.The Gag of development such as China Xiao Yao (Xiao Yao etc., 2000) and the virus-like particle of Gag V3 gene can be induced humoral immunization and cellular immunization to a certain degree on mouse model, but do not see the experimental result on primate.Recombinant DNA vaccine is that the HIV exogenous gene is inserted the viral vector of living, and by virus exogenous gene is expressed in host cell, produces wider host immune response thus.These carriers have increased vaccine safety and have not weakened its immunocompetence (Bruyn Get al., 2004).Be further to increase the safety of vaccine, development attenuated vaccinia virus carrier get along with (Qing F, et al., 2005).Dna vaccination is used the plasmid immune animal that contains the HIV gene, but the generation of inducing specific antibody, and the CTL that also observes T lymphocyte specific simultaneously replys.The plasmid DNA vaccine can be induced the SIV specific CTL Rhesus Macacus, and this vaccine-induced immunity can promote the generation that the second time, CTL replied, and can control the metainfective virus replication of pathogenic SIV (Egan M A, et al., 2000) subsequently.Adopt the plasmid DNA immunogen underway as the early stage experimentation of the human body of HIV candidate vaccine.But the HIV vaccine face huge challenge, global so far AIDS vaccine carried out clinical widely before experimentation, and AIDS vaccine I phase clinical experiment nearly 100 times, the minority vaccine has been finished or has been carried out II phase clinical experiment.Do not show the vaccine that definite protective effect can be provided but still have at present in clinical experiment, completed two AIDS vaccine III phase clinical experiments are all counted out.
According to above-mentioned result of study and both at home and abroad immunologic tolerance and immunodeficiency virus vaccine are not made a breakthrough as yet, and face the severe social need and the present situation of technical challenges, we in time propose a kind of new vaccine development strategy, that is: anti-immunologic tolerance of cocktail type and immunodeficiency virus are developed New Policy, its main points are the features of replying according to the feature of viral infection and body, with multiple antiviral combination of elements in a vaccine.The verified vaccine than single component of cocktail vaccine that makes up has bigger immunocompetence, and can suppress the infection of virus, the immunologic injury of duplicating and causing on a plurality of aspects.Cocktail application method in the treatment of erect image AIDS-treating medicine has been obtained good curative effect like that.In the disclosed invention of this patent, we have designed the recombinant DNA, vaccinia virus and/or the adenovirus that infect comprising of stage of a plurality of antiviral elements at viral difference according to the process of virus infection cell and the characteristics of the organism immune response that causes.Can block in the phase I of poisoning intrusion by the cocktail vaccine that their various combination is constructed; RNA interference sequence and sequence library at the characteristic Design of virus variation can effectively suppress the probability that viral nucleic acid duplicates and makes a variation; Use heat shock protein gp96 is as immunological adjuvant energy enhance immunity activity and activate the antiviral natural immunity and adaptive immunity ability; Conserved sequence design from a plurality of series of variation of virus antigen has the recombiant vaccine of extensive cross-reactivity; The mode of immunity of employing recombinant DNA carrier and viral vector immunity can the effect of the most effective performance antiviral immunity.This cocktail vaccine has the vaccine and the not available global advantage of modified model thereof of single composition, is a kind of strategy of brand-new preparation vaccine.
Summary of the invention
The invention is characterized in that use in conjunction comprises immunological adjuvant such as heat shock protein gp96 or its n terminal fragment, antigen-antibody neutralization reaction, cell immune response, antiviral cell factor, receptor blocking agent, film fusion inhibitor and suppresses the vaccine of the strategies such as RNA interfering of virus replication with development anti immune tolerance and immunodeficiency virus.Antigen is meant regulator gene, virus antigen epitope or its multi-epitope fusion gene, the epitope-MHC-I strand trimer (SCT) of viral structural gene or the structural gene that merges with adjuvant, virus etc.Comprise and suppress virus infection and duplicate relevant element, virus antigen and can activate the body natural immunity and element that adaptive immunity is relevant by use in conjunction, reach prophylaxis of viral infections, the control virus replication also recovers the purpose of body's immunity.
In one aspect of the invention, the invention provides the cocktail vaccine of anti-immunologic tolerance virus and/or togavirus, it comprises two or more element and optional virus antigen recombiant protein and optional pharmaceutically acceptable excipient, described element exists with recombinant DNA and/or recombinant virus form, described element is selected from: (A) neutralizing antibody, receptor blocking agent, membrane fusion inhibitor, cytokine coded sequence, and/or RNA interferes sequence; (B) immunological adjuvant coded sequence; (C) antigen gene, virus antigen and immunological adjuvant fusion gene, virus antigen epitope coded sequence, multi-epitope fusion gene and/or virus antigen epitope-MHC-I SCT gene; (D) coded sequence of two of the togavirus fusion rotein seven peptide repetitive sequence HR1 (SEQ ID NO:17), HR2 (SEQ ID NO:16) and/or its polymer HR212 (SEQ ID NO:18).
Particularly, described anti-immunologic tolerance virus can be selected from, but is not limited to HBV and HCV; Described anti-immunodeficiency virus can be selected from, but is not limited to HIV; Described virus antigen recombiant protein can be selected from, but is not limited to following sequence expressed proteins: the comparison conserved sequence of virus antigen, main epidemic strain sequence or its partial sequence, or the fusion gene sequence of virus antigen and adjuvant fusion; Described membrane fusion inhibitor can be selected from, but is not limited to seven peptide repetitive sequence HR1, HR2 or its polymer that the HIV film merges; Described cytokine can be selected from, but is not limited to, cytokine IFN α (1 and 2) or γ, TNF (tumor necrosis factor) α, IL-2; Described RNA interferes sequence to be selected from, but is not limited to encoding viral sequence or non-coding sequence; Described immunological adjuvant can be heat shock protein gp96 or its N terminal sequence; Described antigen gene can be selected from, but is not limited to the comparison conserved sequence of virus antigen, main epidemic strain sequence or its partial sequence, or the fusion gene sequence of virus antigen and adjuvant fusion; Described virus antigen epitope coded sequence can be selected from, but is not limited to viral structural gene or reconciles the gene epitope sequences; Described multi-epitope fusion gene can be selected from, but is not limited to, and viral Th (helper T lymphocyte) epi-position and MHC-I (major histocompatibility antigen I) restriction epi-position and adjuvant are or/and the ubiquitin fusion sequence; Described virus antigen epitope-MHC-I SCT gene can be selected from, but is not limited to the fusion sequence of HLA-A2, HLA-A11, HLA-A24, HLA-30 and virus antigen and β 2m.Described recombinant DNA can be a recombiant plasmid, and described heavy thin virus can be recombinant adenovirus or vaccinia virus recombinant; Described plasmid can be a carrier for expression of eukaryon.
In another aspect of the present invention, the present invention also provides the purposes that the cocktail vaccine of anti-immunologic tolerance virus of the present invention and/or togavirus is used for preparing the medicine that prevents and/or treats the disease that is caused by immunologic tolerance virus and immunodeficiency virus.
Of the present invention aspect another, the invention provides the method for the disease that causes by immunologic tolerance virus and immunodeficiency virus with described Drug therapy.
Be specific embodiments of the present invention below.
1.HBV therapeutic cocktail vaccine
Among the present invention disclosed HBV therapeutic cocktail vaccine be selected from following two or more combination of elements and the recombinant DNA, adenovirus and/or the vaccinia virus that make up and their compositions (1) effectively reduce RNA interfering (siRNA) that HBV-DNA duplicates and/or IFN (interferon) α ( Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? val=NM_024013.1, serial number NM_024013;
Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=protein﹠amp; Val=11067751, serial number NM_000605) or the γ coded sequence; (2) coded sequence of adjuvant molecule gp96 or its n terminal fragment; (3) fusion gene that merges of the structural antigens HBsAg of HBV, HBcAg or itself and adjuvant, epitope, multi-epitope fusion gene and epitope-MHC-I SCT gene of HBV.Dna vector, viral vector have alternative and good exogenous gene expression and the modification ability of insertion sequence flexibly, also have on the viral vector in addition and bring out autarcetic various active element.Immune step immune with dna vaccination and that viral vaccine is strengthened not only can effectively realize the expression of above-mentioned multiple element, and the CTL that immunity is produced also has the ability to the infection site migration.The cross immunity mode of this external vaccinia virus and adenovirus vector can be avoided the neutralization of body to the time being produced with the immunity once again of a kind of viral vector.
The therapeutic cocktail vaccine of HBV comprises among the present invention:
Vaccine combining form one: multichip carrier HBV therapeutic cocktail vaccine
By the multichip carrier cocktail vaccine (shown in Figure 1) that recombinant DNA, vaccinia virus and adenovirus are formed, wherein carrier one disturbs the recombinant adenovirus of element or comprises the recombinant adenovirus storehouse that a plurality of RNA disturb element for comprising the RNA that suppresses hbv replication.Carrier two is for comprising the fusion gene of HBV structural gene or HBV epitope or structural gene and epi-position and adjuvant and recombinant DNA or the recombinant DNA storehouse that comprises the SCT gene of epitope sequences.Carrier three is vaccinia virus recombinant or recombinant virus storehouse, comprise the structural gene of IFN gene, HBV or structural gene and adjuvant gp96 or its N fragment fusion gene, HBV epitope or multi-epitope recombinant and with the SCT gene of adjuvant gp96 or the segmental fusion gene of its N, HBV epitope.Multichip carrier HBV cocktail vaccine also comprises the combination separated from one another of recombinant DNA, vaccinia virus and adenovirus and HBV surface antigen protein.
Vaccine combining form two: single carrier HBV therapeutic cocktail vaccine
It is characterized by the vaccinia virus recombinant or recombinant adenovirus or vaccinia virus recombinant storehouse or the recombinant adenovirus storehouse (as shown in Figure 7) that comprise following element: the structural gene of IFN gene, HBV or structural gene and adjuvant gp96 or its N fragment fusion gene, HBV epitope or multi-epitope recombinant and with the SCT gene of adjuvant gp96 or the segmental fusion gene of its N, HBV epitope, reconcile the corresponding RNA of gene and non-encoding gene and interfere sequence.Single carrier HBV therapeutic cocktail vaccine also comprises the combination separated from one another by recombinant virus or recombinant virus storehouse and HBV surface antigen.
2.HCV therapeutic cocktail vaccine
Disclosed HCV therapeutic cocktail vaccine is to be selected from following two or more combination of elements and recombinant DNA, vaccinia virus and the adenovirus and their compositions (1) that make up effectively reduce RNA interfering (siRNA) and/or IFN α or the γ that HCV duplicates among the present invention; (2) adjuvant molecule gp96 or its n terminal fragment; (3) structural antigens capsid protein core (SEQ IDNO:58), the E1 (SEQ ID NO:69) of HCV, E2 (SEQ ID NO:70) coded sequence ( Http:// hcv.lanl.gov/components/hcv- Db/combined_search/query_one.comp? se_id=17434) or fusion gene, HCV epitope, multi-epitope fusion gene and epitope-MHC-I SCT gene of merging of itself and adjuvant.The combining form of HCV cocktail vaccine is similar to the HBV cocktail vaccine, is divided into multichip carrier HCV therapeutic cocktail vaccine and single carrier HCV therapeutic cocktail vaccine.
3.HIV-1 preventative cocktail vaccine
The preventative cocktail vaccine of disclosed HIV is to be selected from following two or more combination of elements and the recombinant DNA, vaccinia virus and the adenovirus that make up and their compositions among the present invention:
(1) coded sequence of seven peptide repetitive sequence HR212 of virus capable of blocking and cell fusion; (2) coded sequence of adjuvant molecule gp96 or its n terminal fragment; (3) fusion gene, HIV epitope, multi-epitope fusion gene and epitope-MHC-I SCT gene of merging of the structural antigens gene env of HIV (SEQ ID NO:61), gag (SEQ ID NO:59), pol (SEQ ID NO:60) coded sequence or itself and adjuvant.The combining form of the preventative cocktail vaccine of HIV has following several:
Vaccine combining form one: the preventative cocktail vaccine of multichip carrier HIV that contains the HR212 element of secreting, expressing
It is characterized by recombinant DNA, adenovirus and the poxvirus of the HR212 that comprises the secretion type expression feature.Wherein HR212's is characterized as by sequence and sequence library through the conservative seven peptide repetitive sequences of the HIV of comparison, the main epidemic strain of HIV system.
Vaccine combining form two: the preventative cocktail vaccine of multichip carrier HIV
By the multichip carrier cocktail vaccine that recombinant DNA, vaccinia virus and adenovirus are formed, it is characterized by fusion gene, HIV epitope, multi-epitope fusion gene and epitope-MHC-I SCT gene that structural antigens env, gag, the pol of the HR212 that comprises secreting, expressing and HIV or itself and adjuvant merge.Wherein HR212 be characterized as by through the comparison HIV conservative seven peptide repetitive sequences or the main epidemic strain of HIV be HR212 sequence and sequence library thereof.
Vaccine combining form three: the preventative cocktail vaccine of single vector HIV that contains the HR212 element of secreting, expressing
It is characterized by recombinant DNA (storehouse) or recombinant adenovirus (storehouse) or the poxvirus (storehouse) of the HR212 that comprises the secretion type expression feature.Wherein HR212's is characterized as by HR212 sequence and sequence library through the main epidemic strain of HIV conservative seven peptide repetitive sequences, the HIV of comparison system.
Vaccine combining form four: the preventative cocktail vaccine of single vector HIV
By single carrier cocktail vaccine that recombinant DNA or adenovirus or vaccinia virus are formed, it is characterized by antigen gene, HIV epitope, multi-epitope fusion gene and the epitope-MHC-I SCT gene of the fusion that structural antigens env, gag, the pol of the HR212 that comprises secreting, expressing and HIV or itself and adjuvant merge.Wherein HR212's is characterized as by sequence and sequence library through the main epidemic strain of HIV concordance seven peptide repetitive sequences, the HIV of comparison system.
4.HIV-1 therapeutic cocktail vaccine
Disclosed HIV therapeutic cocktail vaccine is to be selected from following two or more combination of elements and the recombinant DNA, vaccinia virus and the adenovirus that make up and their compositions among the present invention:
(1) seven peptide repetitive sequence polymer HR212 of virus capable of blocking and cell fusion; (2) can effectively suppress RNA interfering (siRNA) and/or IFN α or the γ that HIV duplicates; (3) adjuvant molecule gp96 or its n terminal fragment; (4) fusion gene, HIV epitope, multi-epitope fusion gene and epitope-MHC-I SCT gene of merging of the structural antigens env of HIV, gag, pol or itself and adjuvant.The combining form of HIV cocktail vaccine has following several.
Vaccine combining form one: multichip carrier HIV therapeutic cocktail vaccine
By the multichip carrier cocktail vaccine (shown in Figure 1) that recombinant DNA, vaccinia virus and adenovirus are formed, wherein carrier one is adenovirus or adenovirus storehouse, it is characterized by to comprise the HR212's that suppresses effective RNA interference element that HIV duplicates and/or secreting, expressing.Carrier two is the reorganization dna library, it is characterized by fusion gene, HIV epitope, multi-epitope fusion gene and the epitope-MHC-I SCT gene of structural antigens env, gag, the pol of the HR212, the HIV that comprise secreting, expressing or itself and adjuvant fusion.Wherein HR212's is characterized as by sequence and sequence library through the main epidemic strain of HIV concordance seven peptide repetitive sequences, the HIV of comparison system.
Vaccine combining form two: single vector HIV therapeutic cocktail vaccine
By single carrier cocktail vaccine that recombinant DNA or adenovirus or vaccinia virus are formed, it is characterized by antigen gene, HIV epitope, multi-epitope fusion gene and the epitope-MHC-I SCT gene of the fusion that structural antigens env, gag, the pol of the HR212 that comprises secreting, expressing and HIV or itself and adjuvant merge.Wherein HR212's is characterized as by HR212 sequence and sequence library through the main epidemic strain of HIV conservative seven peptide repetitive sequences, the HIV of comparison system.
The accompanying drawing summary
Fig. 1: the carrier of multichip carrier cocktail vaccine and construction strategy.
Wherein the adenovirus vector of carrier I carries single or multiple RNA interference elements, it also can be the adenovirus storehouse of carrying the different RNA interference element, wherein A, B represent different RNA interference elements, a kind of component of carrier I in can independent or blended composition multichip carrier vaccine.The dna vector of carrier II carries the structural antigens gene of virus or the fusion gene that is merged by structural antigens and adjuvant or the multi-epitope gene that constructed by epi-position or by SCT or its mixture of single epi-position structure, A wherein represents above-mentioned any element, a kind of component of carrier II in can independent or blended composition multichip carrier vaccine.Carrier III comprises cytokine coded sequence and optional carrier II gene, and wherein A, B, C, D represent the different elements that carrier III is entrained respectively, a kind of component of carrier III in can independent or blended composition multichip carrier vaccine.Multichip carrier HBV therapeutic cocktail vaccine comprises two kinds or three kinds of carriers of optional independent packing.Wherein carrier I and carrier II can be separately and carrier III combination make up two carrier cocktail vaccines, carrier I and carrier II also can make up three carrier cocktail vaccines with carrier III combination.
Fig. 2: the recombinant adenoviral vector that carries the RAN interference element.
A: what form in the cell goes out complementary sense and antisense oligos at the dna profiling of the siRNA of HBV according to the sequential design of HBV, is annealed into two strands.Link to each other with SalI/XbaI digested vector pAVU6+27 for convenient, 5 ' end at DNA oligos has added SalI, and 3 ' end has added the XbaI enzyme cutting site, and Intermediate grey is partly for transcribing the ring structure that the back expection forms, for translation is effectively stopped, designed termination signal dT (5) endways.
The level that B:Northern hybridization detects HBV mRNA in the HepG2.2.15 cell is extracted the RNA of HepG2.2.15 cell, is that probe carries out Northern hybridization with HBV DNA, and with GAPDH as internal reference.Phosphorus screen analysis result shows, compared with the control, transfection in 2215 cells of siHBV1 (P1) or siHBV2 (P2) recombinant adenovirus, the mRNA level of 3.5Kb and 2.4Kb/2.1Kb obviously reduces.In matched group, transfection with the cell of irrelevant recombinant virus U6 (mock) of HBV gene or siEGFP (E) in, mRNA in its mRNA level and the non-transfected cells (C) is on close level, and shows that crt gene siEGFP can not influence the HBVmRNA level, has shown the specificity of siRNA effect.C represents 2215 cells of untransfected; Mock, E, P1, P2 have represented transfection respectively and have contained U6+27, U6+27-siEGFP, U6+27-siHBV1,2215 cells of U6+27-siHBV2 recombinant adenovirus.
The C:ELISA method detects the supernatant that the expression (percentage ratio compared with the control) of HBsAg and HBeAg in 2215 cells is got 2215 cells, detects HBsAg, HBeAg level in the cell conditioned medium with the ELISA detection kit.The result shows and to compare with control cells (C), transfection in the cell of siHBV1 (P1) or siHBV2 (P2) recombinant adenovirus HBsAg, HBeAg protein level obviously suppressed.Transfection the cell of siHBV1 recombinant virus, its HBsAg 90.6 ± 0.5% (P=0.00226) that descended, HBeAg 88.4 ± 0.9% (P=0.00011) that descended; Transfection the cell of siHBV2 recombiant plasmid, its HBsAg 92.9 ± 0.6% (P=0.00216) that descended, HBeAg 89.7 ± 1.0% (P=0.0001) that descended, the fall of data show HBsAg is slightly larger than HBeAg.Show that this EVAC is fine to the effect of HBV gene inhibition.C represents 2215 cells of untransfected; Mock, E, P1, P2 have represented transfection respectively and have contained U6+27, U6+27-siEGFP, U6+27-siHBV1,2215 cells of U6+27-siHBV2 recombinant adenovirus.
D:Real time PCR detects the supernatant that the HBV dna content is got 2215 cells, and wherein viral DNA is carried out real time pcr analysis.Be the quantitatively copy number of HBV DNA, the concentration known that increased simultaneously and through the topo-HBV plasmid of serial dilution, produce standard curve, the Ct value of establishing criteria curve and the HBV DNA that records calculates the copy number of HBV DNA.The result shows, contains the cell of the adenovirus infection of siHBV1 (P1) and siHBV2 (P2), HBV dna level in its supernatant and contrast (14.48 ± 2.15 * 10 4Copys/mL) compare and descended 3.7 times (3.91 ± 0.72 * 10 respectively 4Copys/mL; P=0.00821) and 3.3 times (4.41 ± 0.85 * 10 4Copys/mL; P=0.00780).
Fig. 3: the recombinant DNA carrier that carries structural antigens.
A. the construction and expression of fusion gene.(A) fusion gene makes up sketch map.Gene is under the CMV of pcDNA3 promoter.Introducing flexible connexon " GGSGG " in fusion gene guarantees the correct expression of each gene and keeps normal function.N355 w355 aminoacid of acute pyogenic infection of finger tip Mus or people's gp96N end.(B) western blot hybridization analysis.The clone who makes up is through calcium phosphate method transfection 293T cell, two days later, collecting cell, cell lysate detects behind immunoprecipitation.HBcAg (SEQ ID NO:4), HBsAg (SEQ ID NO:5), N355 (SEQ ID NO:2,3), CN355 (fusion gene of HBV cAg and N355) (SEQ ID NO:7), SN355m (fusion gene of the surface antigen S of HBV and Mus gp96N end N355) (SEQ ID NO:8), SN355h (being the surface antigen S of HBV and the fusion gene of people gp96N end N355) (SEQ ID NO:9), S2S (SEQ ID NO:6), S2SN355 (fusion gene of the surface antigen S 2S of HBV and gp96N end N355) (SEQ ID NO:10) and N355S2S (fusion gene of the surface antigen S 2S of HBV and gp96N end N355) (SEQ ID NO:11) are respectively 17kDa etc. the molecular weight of gene outcome, 24kDa, 45kDa, 62kDa, 69kDa, 69kDa, 31kDa, 76kDa and 76kDa.(a) the cAg monoclonal antibody of anti-mice detect truncate cAg and with the fusion product of N355.(b, c, d) the surface antigen monoclonal antibody of anti-mice detect the S of hepatitis B membrane antigen and M albumen and with the fusion product of N355.The band of non-glycosylated (little band) and glycosylation (big band) can both detect.
B.DNA exempts from the detection of humoral immune reaction behind BALB/c Mus and the HLA/A2 transgenic mouse.Immunity is three times altogether, every group of 5 Mus, each intramuscular injection 100 μ g plasmids.For being total to immune group, before the injection each 50 μ g of two kinds of plasmids are mixed.Back 10 days eye sockets of last immunity are got blood, and ELISA serial dilution method is surveyed antibody titer.The result is with light absorption meansigma methods (A 490nm) ± SD represents.(A) surface antibody after 1/1000 dilution of BALB/c Mus serum, (B) surface antibody after 1/1000 dilution of Anti-HBs in HLA/A2 transgenic mouse serum, (C) and (D) be respectively the core antibody after BALB/c Mus and 1/10000 dilution of HLA/A2 transgenic mouse serum, (E) BALB/c Mus and HLA/A2 transgenic mouse serum 1/100 dilution back N355 antibody (IgG).All data are a representative value of three different experiments.
C.ELISPOT detects the reaction of HBV specific cell.Dna vaccination immunity BALB/c Mus and HLA/A2 transgenic mouse three times, every group of 5 Mus, each intramuscular injection 100 μ g plasmids.For being total to immune group, before the injection each 50 μ g of two kinds of plasmids are mixed.Last immunity was got the mouse spleen cell in back 10 days and is analyzed.IFN-γ and CD8 that HBV is special +The speckle number that two positive cells form is with 5 * 10 5Spleen cell calculates.Every group of speckle number (deduct and do not have polypeptide stimulated control speckle) represents that with the mean value standard variance of three numbers all data are a representative value of three different experiments.(A) epi-position HBs362-371 and Ld28-39 analyze with the BALB/c Mus, and epi-position HBs348-357 is used for the analysis of HLA/A2 transgenic mouse.(B) epi-position HBc87-95 is used for the analysis of BALB/c Mus, and epi-position HBc18-27 is used for the analysis of HLA/A2 transgenic mouse.
Fig. 4: the SCT recombinant DNA carrier that carries epitope-MHC-I.
SCT is by with epitope, and β 2m is connected the fusion gene that makes up with the MHC-I quasi-molecule by 15 amino acid whose GS connexons, and its structure is shown in figure A.B, SCT can present epitope at cell surface effectively.C can produce special CTL with SCT reorganization DAN carrier immunity HLA-A2 transgenic mouse, and has the ability that suppresses hbv replication in the HepG2.2.15 cell.pcDNA-SCT-C18(SEQ?ID?NO:13),pcDNA-SCT-C107(SEQID?NO:14),pcDNA-SCT-HIV-gag(SEQ?ID?NO:15)
Fig. 5: the recombinant DNA carrier that carries former epi-position of multi-resistance and adjuvant molecule fusion gene.The ideograph of the fusion gene (SEQ ID NO:12) that makes up by the multi-epitope fusion
Fig. 6: the CTL that vaccinia virus recombinant and multichip carrier therapeutic HBV and HIV vaccine immunity produce.Can bring out the relevant CTL level of HBV cAg with the HBV cAg-SCT recombinant DNA among the embodiment one carrier 2B efficiently with the multichip carrier HBV therapeutic vaccine immunity HLA-A2 transgenic mouse that constitutes as the recombinant vaccinia of figure shown in the A.Gag epi-position with HIV has consistent experimental result.Fig. 6 A, one of structure form of vaccinia virus recombinant.Fig. 6 B, usefulness contains the reorganization DAN carrier of SCT and the CTL that vaccinia virus recombinant immunity HLA-A2 transgenic mouse can efficiently produce HBV cAg and HIVgag antigen-specific.In the present embodiment, the A on the carrier is γ-IFN, and B is S2SN355, and C is HLA-A2-SCT-C18.
Fig. 7 list carrier cocktail vaccine.
In single carrier cocktail vaccine of HBV, the ABC on the carrier is IFN α and β, S, S2S, S1S2S, C, SCT, gp96, the optional combination of Gp96N-S/C.D is that the special RNA of regulator gene and non-encoding gene interferes sequence.Single carrier cocktail vaccine also comprise contain different elements respectively by the mixing of the formed single carrier of same carrier of recombinant DNA, adenovirus or vaccinia virus recombinant.Single vehicle treatment cocktail vaccine of HCV is meant the single vehicle treatment cocktail vaccine among the embodiment 21.Single carrier cocktail vaccine of HIV is meant the preventative and therapeutic vaccine of single vector HIV of four kinds of embodiment three and embodiment.Wherein the ABC on the carrier is IFN α and γ, core, and E1, E2, Gp96, the optional combination of Gp96 or its N end-Core/E1/E2, D is that the special RNA of regulator gene and non-encoding gene interferes sequence.Single carrier cocktail vaccine of HIV is meant the preventative and therapeutic vaccine of single vector HIV of four kinds of embodiment three and embodiment, wherein the ABC on the carrier is IFN α and γ, gag, pol, env, Gp96, the optional combination of Gp96 or its N end-gag/pol/env, D is that the special RNA of regulator gene and non-encoding gene interferes sequence.
Fig. 8: the construction strategy of the Enveloped Virus-cell Membrane Fusion suppressor gene of eukaryotic expression system.
The construction strategy of the membrane fusion inhibitor of A band tPA signal peptide
The construction strategy of the membrane fusion inhibitor of B band IgG signal peptide
The RNA of Fig. 9: HBV interferes sequence.
A: the intervening rna sequence corresponding with HBV X antigen changes into is inserted into sequence on the pAVU6+27 carrier.
B: the intervening rna sequence corresponding with HBV S antigen changes into is inserted into sequence on the pAVU6+27 carrier.
C: the intervening rna sequence corresponding with HBV C antigen changes into is inserted into sequence on the pAVU6+27 carrier.
D: the intervening rna sequence corresponding with HBV P antigen changes into is inserted into sequence on the pAVU6+27 carrier.
Embodiment
Be some embodiments of the present invention below, but described embodiment only is an illustrative, and scope of the present invention is not played the qualification effect.
Embodiment one: the therapeutic cocktail vaccine of HBV
The characteristics of the immunologic tolerance that infection causes according to HBV, its therapeutic cocktail vaccine is single carrier bacterin or the multichip carrier combination-vaccine that comprises a plurality of elements.Cocktail vaccine has and is better than the therapeutic purposes combination advantage that discrete component is realized.Present embodiment provides a kind of structure of multichip carrier cocktail vaccine and the building mode of compound mode and a kind of single carrier cocktail vaccine, but the content that this patent comprised is not limited to the compound mode of the disclosed cocktail vaccine of present embodiment.
1. multichip carrier HBV therapeutic cocktail vaccine (Fig. 1)
The composition and the application of multichip carrier HBV therapeutic cocktail vaccine
Carrier 1: the recombinant adenovirus that contains the RNA interference sequence
The virus replication that continues is a key character of immunologic tolerance virosis, and duplicating of virus not only replenished the storage capacity of virus in the host, produces a large amount of mutants, also caused immune exhaustion and functional defect.Therefore, an important behave of immunologic tolerance virosis treatment is titre and the levels of replication that at first suppresses virus.For RNA or have the virus of RNA intermediate, select virus genomic particular sequence, the RNA that carries by suitable carriers in relate to inhibition virus that element can be very efficient special continue duplicate and antigenic continuous expression.In the present embodiment, we are example with the levels of replication that the recombinant adenovirus that carries the HBV interference sequence reduces HBV, provide preparation and have the method that the RNA that suppresses the HBV virus replication interferes vaccine.But the content of the carrier one that this patent comprised is not limited only to this disclosed sequence and carrier.
A: the RNA at the single site of hepatitis B virus on the single adenovirus vector disturbs
At the antigenic mRNA sequence of hepatitis B virus (HBV) X, utilize the software of the online design siRNA sequence of Dharmacon company, a plurality of siRNA sequences have been designed, and convert them to complementary two DNA sequence (Fig. 2 A) separately, wherein link to each other for U6+27 promoter convenient and that SalI/XbaI digested vector pAVU6+27 obtains, 5 ' end at DNA oligos has added SalI, and 3 ' end has added the XbaI enzyme cutting site.For translation is effectively stopped, also designed termination signal dT (5) endways.After Yu Boya company was synthetic, 100 ℃ were boiled 3 minutes, were cooled to room temperature automatically and carried out two strands annealing, were cloned among the carrier pAVU6+27 (available from Clontech), obtained expressing the expression cassette carrier pAVU6+27-siHBV at the siRNA of HBV.Transient cotransfection pUC19-HBV1.3 (MBI) plasmid and pAVU6+27-siHBV plasmid are in HepG2 (available from ATCC) cell in advance, utilize ELISA to detect relevant S antigen of hepatitis B and the antigenic expression of e, tentatively infer and effectively to suppress the sequence that HBV expresses, Preliminary screening obtains 2 sequences, called after pAVU6+27-siHBV1 (SEQ ID NO:20) and pAVU6+27-siHBV2 (SEQ IDNO:21) (shown in Fig. 2 A) further will comprise in the pAVU6+27-siHBV1 of ordered sequence (seeing shown in Fig. 2 A) and the pAVU6+27-siHBV2 carrier expression cassette and be cloned in pshuttle (available from the Stratagene) carrier of adenovirus.Method is: utilize HindIII to downcut the expression cassette of the siRNA among the carrier pAVU6+27-siHBV, parallel glue reclaims.The pshuttle carrier is utilized the HindIII enzyme action, and dephosphorylation, be connected with the expression cassette of the siRNA that reclaims, obtain purpose plasmid pshuttle-U6+27-siHBV1 and pshuttle-U6+27-siHBV2.With this purpose plasmid pmeI linearisation, utilize ethanol precipitation to reclaim, forward among the competent cell BJ5183 (available from Stratagene) with the common electricity of pAdEasy-1 (available from Stratagene) plasmid, carry out homologous recombination, obtain containing the recombinant of siRNA expression cassette.Further recombinant is utilized pacI linearisation enzyme action to reclaim back transfection virus package cell line GP-293 (available from ATCC), after 7-10 days, collect virus.
The GP-293 cell is paved into monolayer, adds the viral liquid of serial dilution according to gradient, cultivated 48 hours with 1% semisolid culturemedium then, counting forms has a liking for the speckle number, according to the viral dilution ratio with have a liking for the titre that the speckle number is determined recombinant adenovirus.Infection multiplicity according to 0.1MOI infects HepG2.2.15 cell (available from ATCC) (each antigen of stably express HBV, and energy Packing Intact virion), detect HBV surface antigen S and the antigenic expression of e with the ELISA method after 72 hours, (C) compares with control cells, transfection in the cell of siHBV1 (P1) or siHBV2 (P2) recombinant virus the antigenic level of S, e obviously suppressed.Infected the cell of siHBV1 virus, its S antigen 90.6 ± 0.5% (P=0.00226) that descended, e antigen 88.4 ± 0.9% (P=0.00011) that descended; Infected the cell of siHBV2 adenovirus, its S antigen 92.9 ± 0.6% (P=0.00216) that descended, e antigen 89.7 ± 1.0% (P=0.0001) (Fig. 2 B) that descended.The Northern results of hybridization shows that in the cell of viral infection, the mRNA level of 3.5Kb and 2.4Kb/2.1Kb obviously reduces (Fig. 2 C).Fluorescence quantitative PCR detection result shows, infected cells, HBV dna level in its supernatant and contrast (14.48 ± 2.15 * 10 4Copys/mL) compare 3.7 times of (3.91+0.72 * 10 that descended respectively 4Copys/mL; P=0.00821) and 3.3 times (4.41 ± 0.85 * 10 4Copys/mL; P=0.00780) (Fig. 2 D).These results show that the RNAi method of the special target HBV gene that this is adenovirus mediated can effectively suppress HBV expression of gene in the HepG2.2.15 cell, and final effectively duplicating of inhibition HBV.
B: the RNA at a plurality of sites of hepatitis B virus on the single adenovirus vector disturbs
Utilize the method among the A, we have screened respectively at the antigenic siRNA sequence of S, X, C or polymerase P of HBV, have obtained 6 pAVU6+27-siHBV recombiant plasmid that can effectively suppress hbv replication.Wherein:
The X interference sequence is 1.5 ' GAGGACTCTTGGACTCTCA-3 ' (SEQ IDNO:62); ' 2.5 CCGTGTGCACTTCGCTTCACCTCTG-3 ' (SEQ IDNO:63); It is inserted into structure among the pAVU6+27 shown in Fig. 9 A.
S interference sequence 1.5 ' CATCACATCAGGATTCCTA-3 ' (SEQ ID NO:64); ' 2.5 CTCAGTTTACTAGTGCCATTTGTTC-3 ' (SEQ ID NO:65); It is inserted into structure among the pAVU6+27 shown in Fig. 9 B.
C interference sequence 5 ' CATCACATCAGGATTCCTAAA-3 ' (SEQ IDNO:66); It is inserted into structure among the pAVU6+27 shown in Fig. 9 C.
P polymerase interference sequence, 5 ' AGAAGATCTCAATCTCGGGAATCTC-3 ' (SEQ ID NO:67).It is inserted into structure among the pAVU6+27 shown in Fig. 9 D.
Further respectively with in the carrier that builds at S, X, the expression cassette of the siRNA of C or P in twos combination clone in the pshuttle carrier, that is: utilize the expression cassette among the pAVU6+27-siHBV under the HindIII enzyme action earlier, glue is linked in the pshuttle of same processing after reclaiming, obtain purpose clone pshuttle-siHBV1, further will downcut and reclaim with ECoRV and stuI at the expression cassette of another antigenic pAVU6+27-siHBV, put down end with ECoRV enzyme action pshuttle-siHBV1 and dephosphorylation with the expression cassette of the siRNA that downcuts with ECoRV and stuI and be connected, obtained being connected with on the pshuttle carrier expression cassette of two siRNA like this.Same method makes up all expression cassettes at the siRNA of HBV that obtain in twos.The method of utilizing the adenovirus described in the A to set up has obtained X, S, three kinds of adenovirus particles that make up in twos of P and C.After infecting HepG2.2.15, obtained and above similar result.Further with resulting adenovirus as a storehouse, infect the HepG2.2.15 cell after, the inhibition efficient of HBV is better than infection with single adenovirus particles.
Carrier 2: the recombinant DNA carrier that contains HBV structural antigens, adjuvant, adjuvant and structural antigens fusion gene, epi-position and multi-epitope fusion gene, epi-position and adjuvant fusion gene and epi-position-MHC-SCT
A. the recombinant DNA carrier that contains HBV structural antigens, adjuvant, adjuvant and structural antigens fusion gene
Structural protein cAg HbcAg (C) coded sequence (SEQ ID NO:4) with HBV, N end 355 aminoacid (mature peptide does not contain signal peptide) coded sequence N355 of the gp96 of the small protein HBsAg (S) of membrane antigen (SEQ ID NO:5) and middle Protein S 2S (SEQ ID NO:6), people or mice h(SEQ ID NO:2) and N355 m(SEQ ID NO:1) and said structure albumen and gp96N end gene fusion construct.During gene fusion construct, introduce connexon " GGSGG ", its coded sequence is " GGT TCC TCT GGA GGT ".The amplification method of fusion gene is: earlier hold and antigen sequence two end fragments that purification obtains with both sides primer and the middle primer gp96N that increases respectively respectively; Mix two fragments then as template, with the primer amplification fusion gene of both sides.All amplimers are listed in the table 1, and wherein tilted letter is a restriction enzyme site, the connexon of bold-faced letter for introducing.The sketch map of the dna vaccination that makes up as shown in Figure 3A.
Table 1 makes up the primer sequence of HBV structural gene and fusion gene vaccine
Title Primer sequence 5 '-3 '
pcDNA-S (a)+gcggatccatggagaacatcacatcaggat
(b)-gcgctcgagttaaatgtatacccaaagac
pcDNA-HBcAg(C) (c)+gcggatccatggacattgacccgtataaag
(d)-gcgctcgagttaaataacacaagtctccgg
pcDNA-N355 m (e)+gcggatccatggatgatgaagtcgacgt
(f)-gcgctcgagttaagtgaagtggatataagccat
pcDNA-S2S (g)+gcggtaccatgcagtggaattccacaacct
(h)-tagcggccgcttaaatgtatacccaaagac
pcDNA-SN355 mWith (a)+(i) and two terminal sequences that (j)+(f) increase, use (a)+f) amplification fusion gene during amplification (a)+(i)acctccagaggaaccaatgtatacccaaagac
ggttcctctggaggtgatgatgaagtcgacgt(j)+(f)
(a)+(f)
pcDNA-SN355 h (a)+(i)
ggttcctctggaggtgacgatgaagttgatgt+ gcgctcgagttaagtaaagtgaatataagcc(k)
(a)+(k)
pcDNA-CN355 m (c)+acctccagaggaaccaataacacaagtctccg
(j)+(f)
(c)+(f)
pcDNA-S2SN355 m (g)+acctccagaggaaccaatgtatacccaaag
ggttcctctggaggtgatgatgaagtcgac+ tagcggccgcttaagtgaagtggatataag(l)
(g)+(l)
pcDNA-N355 mS2S taggtaccatggatgatgaagtcgacgtgg(m)+ acctccagaggaaccaatgtatacccaaag
ggttcctctggaggtgatgatgaagtcgac+(h)
(m)+(h)
Annotate: " N355 m" represent the N355 (SEQ ID NO:1) in Mus source, " N355 h" N355 (SEQ IDNO:2) in representative source.Wherein (a) is SEQ ID NO:24; (b) be SEQ ID NO:25; (c) be SEQ ID NO:26; (d) be SEQ ID NO:27; (e) be SEQ ID NO:28; (f) be SEQ ID NO:29; (g) be SEQ ID NO:30; (h) be SEQ ID NO:31; (i) be SEQ ID NO:32; (j) be SEQ ID NO:33; SEQ ID NO:34 is ggttcctctggaggtgacgatgaagttgatgt; (k) be SEQ ID NO:35; Acctccagaggaaccaataacacaagtctccg is SEQ ID NO:36; Acctccagaggaaccaatgtatacccaaag is SEQ ID NO:37; Ggttcctctggaggtgatgatgaagtcgac is SEQ ID NO:38; (l) be SEQ ID NO:39; (m) be SEQ ID NO:40; Acctccagaggaaccaatgtatacccaaag is SEQ ID NO:41; Ggttcctctggaggtgatgatgaagtcgac is SEQ ID NO:42.
With the mode immunity BALB/c and the HLA-A2 transgenic mouse (available from Jackson Lab) of intramuscular injection, immune three times, dosage is 100 μ g/ Mus to the dna vaccination that obtains, at interval two weeks respectively.The peripheral blood of getting Mus after two weeks of last immunity prepares serum, detects the antibody titer of related antigen among the HBV.Get the spleen cell of Mus, detect the level of the CTL generation of the HBV antigen-specific that produces in the spleen.Experimental result shows that the form of dna vaccination immunity can produce tangible antibody and CTL level, under the adjuvant effect of gp96N end, produces the neutralizing antibody of Th1 class, and the level of CTL improves 6-8 doubly.Fig. 3 B and Fig. 3 C have explained the level that antibody horizontal and CTL produce.
B. the recombinant DNA that makes up by epitope-MHC-I SCT
Be built into monomolecular SCT on MHC-I and the B2M and can effectively the corresponding epi-position of virus antigen be presented at cell surface by in advance epi-position being fused to, bring out the generation of the relevant CTL of virus antigen very efficiently.Provide the result of the CTL that method that two relevant epi-positions of HBV cAg of HLA-A2 restriction make up and immune mouse produced in the present embodiment.The content that this patent comprised is not limited to the epi-position of HLA-A2 and cAg, and the recombinant DNA carrier that is obtained can be the single carrier that comprises an epi-position, also can be the multi-epitope recombinant DNA mixture that comprises a plurality of epi-positions.
Fig. 4 A is depicted as the ideograph of SCT, wherein connects by 15 amino acid whose GS connexons between each gene.The N end of SCT is a signal peptide, and the epi-position that it comprised can be introduced by the mode of primer sudden change.The SCT dna vector that makes up can be presented the cAg epi-position at cell surface (Fig. 4 B) effectively.The special CTL of epi-position can be produced with SCT dna vector immunity HLA-A2 transgenic mouse, and the expression (Fig. 4 C) of HBV surface antigen and cAg in the HepG2.2.15 cell can be reduced.
Adopt the mode of PCR sudden change to change the entrained epitope of HLA-A2:
The SCT primer:
A2-SCT-up1:CAGCTGTGGAATGTGTGTCAGTTAG(SEQ?ID?NO:43)
A2-SCT-primer2:AATCAGCAAGCTTGGTACCGA(SEQ?ID?NO:44)
The HBcAg18-27 mutant primer:
A2SCT-HBcAg18-27-1(match?1):
AACAGAAGGAAAGAAGTCAGAAGGCAAAAAAGCCTCCAGGCCAGAAAG(SEQ?ID?NO:45)
A2SCT-HBcAg18-27-2(match?2):
TTTTTGCCTTCTGACTTCTTTCCTTCTGTTGGAGGTGGGGGAGGCGGA(SEQ?ID?NO:46)
The mutant primer of HBcAg107-115
A2SCT-HBcAg107-115-1(match?1):
AACAGTTTCTCTTCCAAAAGTAAGACAAGCCTCCAGGCCAGAAAG(SEQ?ID?NO:47)
A2SCT-HBcAg107-115-2(match?2):
TGTCTTACTTTTGGAAGAGAAACTGTTGGAGGTGGGGGAGGCGGA(SEQ?ID?NO:48)
C. merge the multi-epitope recombinant DNA carrier that forms by former epi-position of multi-resistance and adjuvant
Ubiquitin can promote protein degradation in cell, improves the working (machining) efficiency of epi-position, therefore can strengthen cellular immune function.Epi-position can be in body the generation of inducing specific CTL.We have synthesized the t cell epitope of five HBV, be respectively the restrictive epi-position C18-27 of HLA-A2 (FLPSDFFPSV) (SEQ ID NO:72), E335-343 (WLSLLVPFV) (SEQ IDNO:73) and E183-191 (FLLTRILTI) (SEQ ID NO:74), the restrictive epi-position C88-96 of HLA-A11 (YVNVNMGLKL) (SEQ ID NO:75), the restrictive epi-position C117-125 of HLA-A24 (EYLVSFGVW) (SEQ ID NO:76), its polyphone is synthetic, and serial arrangement as shown in Figure 5.Connect the tetanus toxoid general auxiliary epi-position of Th (QYIKANSKFIGITE) (SEQ ID NO:77) at multi-epitope sequence 5 ' end, and be connected in the reading frame, connect by connexon AAY between each epi-position with ubiquitin.The fusion gene sequence called after NHTU (SEQ ID NO:12) of design, serial arrangement as shown in Figure 5.The synthetic DNA of expressing this serial sequence is connected in pcDNA3.1 (available from the Invitrogen) carrier for expression of eukaryon constitutes dna vaccination.With recombinant DNA immunity HLA-A2 transgenic mouse, by muscle immunity three times, can in body, effectively produce specific CTL, thereby body is produced immune protective effect.
Carrier 3: the vaccinia virus recombinant carrier that contains HBV structural antigens, adjuvant, adjuvant and structural antigens fusion gene, epi-position and multi-epitope recombinant and epi-position-MHC-I SCT, γ-IFN
The recombinant vaccinia carrier can carry multiple Expression element, and vaccinia virus can be brought out mucosal immunoreaction efficiently simultaneously, activates natural immune system.The vaccinia virus recombinant that comprises a plurality of elements can be used for independent immunity, also can be with adenovirus or/and recombinant DNA carrier immunity or immunity stage by stage.A kind of building mode that comprises the vaccinia virus recombinant of a plurality of elements is provided in the present embodiment, but element that the content that this patent comprised is not limited in the disclosure to be comprised and combination of elements order.
In the synthetic mode of full gene according to NCBI (National Center for BiotechnologyInformation, http://www.ncbi.nlm.nih.gov/) the 127-627 bit base in the full gene of the synthetic IFN-γ of the sequence shown in the disclosed IFN-γ (NM_000619), wherein the front end of sequence adds 5 '-GCGAAGCTTG-3 ' sequence (SEQ ID NO:49), and the rear end of sequence adds 3 '-ACTCGAGCAT-5 ' sequence (SEQ ID NO:50).Synthetic gene inserts pMD18T carrier (available from TAKARA), carries out sequencing.The correct gene that checks order carries out double digestion with HindIII/Xho I, inserts then in the pcDNA3.0 carrier (available from Invitrogen) of same enzyme action.Make up the carrier for expression of eukaryon of IFN-γ.PcDNA3.0-IFN-γ expression vector is inserted in the recombinant vaccinia carrier (available from Wuhan University) through double digestion and the flush endization of BglII and SmaI, makes up the vaccinia virus recombinant carrier that contains IFN-γ.
Element in present embodiment one carrier 2 is carried out enzyme action and flush endization, be connected to then on the recombinant vaccinia carrier, determine direction of insertion by the mode of enzyme action and order-checking.The recombinant vaccinia carrier 1 μ g and the wild vaccinia virus Tiantan strain (available from Chinese CDC) that obtain (0.1MOI) are carried out homologous recombination in Vero cell (available from ATCC), pass through the expression of EGFP or the expression screening recombinant virus of genes of interest after 24 hours.Recombinant virus carries out purification through six single speckle screenings of taking turns, and the virus of purification increases in the Vero cell, and carries out purification with 36% sucrose pad.Fig. 6 A is an example that comprises some vaccinia virus recombinant carriers of a plurality of elements, and Fig. 6 B utilizes the cocktail vaccine that contains carrier 2 and carrier 3, the recombiant plasmid pcDNA3.0-SCT-C18 intramuscular injection immune transgenic Mus of getting 100 μ g, and all backs are with 2 * 10 6-8The recombinant virus of PFU adopts the mode of intramuscular injection to carry out booster immunization, and the spleen cell of getting mice after the week detects the CTL that mice produced.
The comparative advantages of multichip carrier HBV therapeutic vaccine
With recombinant DNA, adenovirus and the vaccinia virus vector vaccine of above-mentioned structure, the flow process of immune HBV transgenic mouse is: first immunisation uses 2 * 10 6MOI recombinant adenovirus or recombinant adenovirus storehouse RNAi interfere vaccine to reduce hbv replication, after one month with each 50 μ g (pcDNA3.0-SN and pcDNA3.0-CN) intramuscular injection immune transgenic Mus of recombinant DNA vaccine, the recombinant s protein subcutaneous injection immune transgenic Mus that is aided with gp96 and the 50 μ g of 20 μ g simultaneously, 2 of immunity all backs are with 2 * 10 for the second time 6The vaccinia virus recombinant of MOI carries out booster immunization.Experimental result shows: carry out just exempting from the interior HBV titre of back transgenic mouse body with recombinant adenovirus and recombinant adenovirus storehouse RNAi vaccine and reduce more than 90%; begin to occur surface antigen and the special ctl response of cAg after the immune week for the second time; and the S antibody of generation protectiveness; the titre of HBV continues to reduce, and serum HBV DNA presents feminine gender after 2 weeks.After using vaccinia virus recombinant (containing γ-IFN, S antigen and C antigen gene) booster immunization for the third time, the antibody horizontal of surface antigen and cAg correspondence significantly improves in the serum, is accompanied by antigen-specific CD8 +The generation of T cell and γ-IFN and to the liver internal migration slight ALT level occurs and improves in the serum.Treat after three months, serum HBV DNA feminine gender, the ALT level is normal, and the core antigen-positive cell is negative in the liver, and cccDNA detects negative.The HBV transgenic mouse produces the comprehensive therapy effect that obviously is better than the one pack system vaccine behind the cocktail vaccine Comprehensive Treatment.Because the application of adjuvant molecule, the antibody of generation and cellular immunization are Th1; Because the generation of γ-IFN and to the migration of liver inside, the HBV that has obtained the non-cracking performance of liver cell removes ability; Through the combined immunization of dna vaccination and vaccine vaccine, the significant polyclone CTL of generation replys in the body, and produces the memory t cell clone, thereby has further removed the infection cell of the inner trace of liver.Because the generation of protection antibody and multi-epitope CTL and immunological memory, the virion of HBV transgenic mouse inside has obtained effective removing.
2. single carrier HBV therapeutic cocktail vaccine (Fig. 7)
The HBV therapeutic cocktail vaccine that present patent application comprised also comprises single carrier recombinant DNA, adenovirus or the vaccinia virus vector vaccine that the multichip carrier HBV therapeutic vaccine corresponding elements among the embodiment one is made up and make up.Concrete example is a vaccinia virus recombinant as shown in Figure 7, wherein comprise and reduce the needed RNA interference element of virus replication, γ-IFN, bring out the structural antigens gene of neutralizing antibody and polyclone CTL and the structural gene that merges with adjuvant, and the SCT etc. that brings out efficient CTL.
Single carrier HBV therapeutic vaccine that present patent application comprised also comprises the recombiant plasmid that comprises different elements that is made of carrier of the same race or the combination of recombinant virus.
The described single carrier HBV therapeutic vaccine of present patent application has consistent inhibition virus replication and brings out immunoreactive function with multichip carrier HBV therapeutic vaccine.
Embodiment two: the therapeutic cocktail vaccine (Fig. 1) of HCV
1.HCV multichip carrier therapeutic cocktail vaccine
The multichip carrier therapeutic cocktail vaccine of the construction strategy of the multichip carrier therapeutic vaccine of HCV and method and HBV is similar, and as shown in Figure 1, wherein carrier 1 is for containing the recombinant adenovirus or the adenovirus storehouse of RNA interference element.ABC in the carrier 2 and 3 is meant IFN, the structural antigens capsid protein (core) of HCV, E1, E2, SCT and multi-epitope fusion gene that epi-position is relevant, gp96 coded sequence (SEQ ID NO:3), the optional combination of Gp96N-core/E1/E2 fusion gene.
The simple construction strategy of setting forth the recombinant relevant in the present embodiment with wherein part carrier.
Utilize with the same method of embodiment one carrier 1 and obtained suppressing the ordered sequence that HCV expresses: 5 ' CCCGCTCAATGCCTGGAG-3 ' (SEQ ID NO:68), be converted into the sequence of inserting on the pAVU6+27 carrier, acquisition has the ordered sequence (SEQ IDNO:22) that inhibition HCV duplicates, this sequence is at HCV5 ' UTR zone, utilizes above-mentioned method packaging virus standby.With Huh-mono cell (available from ATCC) is model (stably express I389/hyg-ubi/NS3-3 '/5.1 HCV replicons), with this cell of packaged viral infection.The result shows: descended more than 60% by the levels of replication of HCV in the cell of viral infection, the expression of one of them important albumen HCV-NS5B has descended more than 70%.
The vaccinia virus recombinant immunity HLA-A2 transgenic mouse that will comprise the recombinant DNA vaccine of HCV structural gene and comprise γ-IFN, HCV structural gene and HCV epi-position SCT gene can produce the special polyclone ctl response of HCV efficiently.
2.HCV single vehicle treatment cocktail vaccine (Fig. 7)
The HCV therapeutic cocktail vaccine that present patent application comprised also comprises single carrier recombinant DNA, adenovirus or the vaccinia virus vector vaccine that the multichip carrier HCV therapeutic vaccine corresponding elements among the embodiment 21 is made up and make up.Concrete example is a vaccinia virus recombinant as shown in Figure 7, wherein comprise and reduce the needed RNA interference element of virus replication and γ-IFN or α-IFN, bring out the structural antigens gene of neutralizing antibody and polyclone CTL and the structural gene that merges with adjuvant, and the SCT etc. that brings out efficient CTL.
Single carrier HCV therapeutic vaccine that this patent comprised also comprises the recombiant plasmid that comprises different elements that is made of carrier of the same race or the combination of recombinant virus.
The described single carrier HCV therapeutic vaccine of present patent application has consistent inhibition virus replication and brings out immunoreactive function with multichip carrier HCV therapeutic vaccine.
Embodiment three: the preventative cocktail vaccine of HIV-I
The preventative cocktail vaccine of disclosed HIV is to be selected from following combination of elements and the DNA, adenovirus or the vaccinia virus vaccine that make up among the present invention: seven peptide repetitive sequence HR212 of (1) virus capable of blocking and cell fusion; (2) the adjuvant molecule of gp96 or its n terminal fragment; (3) the SCT gene that makes up of epitope, multi-epitope fusion gene and epitope-MHC-I of the fusion gene that merges of the structural antigens env of HIV, gag and pol or itself and adjuvant and/or HIV.Similar with embodiment two to embodiment one, the preventative cocktail vaccine of the disclosed HIV-1 of this patent comprises multichip carrier cocktail vaccine (Fig. 1) and single carrier cocktail vaccine (Fig. 7).The multichip carrier cocktail vaccine is meant the carrier 2 relevant with suppressing the HIV invasion among Fig. 1 and the combination of carrier 3, and wherein ABC is meant IFN α or γ, gag, env, HR212, SCT, gp96, the optional combination of Gp96N-gag/env fusion gene.The preventative cocktail vaccine of single vector HIV-1 is meant the combination that element relevant in the above-mentioned multichip carrier is chosen wantonly and recombinant DNA, adenovirus or the vaccinia virus that makes up, and its main points are to comprise to suppress the relevant structural gene of HR212, neutralizing antibody and CTL that film merges and the fusion gene of structural gene and adjuvant.The preventative cocktail vaccine of single carrier wherein also comprises the combination that the identical carrier that comprises different elements is carried out.
The simple construction strategy of setting forth the recombinant relevant in the present embodiment with wherein part carrier.Utilize the method same with embodiment one, screened the siRNA sequence (SEQ ID NO:23) that suppresses the tat gene of HIV, changing into DNA sequence is: 5 ' AAGTGTTGCTTTCATTGCCAAGTTTGTT-3 ' (SEQ ID NO:71), be cloned among the pshuttle, in the BJ5183 competent cell, obtain recombinant adenoviral vector, frozen standby through the packaged adenovirus of GP-293 in cryogenic refrigerator.With the 293T cell is experimental model, utilizes HIVNL4.3 plasmid transfection 293T cell earlier, and the packaged adenovirus particles of transfection after 6 hours detects the effect that suppresses after 72 hours.Western blotting result shows that the proteic expression of P24 is suppressed 73%, and gag albumen is suppressed more than 60%, and Northern blotting shows that HIV-RNA is obviously suppressed.Illustrate that adenovirus vector-mediated RNA perturbation technique can effectively suppress important proteic expression among the HIV, and further influenced duplicating of virus.The relevant SCT recombinant DNA of HIV gag epi-position that utilizes same principle to make up also can bring out the relevant CTL effect of HIV epi-position (Fig. 4,6) efficiently with vaccinia virus vector.
The film of used HIV-1 fusion inhibition sequence is by making up recombinant DNA, adenovirus and the vaccinia virus (Fig. 8) that the eukaryotic expression system gene makes up in the present embodiment.In a concrete example, we have selected carrier for expression of eukaryon pcDNA3.0 and pSecTag2B.
With pcDNA3.0 (Invitrogen), two carrier for expression of eukaryon of pSecTag2B (Invitrogen) make up the secretion expression carrier that contains tPA and IgG signal peptide sequence respectively.The structure of pcDNA3.0-tPA-HR212 adopts the mode of bridging PCR, and primer sequence is respectively:
Up-tPA-p1:GTCTTCGTTTCGCCCAGCTGGATGGAGTGGGACAG(SEQID?NO:51)
Up-tPA-p2:
GCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCA(SEQID?NO:52)
Up-tPA-p3:GATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGC(SEQ?ID?NO:53)
Up-tPA-p4:CGCAGGATCCGCCACCATGGATGCAATGAAGAGAG(SEQID?NO:54)
Down-HR212-p1:CGCGCTCGAGCTACAATAATTCTTGTTCATTC(SEQID?NO:55)
Constructed gene structure is shown in Fig. 8 A.
The primer sequence of pSecTag2B-HR212 is as follows, and constructed gene structure is shown in Fig. 8 B.
Up-pSec-p1:CGCAGGATCCTGGATGGAGTGGGACAGAG(SEQ?IDNO:56)
Down-HR212-p1:CTCTGTCCCACTCCATCCAGGATCCTGCG(SEQ?IDNO:57)
Gene tPA-HR212 (SEQ ID NO:19) and pSec-HR212 that PCR obtained are carried out double digestion with BamHI/Xho I, be connected into then in the carrier for expression of eukaryon of same enzyme action, make up the eucaryon recombinant DNA of secreting, expressing.
With constructed eukaryotic expression system carrier transfection 293T cell (available from ATCC), cultivate and collect supernatant after 48-72 hour, and concentrate.The pseudovirus of packing HIV, concrete operation method are 24h before the transfection, and digestion 293T cell also reaches in the 10cm ware, and concentration is 4-5 * 10 6/ ware.Behind the 24h, cell about 80% is paved with.With calcium phosphate method with pNL43LucE-R (available from Invitrogen) and two plasmid co-transfection 293T of pMT3-HXB-2 (available from Invitrogen) cell, behind the transfection 48h, cell covers with and becomes circle, and centrifugal (3000rpm * 10min) results contain HIV-1 pseudovirus supernatant.
Merge and suppress preceding 24 hours of experiment, digestion GHOST-CXCR4 (available from ATCC) cell also reaches in 24 orifice plates, and cell concentration is 8 * 10 4/ hole.The supernatant of eukaryotic expression system vector expression is mixed according to 1: 1 with virus, then mixture is inoculated in the GHOST-CXCR4 cell (available from ATCC), after treating virus absorption onto cell 2h, virus mixture is abandoned in suction, adding fresh culture continues to cultivate, each dilution factor is done 2 repetitions, results and cell lysis after 48-72 hour, the activity of Lampyridea luciferase in the mensuration cell conditioned medium.Experimental result is as shown in table 2.
Table 2:HR212 suppresses HIV and infects target cell
Negative Positive Contrast ?pcDNA3.0-tPA- ?HR212 ?pSecTag2B- ?HR212
72 ?26342 ?15674 ?6739 ?2384
82 ?16598 ?22252 ?2009 ?5243
Negative: GHOST-CXCR4 cell non-processor
Positive: as to insert pseudovirus in the GHOST-CXCR4 cell
Contrast: insert pseudovirus and 293 cell conditioned mediums in the GHOST-CXCR4 cell
Insert pseudovirus and transfection in the pcDNA3.0-tPA-HR212:GHOST-CXCR4 cell
293 cell conditioned mediums of pcDNA3.0-TPA-HR212
Insert pseudovirus and transfection in the pSecTag2B-HR212:GHOST-CXCR4 cell
293 cell conditioned mediums of pSecTag2B-HR212
Different with the micromolecule chemicals cocktail vaccine of the HIV that has existed, the disclosed content of present embodiment is the biological cocktail vaccine that designs according to the HIV infection characteristic.The HIV HR212 of the disclosed secreting, expressing of present embodiment can effectively suppress HIV pseudovirus the infecting target cell of packing, in addition, because the eukaryotic vector vaccine can independently duplicate in vivo, express and correct processing purpose albumen, the characteristics of SM in the time of therefore can effectively reducing the HIV treatment.The same with the building mode of embodiment one 1B, C, the SCT DNA-vaccine vaccine that comprises HIV structural gene and comprise the HIV epitope of structure can efficiently bring out HIV in the HLA-A transgenic mouse multi-epitope CTL replys.
Embodiment four: HIV-I therapeutic cocktail vaccine
Similar with embodiment three, HIV-1 therapeutic cocktail vaccine also comprises multichip carrier therapeutic cocktail vaccine and single vehicle treatment cocktail vaccine.Except that a plurality of elements that embodiment three is comprised, also comprise in the present embodiment and can suppress the effective RNA interference element that HIV-1 duplicates and recombinant DNA, adenovirus and the vaccinia virus of structure.
The simple construction strategy of setting forth the recombinant relevant in the present embodiment with wherein part carrier.Utilize the method same with embodiment one, screened the siRNA sequence (SEQ ID NO:23) that suppresses the tat gene of HIV, changing into DNA sequence is: 5 ' AAGTGTTGCTTTCATTGCCAAGTTTGTT-3 ', be cloned among the pshuttle, in the BJ5183 competent cell, obtain recombinant adenoviral vector, frozen standby through the packaged adenovirus of GP-293 in cryogenic refrigerator.With the 293T cell is experimental model, utilizes HIVNL4.3 plasmid transfection 293T cell earlier, and the packaged adenovirus particles of transfection after 6 hours detects the effect that suppresses after 72 hours.Western blotting result shows that the proteic expression of P24 is suppressed 73%, and gag albumen is suppressed more than 60%, and Northern blotting shows that HIV-RNA is obviously suppressed.Illustrate that adenovirus vector-mediated RNA perturbation technique can effectively suppress important proteic expression among the HIV, and further influenced duplicating of virus.The same with the preventative cocktail vaccine of HIV, the recombinant DNA-vaccine vaccine that comprises fusion gene, HIV multi-epitope adjuvant fusion gene and the HIV epi-position-SCT of HIV structural antigens, structural antigens and adjuvant can efficiently bring out multi-epitope CTL effect in the HLA-A2 transgenic mouse.
List of references
Payette,P.J.,X.Ma,et?al.(2006).″Testing?of?CpG-optimized?protein?and?DNAvaccines?against?the?hepatitis?B?virus?in?chimpanzees?for?immunogenicityand?protection?from?challenge.″Intervirology?49(3):144-51.
Seeger,C.and?W.S.Mason(2000).″Hepatitis?B?virus?biology.″Microbiol?MolBiol?Rev?64(1):51-68.
Yang,S.H.,C.G.Lee,et?al.(2006).″Correlation?of?antiviral?T-cell?responseswith?suppression?of?viral?rebound?in?chronic?hepatitis?B?carriers:aproof-of-concept?study.″Gene?Ther.
Zhao,Y.?G.,B.Peng,et?al.(2006).″Anti-HBV?immune?responses?in?rhesusmacaques?elicited?by?electroporation?mediated?DNA?vaccination.″Vaccine?24(7):897-903.
Manns?MP,McHutchinson?JG,Gordon?SC,et?al.Peginterferon?alpha-2b?incombination?with?ribavirin?compared?with?interferon?alpha-2b?plusribavirin?for?initial?treatment?of?chronic?hepatitis?C:results?of?arandomized?trial.Lancet,2001,358:958-965.
Lee?SW,Cho?JH,Sung?YC.Optimal?induction?of?hepatitis?C?virus?envelope-specific?immunity.J?Virol,l998,72:8430-8436.
Foms?X,Emerson?SU,Tobin?GJ,et?al.DNA?immunization?of?mice?andmacaques?with?plasmids?encoding?hepatitis?C?virus?envelope?E2?proteinexpressed?intracellularly?and?on?the?cell?surface.Vaccine,1999,17:1992-2002.Zhou?YH,Shimizu?YK,Esumi?M.Monoclonal?antibodiesto?the?hypervariable?region?1?of?hepatitis?C?virus?capture?virus?and?inhibitvirus?adsorption?to?susceptible?cells?in?vitro.Virology,2000,269(2):276-283
Jiao?X,Wang?RY,Feng?Z,et?al.DNA?immunization?encoding?the?secretednonstructural?protein?3(NS3)of?hepatitis?C?virus?and?enhancing?the?Th1type?immune?response.J?Viral?Hepat,2004,11(1):18-26.
Inchauspe?G,Feinstone?S.Development?of?a?hepatitis?C?virus?vaccine.ClinLiver?Dis,2003,7:243-259
Baba,T.W.,Liska,V.,Khimani,A.H.,et?al.Live?attenuated,multiply?deletedsimian?immunodeficiency?virus?causes?AIDS?in?infant?and?adultmacaques.Nat.Med.,1999,5:194-203.
Bruyn,G.,Rossini,A.J.,Chiu,Y.L.,et?al.Safety?profile?of?recombinantcanarypox?HIV?vaccines.Vaccine,2004,22(5~6):704~713.
Dennis,R.B.,Ronald,C.D.,Robert,W.D.,et?al.Enhanced:A?sound?rationaleneeded?for?phase?III?HIV-1?vaccine?trials.Science,2004,303:316.
Egan,M.A.,Charini,W.A.,Kuroda,M.J.,et?al.Simian?immunodeficiency?virus(SIV)gag?DNA?vaccinated?rhesus?monkeys?develop?secondary?cytotoxicT-lymphocyte?responses?and?control?viral?replication?after?pathogenicSIV?infection.J.Virol.,2000,74:7485-7495.
Qing?Fang,Lin?Yang,Weijun?Zhu,et?al.Host?range,growth?property,andvirulence?of?the?smallpox?vaccine:Vaccinia?Virus?Tian?Tan?strain.Virology,2005,335:242-251.
Xiao Yao, Fan Xiujuan. Chinese HIV-1B hypotype P55 and chimeric P55-V3 virus-like particle candidate vaccine Immunogenicity Study. viral journal, 2000,16 (4): 313~316.
Sequence table
<110〉Institute of Microorganism, Academia Sinica
<120〉cocktail vaccine of a kind of anti immune tolerance and immunodeficiency virus and application thereof
<130>IBO67386
<160>77
<170>PatentIn?version?3.1
<210>1
<211>1071
<212>DNA
<213〉mice
<220>
<221>CDS
<222>(1)..(1071)
<223>
<400>1
atggatgatg?aagtcgacgt?ggatggcaca?gtggaagagg?acctgggtaa?aagccgagaa 60
ggctcaagga?cagatgatga?agttgtgcag?agagaggaag?aagctattca?gttggatggg 120
ttaaacgcat?cacagataag?agaacttaga?gaaaaatctg?aaaagttcgc?cttccaagct 180
gaagtgaaca?ggatgatgaa?acttatcatc?aattctttgt?ataaaaataa?agagattttc 240
ctgagagaac?tgatttcaaa?tgcttctgat?gctttagaca?agataaggct?catctcccta 300
actgatgaaa?atgcactcgc?tggaaatgag?gagttaacgg?tcaagattaa?gtgtgacaaa 360
gagaaaaacc?tgctgcatgt?cacagacacg?ggtgtaggaa?tgactagaga?ggagttggtt 420
aaaaatctcg?gcaccatagc?caaatctgga?acaagcgagt?ttttaaacaa?aatgacagaa 480
gctcaagaag?atggtcagtc?aacctctgaa?ctgattggcc?agtttggtgt?cggtttttat 540
tctgccttcc?ttgtagcaga?taaggtcatt?gtcacatcga?aacacaacaa?tgatacccag 600
cacatctggg?aatcagactc?caatgaattc?tctgtaattg?ctgacccaag?aggaaacaca 660
ctaggtcgtg?gaacaacaat?tactcttgtc?ttaaaagaag?aagcatctga?ttaccttgaa 720
ttggacacaa?ttaaaaatct?cgtcaggaag?tactctcagt?tcatcaactt?tcccatctac 780
gtgtggagta?gcaagacaga?gactgttgag?gagcccttgg?aagaagatga?agcagcaaaa 840
gaagagaaag?aagaatctga?tgatgaagct?gcagtagagg?aggaagaaga?agaaaagaaa 900
ccaaaaacta?agaaagttga?aaaaactgtg?tgggattggg?aacttatgaa?tgatatcaaa 960
ccaatatggc?agagaccatc?caaagaagta?gaagaagacg?aatacaaagc?tttctacaaa 1020
tcattttcaa?aggaaagtga?tgaccccatg?gcttatatcc?acttcactta?a 1071
<210>2
<211>1071
<212>DNA
<213〉people
<220>
<221>CDS
<222>(1)..(1071)
<223>
<400>2
atggacgatg?aagttgatgt?ggatggtaca?gtagaagagg?atctgggtaa?aagtagagaa 60
ggatcaagga?cggatgatga?agtagtacag?agagaggaag?aagctattca?gttggatgga 120
ttaaatgcat?cacaaataag?agaacttaga?gagaagtcgg?aaaagtttgc?cttccaagcc 180
gaagttaaca?gaatgatgaa?acttatcatc?aattcattgt?ataaaaataa?agagattttc 240
ctgagagaac?tgatttcaaa?tgcttctgat?gctttagata?agataaggct?aatatcactg 300
actgatgaaa?atgctctttc?tggaaatgag?gaactaacag?tcaaaattaa?gtgtgataag 360
gagaagaacc?tgctgcatgt?cacagacacc?ggtgtaggaa?tgaccagaga?agagttggtt 420
aaaaaccttg?gtaccatagc?caaatctggg?acaagcgagt?ttttaaacaa?aatgactgaa 480
gcacaggaag?atggccagtc?aacttctgaa?ttgattggcc?agtttggtgt?cggtttctat 540
tccgccttcc?ttgtagcaga?taaggttatt?gtcacttcaa?aacacaacaa?cgatacccag 600
cacatctggg?agtctgactc?caatgaattt?tctgtaattg?ctgacccaag?aggaaacact 660
ctaggacggg?gaacgacaat?tacccttgtc?ttaaaagaag?aagcatctga?ttaccttgaa 720
ttggatacaa?ttaaaaatct?cgtcaaaaaa?tattcacagt?tcataaactt?tcctatttat 780
gtatggagca?gcaagactga?aactgttgag?gagcccatgg?aggaagaaga?agcagccaaa 840
gaagagaaag?aagaatctga?tgatgaagct?gcagtagagg?aagaagaaga?agaaaagaaa 900
ccaaagacta?aaaaagttga?aaaaactgtc?tgggactggg?aacttatgaa?tgatatcaaa 960
ccaatatggc?agagaccatc?aaaagaagta?gaagaagatg?aatacaaagc?tttctacaaa 1020
tcattttcaa?aggaaagtga?tgaccccatg?gcttatattc?actttacttaa 1071
<210>3
<211>2352
<212>DNA
<213〉people
<220>
<221>CDS
<222>(1)..?(2352)
<223>
<400>3
atggacgatg?aagttgatgt?ggatggtaca?gtagaagagg?atctgggtaa?aagtagagaa 60
ggatcaagga?cggatgatga?agtagtacag?agagaggaag?aagctattca?gttggatgga 120
ttaaatgcat?cacaaataag?agaacttaga?gagaagtcgg?aaaagtttgc?cttccaagcc 180
gaagttaaca?gaatgatgaa?acttatcatc?aattcattgt?ataaaaataa?agagattttc 240
ctgagagaac?tgatttcaaa?tgcttctgat?gctttagata?agataaggct?aatatcactg 300
actgatgaaa?atgctctttc?tggaaatgag?gaactaacag?tcaaaattaa?gtgtgataag 360
gagaagaacc?tgctgcatgt?cacagacacc?ggtgtaggaa?tgaccagaga?agagttggtt 420
aaaaaccttg?gtaccatagc?caaatctggg?acaagcgagt?ttttaaacaa?aatgactgaa 480
gcacaggaag?atggccagtc?aacttctgaa?ttgattggcc?agtttggtgt?cggtttctat 540
tccgccttcc?ttgtagcaga?taaggttatt?gtcacttcaa?aacacaacaa?cgatacccag 600
cacatctggg?agtctgactc?caatgaattt?tctgtaattg?ctgacccaag?aggaaacact 660
ctaggacggg?gaacgacaat?tacccttgtc?ttaaaagaag?aagcatctga?ttaccttgaa 720
ttggatacaa?ttaaaaatct?cgtcaaaaaa?tattcacagt?tcataaactt?tcctatttat 780
gtatggagca?gcaagactga?aactgttgag?gagcccatgg?aggaagaaga?agcagccaaa 840
gaagagaaag?aagaatctga?tgatgaagct?gcagtagagg?aagaagaaga?agaaaagaaa 900
ccaaagacta?aaaaagttga?aaaaactgtc?tgggactggg?aacttatgaa?tgatatcaaa 960
ccaatatggc?agagaccatc?aaaagaagta?gaagaagatg?aatacaaagc?tttctacaaa 1020
tcattttcaa?aggaaagtga?tgaccccatg?gcttatattc?actttactgc?tgaaggggaa 1080
gttaccttca?aatcaatttt?atttgtaccc?acatctgctc?cacgtggtct?gtttgacgaa 1140
tatggatcta?aaaagagcga?ttacattaag?ctctatgtgc?gccgtgtatt?catcacagac 1200
gacttccatg?atatgatgcc?taaatacctc?aattttgtca?agggtgtggt?ggactcagat 1260
gatctcccct?tgaatgtttc?ccgcgagact?cttcagcaac?ataaactgct?taaggtgatt 1320
aggaagaagc?ttgttcgtaa?aacgctggac?atgatcaaga?agattgctga?tgataaatac 1380
aatgatactt?tttggaaaga?atttggtacc?aacatcaagc?ttggtgtgat?tgaagaccac 1440
tcgaatcgaa?cacgtcttgc?taaacttctt?aggttccagt?cttctcatca?tccaactgac 1500
attactagcc?tagaccagta?tgtggaaaga?atgaaggaaa?aacaagacaa?aatctacttc 1560
atggctgggt?ccagcagaaa?agaggctgaa?tcttctccat?ttgttgagcg?acttctgaaa 1620
aagggctatg?aagttattta?cctcacagaa?cctgtggatg?aatactgtat?tcaggccctt 1680
cccgaatttg?atgggaagag?gttccagaat?gttgccaagg?aaggagtgaa?gttcgatgaa 1740
agtgagaaaa?ctaaggagag?tcgtgaagca?gttgagaaag?aatttgagcc?tctgctgaat 1800
tggatgaaag?ataaagccct?taaggacaag?attgaaaagg?ctgtggtgtc?tcagcgcctg 1860
acagaatctc?cgtgtgcttt?ggtggccagc?cagtacggat?ggtctggcaa?catggagaga 1920
atcatgaaag?cacaagcgta?ccaaacgggc?aaggacatct?ctacaaatta?ctatgcgagt 1980
cagaagaaaa?catttgaaat?taatcccaga?cacccgctga?tcagagacat?gcttcgacga 2040
attaaggaag?atgaagatga?taaaacagtt?ttggatcttg?ctgtggtttt?gtttgaaaca 2100
gcaacgcttc?ggtcagggta?tcttttacca?gacactaaag?catatggaga?tagaatagaa 2160
agaatgcttc?gcctcagttt?gaacattgac?cctgatgcaa?aggtggaaga?agagcccgaa 2220
gaagaacctg?aagagacagc?agaagacaca?acagaagaca?cagagcaaga?cgaagatgaa 2280
gaaatggatg?tgggaacaga?tgaagaagaa?gaaacagcaa?aggaatctac?agctgaaaaa 2340
gatgaattgt?aa 2352
<210>4
<211>450
<212>DNA
<213〉hepatitis B virus
<220>
<221>CDS
<222>(1)..(450)
<223>
<400>4
atgcaacttt?ttgaattctg?cctaatcatc?tcatgttcat?gtcctactgt?tcaagcctcc 60
aagctgtgcc?ttgggtggct?ttggggcatg?gacattgacc?cgtataaaga?atttggagct 120
tctgtggagt?tactctcttt?tttgccttct?gacttctttc?cttctgttcg?agagcttctc 180
gacaccgcct?ctgctctgta?tcgggaggcc?ttagagtctc?cggaacattg?ttcacctcat 240
catacggcac?tcaggcaagc?tattctgtgt?tgggttgagt?tgatgaatct?agccacctgg 300
gtgggaagta?atttggaaga?cccagcatcc?agggaattgg?tagtcagcca?tgtcaatgtt 360
aatatgggcc?taaaaatcag?acaactattg?tggtttcaca?tttcctgtct?tacttttgga 420
agagaaactg?ttcttgaata?tttggtgtaa 450
<210>5
<211>681
<212>DNA
<213〉hepatitis B virus
<220>
<221>CDS
<222>(1)..(681)
<223>
<400>5
atggagaaca?tcgcatcagg?actcctagga?cccctgctcg?tgttacaggc?ggggtttttc 60
tcgttgacaa?aaatcctcac?aataccacag?agtctagact?cgttgtggac?ttctctcaat 120
tttctagggg?aaacacccgt?gtgtcttggc?caaaattcgc?agtcccaaat?ctccagtcac 180
tcactaactt?gttgtcctcc?aatttgtcct?ggttatcgct?ggatgtgtct?gcggcgtttt 240
atcatcttcc?tctgcatcct?gctgctatgc?ctcatcttct?tgttggttct?tctggactat 300
caaggtatgt?tgcccgtttg?tcctctaatt?ccaggatca?tcaaccaccag?caccggacca 360
tgcagaacct?gcacgactcc?tgctcaagga?acctctatgt?ttccctcatg?ttgctgtaca 420
aaacctacgg?acggaaactg?cacctgtatt?cccatcccat?catcttgggc?tttcgcaaaa 480
tacctatggg?agtgggcctc?agtccgtttc?tcttggctca?gtttactagt?gccatttgtt 540
cagtggttcg?tagggctttc?ccccactgtc?tggctttcag?ttatatggat?gatgtggtat 600
tgggggccaa?gtctgtacaa?catcttgagt?ccctttatgc?cgctgttacc?aattttcttt 660
tgtctttggg?tatacattta?a 681
<210>6
<211>846
<212>DNA
<213〉hepatitis B virus
<220>
<221>CDS
<222>(1)..(846)
<223>
<400>6
atgcagtgga?attccacaac?ctttcaccaa?actctgcaag?atcccagagt?gagaggcctg 60
tatttccctg?ctggtggctc?cagttcagga?gcagtaaacc?ctgttccgac?tactgcctct 120
cccttatcgt?caatcttctc?gaggattggg?gaccctgcgc?tgaacatgga?gaacatcaca 180
tcaggattcc?taggacccct?tctcgtgtta?caggcggggt?ttttcttgtt?gacaagaatc 240
ctcacaatac?cgcagagtct?agactcgtgg?tggacttctc?tcaattttct?agggggaact 300
accgtgtgtc?ttggccaaaa?ttcgcagtcc?ccaacctcca?atcactcacc?aacctcctgt 360
cctccaactt?gtcctggtta?tcgctggatg?tgtctgcggc?gttttatcat?cttcctcttc 420
atcctgctgc?tatgcctcat?cttcttgttg?gttcttctgg?actatcaagg?tatgttgccc 480
gtttgtcctc?taattccagg?atcctcaacc?accagcacgg?gaccatgccg?aacctgcatg 540
actactgctc?aaggaacctc?tatgtatccc?tcctgttgct?gtaccaaacc?ttcggacgga 600
aattgcacct?gtattcccat?cccatcatcc?tgggctttcg?gaaaattcct?atgggagtgg 660
gcctcagccc?gtttctcctg?gctcagttta?ctagtgccat?ttgttcagtg?gttcgtaggg 720
ctttccccca?ctgtttggct?ttcagttata?tggatgatgt?ggtattgggg?gccaagtctg 780
tacagcatct?tgagtccctt?tttaccgctg?ttaccaattt?tcttttgtct?ttgggtatac 840
atttaa 846
<210>7
<211>1530
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1530)
<223>
<400>7
atgcaacttt?ttgaattctg?cctaatcatc?tcatgttcat?gtcctactgt?tcaagcctcc 60
aagctgtgcc?ttgggtggct?ttggggcatg?gacattgacc?cgtataaaga?atttggagct 120
tctgtggagt?tactctcttt?tttgccttct?gacttctttc?cttctgttcg?agagcttctc 180
gacaccgcct?ctgctctgta?tcgggaggcc?ttagagtctc?cggaacattg?ttcacctcat 240
catacggcac?tcaggcaagc?tattctgtgt?tgggttgagt?tgatgaatct?agccacctgg 300
gtgggaagta?atttggaaga?cccagcatcc?agggaattgg?tagtcagcca?tgtcaatgtt 360
aatatgggcc?taaaaatcag?acaactattg?tggtttcaca?tttcctgtct?tacttttgga 420
agagaaactg?ttcttgaata?tttggtgggt?tcctctggag?gtgatgatga?agtcgacgtg 480
gatggcacag?tggaagagga?cctgggtaaa?agccgagaag?gctcaaggac?agatgatgaa 540
gttgtgcaga?gagaggaaga?agctattcag?ttggatgggt?taaacgcatc?acagataaga 600
gaacttagag?aaaaatctga?aaagttcgcc?ttccaagctg?aagtgaacag?gatgatgaaa 660
cttatcatca?attctttgta?taaaaataaa?gagattttcc?tgagagaact?gatttcaaat 720
gcttctgatg?ctttagacaa?gataaggctc?atctccctaa?ctgatgaaaa?tgcactcgct 780
ggaaatgagg?agttaacggt?caagattaag?tgtgacaaag?agaaaaacct?gctgcatgtc 840
acagacacgg?gtgtaggaat?gactagagag?gagttggtta?aaaatctcgg?caccatagcc 900
aaatctggaa?caagcgagtt?tttaaacaaa?atgacagaag?ctcaagaaga?tggtcagtca 960
acctctgaac?tgattggcca?gtttggtgtc?ggtttttatt?ctgccttcct?tgtagcagat 1020
aaggtcattg?tcacatcgaa?acacaacaat?gatacccagc?acatctggga?atcagactcc 1080
aatgaattct?ctgtaattgc?tgacccaaga?ggaaacacac?taggtcgtgg?aacaacaatt 1140
actcttgtct?taaaagaaga?agcatctgat?taccttgaat?tggacacaat?taaaaatctc 1200
gtcaggaagt?actctcagtt?catcaacttt?cccatctacg?tgtggagtag?caagacagag 1260
actgttgagg?agcccttgga?agaagatgaa?gcagcaaaag?aagagaaaga?agaatctgat 1320
gatgaagctg?cagtagagga?ggaagaagaa?gaaaagaaac?caaaaactaa?gaaagttgaa 1380
aaaactgtgt?gggattggga?acttatgaat?gatatcaaac?caatatggca?gagaccatcc 1440
aaagaagtag?aagaagacga?atacaaagct?ttctacaaat?cattttcaaa?ggaaagtgat 1500
gaccccatgg?cttatatcca?cttcacttaa 1530
<210>8
<211>1761
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1761)
<223>
<400>8
atggagaaca?tcgcatcagg?actcctagga?cccctgctcg?tgttacaggc?ggggtttttc 60
tcgttgacaa?aaatcctcac?aataccacag?agtctagact?cgttgtggac?ttctctcaat 120
tttctagggg?aaacacccgt?gtgtcttggc?caaaattcgc?agtcccaaat?ctccagtcac 180
tcactaactt?gttgtcctcc?aatttgtcct?ggttatcgct?ggatgtgtct?gcggcgtttt 240
atcatcttcc?tctgcatcct?gctgctatgc?ctcatcttct?tgttggttct?tctggactat 300
caaggtatgt?tgcccgtttg?tcctctaatt?ccaggatcat?caaccaccag?caccggacca 360
tgcagaacct?gcacgactcc?tgctcaagga?acctctatgt?ttccctcatg?ttgctgtaca 420
aaacctacgg?acggaaactg?cacctgtatt?cccatcccat?catcttgggc?tttcgcaaaa 480
tacctatggg?agtgggcctc?agtccgtttc?tcttggctca?gtttactagt?gccatttgtt 540
cagtggttcg?tagggctttc?ccccactgtc?tggctttcag?ttatatggat?gatgtggtat 600
tgggggccaa?gtctgtacaa?catcttgagt?ccctttatgc?cgctgttacc?aattttcttt 660
tgtctttggg?tatacattgg?ttcctctgga?ggtgatgatg?aagtcgacgt?ggatggcaca 720
gtggaagagg?acctgggtaa?aagccgagaa?ggctcaagga?cagatgatga?agttgtgcag 780
agagaggaag?aagctattca?gttggatggg?ttaaacgcat?cacagataag?agaacttaga 840
gaaaaatctg?aaaagttcgc?cttccaagct?gaagtgaaca?ggatgatgaa?acttatcatc 900
aattctttgt?ataaaaataa?agagattttc?ctgagagaac?tgatttcaaa?tgcttctgat 960
gctttagaca?agataaggct?catctcccta?actgatgaaa?atgcactcgc?tggaaatgag 1020
gagttaacgg?tcaagattaa?gtgtgacaaa?gagaaaaacc?tgctgcatgt?cacagacacg 1080
ggtgtaggaa?tgactagaga?ggagttggtt?aaaaatctcg?gcaccatagc?caaatctgga 1140
acaagcgagt?ttttaaacaa?aatgacagaa?gctcaagaag?atggtcagtc?aacctctgaa 1200
ctgattggcc?agtttggtgt?cggtttttat?tctgccttcc?ttgtagcaga?taaggtcatt 1260
gtcacatcga?aacacaacaa?tgatacccag?cacatctggg?aatcagactc?caatgaattc 1320
tctgtaattg?ctgacccaag?aggaaacaca?ctaggtcgtg?gaacaacaat?tactcttgtc 1380
ttaaaagaag?aagcatctga?ttaccttgaa?ttggacacaa?ttaaaaatct?cgtcaggaag 1440
tactctcagt?tcatcaactt?tcccatctac?gtgtggagta?gcaagacaga?gactgttgag 1500
gagcccttgg?aagaagatga?agcagcaaaa?gaagagaaag?aagaatctga?tgatgaagct 1560
gcagtagagg?aggaagaaga?agaaaagaaa?ccaaaaacta?agaaagttga?aaaaactgtg 1620
tgggattggg?aacttatgaa?tgatatcaaa?ccaatatggc?agagaccatc?caaagaagta 1680
gaagaagacg?aatacaaagc?tttctacaaa?tcattttcaa?aggaaagtga?tgaccccatg 1740
gcttatatcc?acttcactta?a 1761
<210>9
<211>1761
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1761)
<223>
<400>9
atggagaaca?tcgcatcagg?actcctagga?cccctgctcg?tgttacaggc?ggggtttttc 60
tcgttgacaa?aaatcctcac?aataccacag?agtctagact?cgttgtggac?ttctctcaat 120
tttctagggg?aaacacccgt?gtgtcttggc?caaaattcgc?agtcccaaat?ctccagtcac 180
tcactaactt?gttgtcctcc?aatttgtcct?ggttatcgct?ggatgtgtct?gcggcgtttt 240
atcatcttcc?tctgcatcct?gctgctatgc?ctcatcttct?tgttggttct?tctggactat 300
caaggtatgt?tgcccgtttg?tcctctaatt?ccaggatcat?caaccaccag?caccggacca 360
tgcagaacct?gcacgactcc?tgctcaagga?acctctatgt?ttccctcatg?ttgctgtaca 420
aaacctacgg?acggaaactg?cacctgtatt?cccatcccat?catcttgggc?tttcgcaaaa 480
tacctatggg?agtgggcctc?agtccgtttc?tcttggctca?gtttactagt?gccatttgtt 540
cagtggttcg?tagggctttc?ccccactgtc?tggctttcag?ttatatggat?gatgtggtat 600
tgggggccaa?gtctgtacaa?catcttgagt?ccctttatgc?cgctgttacc?aattttcttt 660
tgtctttggg?tatacattgg?ttcctctgga?ggtgacgatg?aagttgatgt?ggatggtaca 720
gtagaagagg?atctgggtaa?aagtagagaa?ggatcaagga?cggatgatga?agtagtacag 780
agagaggaag?aagctattca?gttggatgga?ttaaatgcat?cacaaataag?agaacttaga 840
gagaagtcgg?aaaagtttgc?cttccaagcc?gaagttaaca?gaatgatgaa?acttatcatc 900
aattcattgt?ataaaaataa?agagattttc?ctgagagaac?tgatttcaaa?tgcttctgat 960
gctttagata?agataaggct?aatatcactg?actgatgaaa?atgctctttc?tggaaatgag 1020
gaactaacag?tcaaaattaa?gtgtgataag?gagaagaacc?tgctgcatgt?cacagacacc 1080
ggtgtaggaa?tgaccagaga?agagttggtt?aaaaaccttg?gtaccatagc?caaatctggg 1140
acaagcgagt?ttttaaacaa?aatgactgaa?gcacaggaag?atggccagtc?aacttctgaa 1200
ttgattggcc?agtttggtgt?cggtttctat?tccgccttcc?ttgtagcaga?taaggttatt 1260
gtcacttcaa?aacacaacaa?cgatacccag?cacatctggg?agtctgactc?caatgaattt 1320
tctgtaattg?ctgacccaag?aggaaacact?ctaggacggg?gaacgacaat?tacccttgtc 1380
ttaaaagaag?aagcatctga?ttaccttgaa?ttggatacaa?ttaaaaatct?cgtcaaaaaa 1440
tattcacagt?tcataaactt?tcctatttat?gtatggagca?gcaagactga?aactgttgag 1500
gagcccatgg?aggaagaaga?agcagccaaa?gaagagaaag?aagaatctga?tgatgaagct 1560
gcagtagagg?aagaagaaga?agaaaagaaa?ccaaagacta?aaaaagttga?aaaaactgtc 1620
tgggactggg?aacttatgaa?tgatatcaaa?ccaatatggc?agagaccatc?aaaagaagta 1680
gaagaagatg?aatacaaagc?tttctacaaa?tcattttcaa?aggaaagtga?tgaccccatg 1740
gcttatattc?actttactta?a 1761
<210>10
<211>1926
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1926)
<223>
<400>10
atgcagtgga?attccacaac?ctttcaccaa?actctgcaag?atcccagagt?gagaggcctg 60
tatttccctg?ctggtggctc?cagttcagga?gcagtaaacc?ctgttccgac?tactgcctct 120
cccttatcgt?caatcttctc?gaggattggg?gaccctgcgc?tgaacatgga?gaacatcaca 180
tcaggattcc?taggacccct?tctcgtgtta?caggcggggt?ttttcttgtt?gacaagaatc 240
ctcacaatac?cgcagagtct?agactcgtgg?tggacttctc?tcaattttct?agggggaact 300
accgtgtgtc?ttggccaaaa?ttcgcagtcc?ccaacctcca?atcactcacc?aacctcctgt 360
cctccaactt?gtcctggtta?tcgctggatg?tgtctgcggc?gttttatcat?cttcctcttc 420
atcctgctgc?tatgcctcat?cttcttgttg?gttcttctgg?actatcaagg?tatgttgccc 480
gtttgtcctc?taattccagg?atcctcaacc?accagcacgg?gaccatgccg?aacctgcatg 540
actactgctc?aaggaacctc?tatgtatccc?tcctgttgct?gtaccaaacc?ttcggacgga 600
aattgcacct?gtattcccat?cccatcatcc?tgggctttcg?gaaaattcct?atgggagtgg 660
gcctcagccc?gtttctcctg?gctcagttta?ctagtgccat?ttgttcagtg?gttcgtaggg 720
ctttccccca?ctgtttggct?ttcagttata?tggatgatgt?ggtattgggg?gccaagtctg 780
tacagcatct?tgagtccctt?tttaccgctg?ttaccaattt?tcttttgtct?ttgggtatac 840
attggttcct?ctggaggtga?tgatgaagtc?gacgtggatg?gcacagtgga?agaggacctg 900
ggtaaaagcc?gagaaggctc?aaggacagat?gatgaagttg?tgcagagaga?ggaagaagct 960
attcagttgg?atgggttaaa?cgcatcacag?ataagagaac?ttagagaaaa?atctgaaaag 1020
ttcgccttcc?aagctgaagt?gaacaggatg?atgaaactta?tcatcaattc?tttgtataaa 1080
aataaagaga?ttttcctgag?agaactgatt?tcaaatgctt?ctgatgcttt?agacaagata 1140
aggctcatct?ccctaactga?tgaaaatgca?ctcgctggaa?atgaggagtt?aacggtcaag 1200
attaagtgtg?acaaagagaa?aaacctgctg?catgtcacag?acacgggtgt?aggaatgact 1260
agagaggagt?tggttaaaaa?tctcggcacc?atagccaaat?ctggaacaag?cgagttttta 1320
aacaaaatga?cagaagctca?agaagatggt?cagtcaacct?ctgaactgat?tggccagttt 1380
ggtgtcggtt?tttattctgc?cttccttgta?gcagataagg?tcattgtcac?atcgaaacac 1440
aacaatgata?cccagcacat?ctgggaatca?gactccaatg?aattctctgt?aattgctgac 1500
ccaagaggaa?acacactagg?tcgtggaaca?acaattactc?ttgtcttaaa?agaagaagca 1560
tctgattacc?ttgaattgga?cacaattaaa?aatcttgtca?ggaagtactc?tcagttcatc 1620
aactttccca?tctacgtgtg?gagtagcaag?acagagactg?ttgaggagcc?cttggaagaa 1680
gatgaagcag?caaaagaaga?gaaagaagaa?tctgatgatg?aagctgcagt?agaggaggaa 1740
gaagaagaaa?agaaaccaaa?aactaagaaa?gttgaaaaaa?ctgtgtggga?ttgggaactt 1800
atgaatgata?tcaaaccaat?atggcagaga?ccatccaaag?aagtagaaga?agacgaatac 1860
aaagctttct?acaaatcatt?ttcaaaggaa?agtgatgacc?ccatggctta?tatccacttc 1920
acttaa 1926
<210>11
<211>1926
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1926)
<223>
<400>11
atggatgatg?aagtcgacgt?ggatggcaca?gtggaagagg?acctgggtaa?aagccgagaa 60
ggctcaagga?cagatgatga?agttgtgcag?agagaggaag?aagctattca?gttggatggg 120
ttaaacgcat?cacagataag?agaacttaga?gaaaaatctg?aaaagttcgc?cttccaagct 180
gaagtgaaca?ggatgatgaa?acttatcatc?aattctttgt?ataaaaataa?agagattttc 240
ctgagagaac?tgatttcaaa?tgcttctgat?gctttagaca?agataaggct?catctcccta 300
actgatgaaa?atgcactcgc?tggaaatgag?gagttaacgg?tcaagattaa?gtgtgacaaa 360
gagaaaaacc?tgctgcatgt?cacagacacg?ggtgtaggaa?tgactagaga?ggagttggtt 420
aaaaatctcg?gcaccatagc?caaatctgga?acaagcgagt?ttttaaacaa?aatgacagaa 480
gctcaagaag?atggtcagtc?aacctctgaa?ctgattggcc?agtttggtgt?cggtttttat 540
tctgccttcc?ttgtagcaga?taaggtcatt?gtcacatcga?aacacaacaa?tgatacccag 600
cacatctggg?aatcagactc?caatgaattc?tctgtaattg?ctgacccaag?aggaaacaca 660
ctaggtcgtg?gaacaacaat?tactcttgtc?ttaaaagaag?aagcatctga?ttaccttgaa 720
ttggacacaa?ttaaaaatct?tgtcaggaag?tactctcagt?tcatcaactt?tcccatctac 780
gtgtggagta?gcaagacaga?gactgttgag?gagcccttgg?aagaagatga?agcagcaaaa 840
gaagagaaag?aagaatctga?tgatgaagct?gcagtagagg?aggaagaaga?agaaaagaaa 900
ccaaaaacta?agaaagttga?aaaaactgtg?tgggattggg?aacttatgaa?tgatatcaaa 960
ccaatatggc?agagaccatc?caaagaagta?gaagaagacg?aatacaaagc?tttctacaaa 1020
tcattttcaa?aggaaagtga?tgaccccatg?gcttatatcc?acttcactgg?ttcctctgga 1080
ggtcagtgga?attccacaac?ctttcaccaa?actctgcaag?atcccagagt?gagaggcctg 1140
tatttccctg?ctggtggctc?cagttcagga?gcagtaaacc?ctgttccgac?tactgcctct 1200
cccttatcgt?caatcttctc?gaggattggg?gaccctgcgc?tgaacatgga?gaacatcaca 1260
tcaggattcc?taggacccct?tctcgtgtta?caggcggggt?ttttcttgtt?gacaagaatc 1320
ctcacaatac?cgcagagtct?agactcgtgg?tggacttctc?tcaattttct?agggggaact 1380
accgtgtgtc?ttggccaaaa?ttcgcagtcc?ccaacctcca?atcactcacc?aacctcctgt 1440
cctccaactt?gtcctggtta?tcgctggatg?tgtctgcggc?gttttatcat?cttcctcttc 1500
atcctgctgc?tatgcctcat?cttcttgttg?gttcttctgg?actatcaagg?tatgttgccc 1560
gtttgtcctc?taattccagg?atcctcaacc?accagcacgg?gaccatgccg?aacctgcatg 1620
actactgctc?aaggaacctc?tatgtatccc?tcctgttgct?gtaccaaacc?ttcggacgga 1680
aattgcacct?gtattcccat?cccatcatcc?tgggctttcg?gaaaattcct?atgggagtgg 1740
gcctcagccc?gtttctcctg?gctcagttta?ctagtgccat?ttgttcagtg?gttcgtaggg 1800
ctttccccca?ctgtttggct?ttcagttata?tggatgatgt?ggtattgggg?gccaagtctg 1860
tacagcatct?tgagtccctt?tttaccgctg?ttaccaattt?tcttttgtct?ttgggtatac 1920
atttaa 1926
<210>12
<211>1440
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1440)
<223>
<400>12
atggacgatg?aagttgatgt?ggatggtaca?gtagaagagg?atctgggtaa?aagtagagaa 60
ggatcaagga?cggatgatga?agtagtacag?agagaggaag?aagctattca?gttggatgga 120
ttaaatgcat?cacaaataag?agaacttaga?gagaagtcgg?aaaagtttgc?cttccaagcc 180
gaagttaaca?gaatgatgaa?acttatcatc?aattcattgt?ataaaaataa?agagattttc 240
ctgagagaac?tgatttcaaa?tgcttctgat?gctttagata?agataaggct?aatatcactg 300
actgatgaaa?atgctctttc?tggaaatgag?gaactaacag?tcaaaattaa?gtgtgataag 360
gagaagaacc?tgctgcatgt?cacagacacc?ggtgtaggaa?tgaccagaga?agagttggtt 420
aaaaaccttg?gtaccatagc?caaatcaggg?acaagcgagt?ttttaaacaa?aatgactgaa 480
gcacaggaag?atggccagtc?aacttctgaa?ttgattggcc?agtttggtgt?cggtttctat 540
tccgccttcc?ttgtagcaga?taaggttatt?gtcacttcaa?aacacaacaa?cgatacccag 600
cacatctggg?agtctgactc?caatgaattt?tctgtaattg?ctgacccaag?aggaaacact 660
ctaggacggg?gaacgacaat?tacccttgtc?ttaaaagaag?aagcatctga?ttaccttgaa 720
ttggatacaa?ttaaaaatct?cgtcaaaaaa?tattcacagt?tcataaactt?tcctatttat 780
gtatggagca?gcaagactga?aactgttgag?gagcccatgg?aggaagaaga?agcagccaaa 840
gaagagaaag?aagaatctga?tgatgaagct?gcagtagagg?aagaagaaga?agaaaagaaa 900
ccaaagacta?aaaaagttga?aaaaactgtc?tgggactggg?aacttatgaa?gcttgctgct 960
tactttttgc?catctgactt?ctttccatcc?gtagctgctt?actatgtcaa?tgttaatatg 1020
ggcctaaaat?tagctgctta?ctggctcagt?ttactagtgc?catttgttgc?tgcttacgaa 1080
tatttggtgt?cttttggagt?gtgggctgct?tacttcttgt?tgacaagaat?cctcacaata 1140
gctgcttacg?atgctgctta?ccagtatata?aaagcaaatt?ctaaatttat?aggtataact 1200
gaatctagaa?tgcagatctt?cgtgaagact?ctgactggta?agaccatcac?cctcgaggtg 1260
gagcccagtg?acaccatcga?gaatgtcaag?gcaaagatcc?aagataagga?aggcattcca 1320
ccagatcagc?agaggttgat?ctttgccgga?aaacagctgg?aagatggtcg?taccctgtct 1380
gactacaaca?tccagaaaga?gtccaccttg?cacctggtac?tccgtctcag?aggtgggtaa 1440
<210>13
<211>1503
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1503)
<223>
<400>13
atgtctcgct?ccgtggcctt?agctgtgctc?gcgatactct?ctctttctgg?cctggaggct 60
tttttgcctt?ctgacttctt?tccttctgtt?ggaggtgggg?gaggcggatc?aggaggctca 120
ggtgggtcag?gaggcatcca?gcgtactcca?aagattcagg?tttactcacg?tcatccagca 180
gagaatggaa?agtcaaattt?cctgaattgc?tatgtgtctg?ggtttcatcc?atccgacatt 240
gaagttgact?tactgaagaa?tggagagaga?attgaaaaag?tggagcattc?agacttgtct 300
ttcagcaagg?actggtcttt?ctatctcttg?tactacactg?aattcacccc?cactgaaaaa 360
gatgagtatg?cctgccgtgt?gaaccatgtg?actttgtcac?agcccaagat?agttaagtgg 420
gatcgagaca?tgggaggtgg?cggttcgggc?ggaggcggct?cgggtggcgg?cggctctggc 480
tctcactcca?tgaggtattt?cttcacatcc?gtgtcccggc?ccggccgcgg?ggagccccgc 540
ttcatcgcag?tgggctacgt?ggacgacacg?cagttcgtgc?ggttcgacag?cgacgccgcg 600
agccagagga?tggagccgcg?ggcgccgtgg?atagagcagg?agggtccgga?gtattgggac 660
ggggagacac?ggaaagtgaa?ggcccactca?cagactcacc?gagtggacct?ggggaccctg 720
cgcggctact?acaaccagag?cgaggccggt?tctcacaccg?tccagaggat?gtatggctgc 780
gacgtggggt?cggactggcg?cttcctccgc?gggtaccacc?agtacgccta?cgacggcaag 840
gattacatcg?ccctgaaaga?ggacctgcgc?tcttggaccg?cggcggacat?ggcagctcag 900
accaccaagc?acaagtggga?ggcggcccat?gtggcggagc?agttgagagc?ctacctggag 960
ggcacgtgcg?tggagtggct?ccgcagatac?ctggagaacg?ggaaggagac?gctgcagcgc 1020
acggacgccc?ccaaaacgca?tatgactcac?cacgctgtct?ctgaccatga?agccaccctg 1080
aggtgctggg?ccctgagctt?ctaccctgcg?gagatcacac?tgacctggca?gcgggatggg 1140
gaggaccaga?cccaggacac?ggagctcgtg?gagaccaggc?ctgcagggga?tggaaccttc 1200
cagaagtggg?cggctgtggt?ggtgccttct?ggacaggagc?agagatacac?ctgccatgtg 1260
cagcatgagg?gtttgcccaa?gcccctcacc?ctgagatggg?agccgtcttc?ccagcccacc 1320
atccccatcg?tgggcatcat?tgctggcctg?gttctctttg?gagctgtgat?cactggagct 1380
gtggtcgctg?ctgtgatgtg?gaggaggaag?agctcagata?gaaaaggagg?gagctactct 1440
caggctgcaa?gcagtgacag?tgcccagggc?tctgatgtgt?ctctcacagc?ttgtaaagtg 1500
tga 1503
<210>14
<211>1500
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1500)
<223>
<400>14
atgtctcgct?ccgtggcctt?agctgtgctc?gcgatactct?ctctttctgg?cctggaggct 60
tgtcttactt?ttggaagaga?aactgttgga?ggtgggggag?gcggatcagg?aggctcaggt 120
gggtcaggag?gcatccagcg?tactccaaag?attcaggttt?actcacgtca?tccagcagag 180
aatggaaagt?caaatttcct?gaattgctat?gtgtctgggt?ttcatccatc?cgacattgaa 240
gttgacttac?tgaagaatgg?agagagaatt?gaaaaagtgg?agcattcaga?cttgtctttc 300
agcaaggact?ggtctttcta?tctcttgtac?tacactgaat?tcacccccac?tgaaaaagat 360
gagtatgcct?gccgtgtgaa?ccatgtgact?ttgtcacagc?ccaagatagt?taagtgggat 420
cgagacatgg?gaggtggcgg?ttcgggcgga?ggcggctcgg?gtggcggcgg?ctctggctct 480
cactccatga?ggtatttctt?cacatccgtg?tcccggcccg?gccgcgggga?gccccgcttc 540
atcgcagtgg?gctacgtgga?cgacacgcag?ttcgtgcggt?tcgacagcga?cgccgcgagc 600
cagaggatgg?agccgcgggc?gccgtggata?gagcaggagg?gtccggagta?ttgggacggg 660
gagacacgga?aagtgaaggc?ccactcacag?actcaccgag?tggacctggg?gaccctgcgc 720
ggctactaca?accagagcga?ggccggttct?cacaccgtcc?agaggatgta?tggctgcgac 780
gtggggtcgg?actggcgctt?cctccgcggg?taccaccagt?acgcctacga?cggcaaggat 840
tacatcgccc?tgaaagagga?cctgcgctct?tggaccgcgg?cggacatggc?agctcagacc 900
accaagcaca?agtgggaggc?ggcccatgtg?gcggagcagt?tgagagccta?cctggagggc 960
acgtgcgtgg?agtggctccg?cagatacctg?gagaacggga?aggagacgct?gcagcgcacg 1020
gacgccccca?aaacgcatat?gactcaccac?gctgtctctg?accatgaagc?caccctgagg 1080
tgctgggccc?tgagcttcta?ccctgcggag?atcacactga?cctggcagcg?ggatggggag 1140
gaccagaccc?aggacacgga?gctcgtggag?accaggcctg?caggggatgg?aaccttccag 1200
aagtgggcgg?ctgtggtggt?gccttctgga?caggagcaga?gatacacctg?ccatgtgcag 1260
catgagggtt?tgcccaagcc?cctcaccctg?agatgggagc?cgtcttccca?gcccaccatc 1320
cccatcgtgg?gcatcattgc?tggcctggtt?ctctttggag?ctgtgatcac?tggagctgtg 1380
gtcgctgctg?tgatgtggag?gaggaagagc?tcagatagaa?aaggagggag?ctactctcag 1440
gctgcaagca?gtgacagtgc?ccagggctct?gatgtgtctc?tcacagcttg?taaagtgtga 1500
<210>15
<211>1500
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(1500)
<223>
<400>15
atgtctcgct?ccgtggcctt?agctgtgctc?gcgatactct?ctctttctgg?cctggaggct 60
agcctgtaca?acaccgtcgc?gaccctagga?ggtgggggag?gcggatcagg?aggctcaggt 120
gggtcaggag?gcatccagcg?tactccaaag?attcaggttt?actcacgtca?tccagcagag 180
aatggaaagt?caaatttcct?gaattgctat?gtgtctgggt?ttcatccatc?cgacattgaa 240
gttgacttac?tgaagaatgg?agagagaatt?gaaaaagtgg?agcattcaga?cttgtctttc 300
agcaaggact?ggtctttcta?tctcttgtac?tacactgaat?tcacccccac?tgaaaaagat 360
gagtatgcct?gccgtgtgaa?ccatgtgact?ttgtcacagc?ccaagatagt?taagtgggat 420
cgagacatgg?gaggtggcgg?ttcgggcgga?ggcggctcgg?gtggcggcgg?ctctggctct 480
cactccatga?ggtatttctt?cacatccgtg?tcccggcccg?gccgcgggga?gccccgcttc 540
atcgcagtgg?gctacgtgga?cgacacgcag?ttcgtgcggt?tcgacagcga?cgccgcgagc 600
cagaggatgg?agccgcgggc?gccgtggata?gagcaggagg?gtccggagta?ttgggacggg 660
gagacacgga?aagtgaaggc?ccactcacag?actcaccgag?tggacctggg?gaccctgcgc 720
ggctactaca?accagagcga?ggccggttct?cacaccgtcc?agaggatgta?tggctgcgac 780
gtggggtcgg?actggcgctt?cctccgcggg?taccaccagt?acgcctacga?cggcaaggat 840
tacatcgccc?tgaaagagga?cctgcgctct?tggaccgcgg?cggacatggc?agctcagacc 900
accaagcaca?agtgggaggc?ggcccatgtg?gcggagcagt?tgagagccta?cctggagggc 960
acgtgcgtgg?agtggctccg?cagatacctg?gagaacggga?aggagacgct?gcagcgcacg 1020
gacgccccca?aaacgcatat?gactcaccac?gctgtctctg?accatgaagc?caccctgagg 1080
tgctgggccc?tgagcttcta?ccctgcggag?atcacactga?cctggcagcg?ggatggggag 1140
gaccagaccc?aggacacgga?gctcgtggag?accaggcctg?caggggatgg?aaccttccag 1200
aagtgggcgg?ctgtggtggt?gccttctgga?caggagcaga?gatacacctg?ccatgtgcag 1260
catgagggtt?tgcccaagcc?cctcaccctg?agatgggagc?cgtcttccca?gcccaccatc 1320
cccatcgtgg?gcatcattgc?tggcctggtt?ctctttggag?ctgtgatcac?tggagctgtg 1380
gtcgctgctg?tgatgtggag?gaggaagagc?tcagatagaa?aaggagggag?ctactctcag 1440
gctgcaagca?gtgacagtgc?ccagggctct?gatgtgtctc?tcacagcttg?taaagtgtga 1500
<210>16
<211>102
<212>DNA
<213〉human immunodeficiency virus
<220>
<221>CDS
<222>(1)..(102)
<223>
<400>16
atggagtggg?acagagaaat?taacaattac?acaagcttaa?tacactcctt?aattgaagaa 60
tcgcaaaacc?agcaagaaaa?gaatgaacaa?gaattattgt?aa 102
<210>17
<211>108
<212>DNA
<213〉human immunodeficiency virus
<220>
<221>CDS
<222>(1)..(108)
<223>
<400>17
atgtctggta?tagtgcagca?gcagaacaat?ttgctgaggg?ctattgaggc?gcaacagcat 60
ctgttgcaac?tcacagtctg?gggcatcaag?cagctccagg?caagataa 108
<210>18
<211>336
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(336)
<223>
<400>18
atggagtggg?acagagaaat?taacaattac?acaagcttaa?tacactcctt?aattgaagaa 60
tcgcaaaacc?agcaagaaaa?gaatgaacaa?gaattattgg?gtggttctgg?tggatctggt 120
atagtgcagc?agcagaacaa?tttgctgagg?gctattgagg?cgcaacagca?tctgttgcaa 180
ctcacagtct?ggggcatcaa?gcagctccag?gcaagaggtt?cctctggagg?ttggatggag 240
tgggacagag?aaattaacaa?ttacacaagc?ttaatacact?ccttaattga?agaatcgcaa 300
aaccagcaag?aaaagaatga?acaagaatta?ttgtag 336
<210>19
<211>405
<212>DNA
<213〉artificial sequence
<220>
<221>CDS
<222>(1)..(405)
<223>
<400>19
atggatgcaa?tgaagagagg?gctctgctgt?gtgctgctgc?tgtgtggagc?agtcttcgtt 60
tcgcccagca?tggagtggga?cagagaaatt?aacaattaca?caagcttaat?acactcctta 120
attgaagaat?cgcaaaacca?gcaagaaaag?aatgaacaag?aattattggg?tggttctggt 180
ggatctggta?tagtgcagca?gcagaacaat?ttgctgaggg?ctattgaggc?gcaacagcat 240
ctgttgcaac?tcacagtctg?gggcatcaag?cagctccagg?caagaggttc?ctctggaggt 300
tggatggagt?gggacagaga?aattaacaat?tacacaagct?taatacactc?cttaattgaa 360
gaatcgcaaa?accagcaaga?aaagaatgaa?caagaattat?tgtag 405
<210>20
<211>56
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(56)
<223>
<400>20
tcgagaggac?tcttggactc?tcattcaaga?gatgagagtc?caagagtcct?cttttt 56
<210>21
<211>68
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(68)
<223>
<400>21
tcgaccgtgt?gcacttcgct?tcacctctgt?tcaagagaca?gaggtgaagc?gaagtgcaca 60
cggttttt 68
<210>22
<211>56
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(56)
<223>
<400>22
tcgacccgct?caatgcctgg?agattcaaga?gatctccagg?cattgagcgg?gttttt 56
<210>23
<211>74
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(74)
<223>
<400>23
tcgaaagtgt?tgctttcatt?gccaagtttg?ttttcaagag?aaacaaactt?ggcaatgaaa 60
gcaacacttt?tttt 74
<210>24
<211>30
<212>DNA
<213〉artificial sequence
<400>24
gcggatccat?ggagaacatc?acatcaggat 30
<210>25
<211>29
<212>DNA
<213〉artificial sequence
<400>25
gcgctcgagt?taaatgtata?cccaaagac 29
<210>26
<211>30
<212>DNA
<213〉artificial sequence
<400>26
gcggatccat?ggacattgac?ccgtataaag 30
<210>27
<211>30
<212>DNA
<213〉artificial sequence
<400>27
gcgctcgagt?taaataacac?aagtctccgg 30
<210>28
<211>28
<212>DNA
<213〉artificial sequence
<400>28
gcggatccat?ggatgatgaa?gtcgacgt 28
<210>29
<211>33
<212>DNA
<213〉artificial sequence
<400>29
gcgctcgagt?taagtgaagt?ggatataagc?cat 33
<210>30
<211>30
<212>DNA
<213〉artificial sequence
<400>30
gcggtaccat?gcagtggaat?tccacaacct 30
<210>31
<211>30
<212>DNA
<213〉artificial sequence
<400>31
tagcggccgc?ttaaatgtat?acccaaagac 30
<210>32
<211>32
<212>DNA
<213〉artificial sequence
<400>32
acctccagag?gaaccaatgt?atacccaaag?ac 32
<210>33
<211>32
<212>DNA
<213〉artificial sequence
<400>33
ggttcctctg?gaggtgatga?tgaagtcgac?gt 32
<210>34
<211>32
<212>DNA
<213〉artificial sequence
<400>34
ggttcctctg?gaggtgacga?tgaagttgat?gt 32
<210>35
<211>31
<212>DNA
<213〉artificial sequence
<400>35
gcgctcgagt?taagtaaagt?gaatataagc?c 31
<210>36
<211>32
<212>DNA
<213〉artificial sequence
<400>36
acctccagag?gaaccaataa?cacaagtctc?cg 32
<210>37
<211>30
<212>DNA
<213〉artificial sequence
<400>37
acctccagag?gaaccaatgt?atacccaaag 30
<210>38
<211>30
<212>DNA
<213〉artificial sequence
<400>38
ggttcctctg?gaggtgatga?tgaagtcgac 30
<210>39
<211>30
<212>DNA
<213〉artificial sequence
<400>39
tagcggccgc?ttaagtgaag?tggatataag 30
<210>40
<211>30
<212>DNA
<213〉artificial sequence
<400>40
taggtaccat?ggatgatgaa?gtcgacgtgg 30
<210>41
<211>30
<212>DNA
<213〉artificial sequence
<400>41
acctccagag?gaaccaatgt?atacccaaag 30
<210>42
<211>30
<212>DNA
<213〉artificial sequence
<400>12
ggttcctctg?gaggtgatga?tgaagtcgac 30
<210>43
<211>25
<212>DNA
<213〉artificial sequence
<400>43
cagctgtgga?atgtgtgtca?gttag 25
<210>44
<211>21
<212>DNA
<213〉artificial sequence
<400>44
aatcagcaag?cttggtaccg?a 21
<210>45
<211>48
<212>DNA
<213〉artificial sequence
<400>45
aacagaagga?aagaagtcag?aaggcaaaaa?agcctccagg?ccagaaag 48
<210>46
<211>8
<212>DNA
<213〉artificial sequence
<400>46
tttttgcctt?ctgacttctt?tccttctgtt?ggaggtgggg?gaggcgga 48
<210>47
<211>45
<212>DNA
<213〉artificial sequence
<400>47
aacagtttct?cttccaaaag?taagacaagc?ctccaggcca?gaaag 45
<210>48
<211>45
<212>DNA
<213〉artificial sequence
<400>48
tgtcttactt?ttggaagaga?aactgttgga?ggtgggggag?gcgga 45
<210>49
<211>10
<212>DNA
<213〉artificial sequence
<400>49
gcgaagcttg 10
<210>50
<211>10
<212>DNA
<213〉artificial sequence
<400>50
actcgagcat 10
<210>51
<211>35
<212>DNA
<213〉artificial sequence
<400>51
gtcttcgttt?cgcccagctg?gatggagtgg?gacag 35
<210>52
<211>42
<212>DNA
<213〉artificial sequence
<400>52
gctgtgtgct?gctgctgtgt?ggagcagtct?tcgtttcgcc?ca 42
<210>53
<211>37
<212>DNA
<213〉artificial sequence
<400>53
gatgcaatga?agagagggct?ctgctgtgtg?ctgctgc 37
<210>54
<211>35
<212>DNA
<213〉artificial sequence
<400>54
cgcaggatcc?gccaccatgg?atgcaatgaa?gagag 35
<210>55
<211>32
<212>DNA
<213〉artificial sequence
<400>55
cgcgctcgag?ctacaataat?tcttgttcat?tc 32
<210>56
<211>29
<212>DNA
<213〉artificial sequence
<400>56
cgcaggatcc?tggatggagt?gggacagag 29
<210>57
<211>29
<212>DNA
<213〉artificial sequence
<400>57
ctctgtccca?ctccatccag?gatcctgcg 29
<210>58
<211>3027
<212>DNA
<213〉hepatitis C virus
<220>
<221>CDS
<222>(1)..(3027)
<223>
<400>58
atgagcacaa?atcctaaacc?tcaaagaaaa?accaaacgta?acaccaaccg?ccgcccacag 60
gacgtcaagt?tcccgggcgg?tggtcagatc?gttggtggag?tttacctgtt?gccgcgcagg 120
ggccccaggt?tgggtgtgcg?cgcgactagg?aagacttccg?agcggtcgca?acctcgtgga 180
aggcgacaac?ctatccccaa?ggctcgccgg?cccgagggca?gggcctgggc?tcagcccggg 240
tacccttggc?ccctctatgg?caatgagggc?ttggggtggg?caggatggct?cctgtcaccc 300
cgtggctctc?ggcctagttg?gggccccaca?gacccccggc?gtaggtcgcg?taatttgggt 360
aaggtcatcg?ataccctcac?atgcggcttc?gccgacctca?tggggtacat?tccgcttgtc 420
ggcgcccccc?tagggggcgc?tgccagggcc?ctggcgcatg?gcgtccgggt?tctggaggac 480
ggcgtgaact?atgcaacagg?gaatttgccc?ggttgctctt?tctctatctt?cctcttggct 540
ttgctgtctt?gtctgaccat?cccagcttcc?gcttatgaag?tgcgcaacgt?gtccggggtg 600
taccatgtca?cgaacgactg?ctccaactca?agtattgtgt?atgaggcagc?ggacatgatc 660
atgcacaccc?ccgggtgcgt?accctgcgtc?cgggaggcca?actcctcccg?ctgctgggta 720
gcgctcactc?ccacgctcgc?ggccaggaac?agcagcgtcc?ccactacgac?aatacgacgc 780
cacgtcgatt?tgctcgttgg?ggcagctgct?ctctgctccg?ccatgtacgt?gggagatctc 840
tgcggatctg?ttttcctcat?ctcccagctg?ttcaccttct?cacctcgcca?gtatgagacg 900
gtacaagact?gcaattgctc?actctatccc?ggccacgtat?caggtcaccg?catggcttgg 960
gatatgatga?tgaactggtc?acctacaaca?gccctggtgg?tatcgcagtt?gctccggatc 1020
ccacaagccg?tcgtggacat?ggtggcgggg?gcccactggg?gagtcctagc?gggccttgcc 1080
tactattcca?tggtggggaa?ctgggctaag?gttctgattg?tgatgctact?ctttgccggc 1140
gtcgacgggg?ctacctacac?gtcagggggg?acggtaggcc?ggaccaccag?aggccttacg 1200
tccttctttg?cacctggccc?gtctcagaag?atccaactta?tcaacaccaa?cggcagctgg 1260
cacatcaata?ggactgccct?gaattgcaat?gactccctcc?acaccgggtt?ccttgctgcg 1320
ctgttctaca?cgcacaagtt?caactcgtcc?ggatgctcgg?agcgtatggc?cagctgccgc 1380
cccattgaca?agttcgccca?ggggtggggt?cccatcactc?atggtgtgcc?tgacgacccg 1440
gaccagaggc?cctactgttg?gcactacgcg?cctcggccgt?gcggtatcgt?acccgcgtcg 1500
caggtgtgtg?gtccagtgta?ttgcttcacc?ccgagccctg?tcgtggtggg?gacgaccgat 1560
cgttccggcg?cccccacgta?cacctggggg?gagaatgaga?cggacgtgct?actccttaac 1620
aacacgcgac?cgccgcaagg?caactggttc?ggttgcacat?gggtgaacag?caccgggttc 1680
accaaaacgt?gcgggggccc?cccatgcaac?attggagggg?tcggcaacaa?caccttgacc 1740
tgccccacgg?actgcttccg?gaagcacccc?gaggccactt?acaccaaatg?cggctcgggg 1800
ccatggttga?cacccaggtg?catggttgac?tacccataca?gactctggca?ctacccttgc 1860
actgtcaatt?ttaccatctt?caaggtcagg?atgtatgtag?ggggtgtgga?gcacaggctc 1920
gacgccgcgt?gcaattggac?ccgaggagag?cgttgcaatg?tggaggacag?ggatagatca 1980
gagcttagcc?cactgctact?gtccacaaca?gagtggcagg?tactgccctg?ttctttcacc 2040
accctaccgg?ctctgtccac?tggtttgatc?cacctccacc?agaacatcgt?ggacgtgcaa 2100
tacctgtacg?gtgtggggtc?agtggttgtc?tccgttgtaa?tcagatggga?gtatgtcgtg 2160
ctgctcttcc?ttctcctggc?ggacgcacgc?gtctgcgcct?gcttgtggat?gatgctgctg 2220
gtagcccagg?ctgaggccgc?cttagagaac?ttggtggtcc?tcaatgcggc?atctgtagcc 2280
ggagcgcatg?gcattctctc?cttccttgtg?ttcttctgtg?ctgcctggta?catcaagggc 2340
aagctggtcc?ctggggcggc?atatgccttc?tatggcgtat?ggccgctgct?cctgctcctg 2400
ctggcgttac?caccacgagc?atacgccatg?gaccgggaga?tggctgcatc?atgcggaggc 2460
gcggttttcg?taggtctggc?actcttgacc?ttgtcaccac?actataaagt?gttcctcgcc 2520
aggctcatat?ggtggttgca?ataccttatc?accagggccg?aggcgctgct?gcaagtgtgg 2580
atcccccctc?tcaacgttcg?ggggggccgc?gatgccatca?tcctcctcac?gtgcgcggtc 2640
catccagagc?taatctttga?catcaccaaa?atcttgctcg?ccatatttgg?tccgctcatg 2700
gtgctccagg?ctggcttaac?cagagtgccg?tacttcgtgc?gcgctcaggg?gctcatccgt 2760
gtgtgcatgt?tggtgcggaa?ggtcgctggg?ggtcactacg?tccagatggc?tctcatgagg 2820
ctcgccgcac?tgacgggcac?gtacgtttac?gaccatctca?ctccgctgcg?gggctgggcc 2880
catgcgggcc?tgcgggacct?tgcggtggca?gttgagcccg?tcgtcttctc?tgacatggag 2940
accaagatca?tcacctgggg?ggcagacacc?gcggcgtgtg?gggacatcat?cctgggtcta 3000
cccgtctccg?cccgaagggg?gaggtaa 3027
<210>59
<211>1524
<212>DNA
<213〉human immunodeficiency virus
<220>
<221>CDS
<222>(1)..(1524)
<223>
<400>59
atgggtgcga?gagcgtcagt?attaagcggg?ggaaaattag?ataaatggga?aaaaattcgg 60
ttacggccag?gaggaaagaa?acaatataga?ttaaaacatt?tagtatgggc?aagcagggaa 120
ttagaacgat?tcgcagttaa?tcctggcctt?ttagagacag?cagaaggctg?taaacaaatt 180
ctggaacagc?tacaaccatc?ccttcagaca?ggatcagaag?aacttaggtc?attatttaat 240
acagtagcaa?ccctctattg?tgtccatcag?aggatagagg?taagagacac?caaggaagcc 300
ttagataagg?tagaggagga?gcaaaacaac?agtaagaaaa?aggcacagca?agcagcagct 360
ggcacaggaa?atagcagcca?ggccagccaa?aattacccta?tagtgcagaa?catgcagggg 420
caaatggtac?atcaggcaat?atcacccaga?actttaaatg?catgggtaaa?agtagtagaa 480
gaaaagggtt?ttagcccaga?agtaataccc?atgttttcag?cattatcaga?aggaggcacc 540
ccacaagatt?taaacaccat?gctaaacaca?atagggggac?atcaagcagc?catgcaaatg 600
ttaaaagaca?ccatcaatga?ggaagctgca?gaatgggaca?gattacatcc?agtgcatgca 660
ggacctatcc?caccaggcca?gatgagggaa?cctaggggaa?gtgatatagc?tggatccact 720
agtacccttc?aggaacaaat?acaatggatg?acaagcaacc?cacctgtccc?agtgggagaa 780
atctataaaa?gatggattat?cctaggatta?aataaaatag?taagaatgta?tagccctgtc 840
agcattttgg?acataaaaca?agggccaaag?gaacccttta?gagactacgt?agacaggttc 900
tttaaaaccc?taagagctga?gcaagctaca?caggaagtaa?agggttggat?gacagacacc 960
ttgttggtcc?aaaatgcgaa?cccagattgt?aagaccattt?taaaagcact?gggaccaggg 1020
gcttcactag?aagaaatgat?gacagcatgt?cagggagtag?gaggacccgg?ccataaagca 1080
agagttttgg?ctgaagcaat?gagccacatg?acaaattcag?ctgctataat?gatgcagaaa 1140
ggcaatttta?ggaatcaaag?aaaaactgtc?aagtgtttca?attgtggcaa?agaagggcac 1200
atagccagaa?attgcagggc?ccctagaaaa?aagggctgtt?ggaaatgtgg?aagagaagga 1260
caccaaatga?aagactgcac?tgaaagacag?gctaattttt?tagggagaat?gtggccttcc 1320
cacaagggga?ggccgggaaa?tttccttcag?aacaggccag?agccaacagc?cccaccagca 1380
gagagcttcg?ggttcggaga?ggagataacc?ccatctccga?aggtgacccc?ctctccgaag 1440
caggagcaga?agcaggagca?gaaggacgag?ggactgtacc?ctcccttagc?ttccctcaaa 1500
tcactctttg?gcaacgacca?ctag 1524
<210>60
<211>3045
<212>DNA
<213〉human immunodeficiency virus
<220>
<221>CDS
<222>(1)..(3045)
<223>
<400>60
atgtttttta?gggagaatgt?ggccttccca?caaggggagg?ccgggaaatt?tccttcagaa 60
caggccagag?ccaacagccc?caccagcaga?gagcttcggg?ttcggagagg?agataacccc 120
atctccgaag?gtgaccccct?ctccgaagca?ggagcagaag?caggagcaga?aggacgaggg 180
actgtaccct?cccttagctt?ccctcaaatc?actctttggc?aacgaccact?agttacaata 240
aaaatagagg?gacagctaat?ggaagctcta?ttagatacag?gagcagatga?tacagtatta 300
gaagatgtaa?atttgccagg?aaaatggaga?ccaaaaatga?tagggggaat?tggaggtttt 360
atcaaagtaa?aacagtatga?taacatactc?atagaaattt?gtggacataa?ggctataggt 420
acagtgttgg?taggacctac?gcctgtcaac?ataattggaa?gaaacatgat?gactcagatt 480
ggttgtactt?taaattttcc?aattagtcct?attaggactg?taccagtaaa?attgaagcca 540
ggaatggatg?gcccaaaggt?taaacaatgg?ccattgacag?aagaaaaaat?aaaagcatta 600
acagaaatat?gtttggaaat?ggaaaaagaa?ggaaaaattt?caaaaattgg?gcctgaaaat 660
ccatacaata?ctccagtatt?tgccataaag?aaaaaagaca?gtactaaatg?gagaaaatta 720
gtagatttca?gagaacttaa?taaaagaact?caagattttt?gggaaattca?attaggaata 780
ccacatcctg?cagggttaaa?aaagaaaaag?tcagtgacag?tactggatgt?gggagatgca 840
tatttttcaa?ttcccttaga?tgaggatttc?aggaagtaca?ctgcattcac?catacctagt 900
atcaacaatg?agacaccagg?aattaggtac?cagtacaatg?tgcttccaca?aggatggaaa 960
ggatcaccag?caatattcca?atgtagcatg?acaaaaatct?tagacccctt?tagaacaaaa 1020
aatccagaca?tagtgatctg?ccaatacatg?gatgatttgt?atgtagggtc?tgacttagaa 1080
atagggcaac?atagagcaaa?aatagaggag?ttaagagaac?atctattgaa?atggggactt 1140
actacaccag?acaaaaaata?tcaaaaggag?cctccattcc?actggatggg?gtatgaactc 1200
catcctgata?agtggacagt?gcagcctata?caattgccag?acaaggacag?ctggactgtc 1260
aatgatatac?agaagttagt?aggaaaacta?aattgggcaa?gtcagattta?tccagggatt 1320
aaagtaaagc?aattatgtaa?actccttagg?ggagccaagg?cattaacaga?aatagtgcca 1380
ctgactgcag?aagcagagtt?agaattagca?gagaataggg?aaattctaaa?agaaccagta 1440
catggggtat?attatgaccc?atcaaaagac?ttaatagcag?aaatacagaa?acaagggcaa 1500
gggcagtgga?catatcaaat?ttatcaagag?ccatttaaaa?atctaaaaac?aggaaagtat 1560
gcaaaaatga?ggtctgccca?cactaatgat?gtaaaacaat?taacagaagc?agtgcaaaag 1620
atagctctag?aaagcatagt?catatgggga?aagactccta?agtttagact?acccatatta 1680
aaagagacat?gggatacatg?gtggacagag?tattggcaag?ccacctggat?tcctgaatgg 1740
gagtttgtca?ataccccccc?tctagtaaaa?ctatggtatc?agctagaaac?agagcccata 1800
gcaggagcag?aaaccttcta?tgtagatggg?gcatctaata?gggagaccaa?aaaaggaaaa 1860
gcaggatatg?ttactgacag?aggaagacaa?aaagctgttt?ccctaactga?gaccacaaat 1920
cagaaagccg?agttacaggc?aattcagtta?gctttacagg?attcaggatc?aaaagtgaac 1980
atagtaacag?actcacagta?tgtgttagga?atcattcaag?cacaaccaga?taagagtgac 2040
tcagaattag?tcaatcaaat?aatagagcaa?ttaatagaaa?aggaaaaggt?ctacctgtca 2100
tgggtaccag?cacacaaagg?aattggagga?aatgaacaag?tagataaatt?agtcagtgct 2160
gggatcagga?aagtattatt?tttagatgga?atagataagg?cccaagagga?acatgaaaaa 2220
tatcacaata?attggagatc?aatggctagt?gattttaatc?tgccacctgt?agtagcaaaa 2280
gaaatagtgg?ccagttgtga?taaatgtcag?ctaaaaggag?aagccatgca?tggacaagta 2340
gactgtagtc?caggaatatg?gcagctagat?tgtacacact?tagaaggaaa?aattatcctg 2400
gtagcagttc?atgtagccag?tggatatata?gaagcagaag?ttattccagc?agaaacaggg 2460
caagaaacag?catacttcat?cttaaaatta?gcaggaagat?ggccagtaaa?aataatacat 2520
acagacaatg?gcagcaattt?caccagtact?acagttaagg?ccgcctgttg?gtgggcgggg 2580
atcaaacagg?aatttggcat?tccctacaat?ccccaaagtc?agggagtaat?agaatctatg 2640
aataaggagt?taaagaaaat?tataggacag?gtaagagatc?aggctgaaca?tcttaagaca 2700
gcagtacaaa?tggcagtatt?catccacaat?tttaaaagaa?aaggggggat?tggggggtac 2760
agtgcagggg?aaagaataat?agacataata?gcaacagaca?tacaaactaa?agaattacaa 2820
aaacaaatta?caaaaattca?aaatttccgg?gtttattaca?gggacagcag?agacccagtt 2880
tggaaaggac?cagcaaagct?actctggaaa?ggtgaagggg?cagtagtcat?acaagacaat 2940
agtgaaataa?aagtagtacc?aagaagaaaa?gcaaagatca?ttagggatta?tggaaaacag 3000
atggcaggtg?atgattgtgt?ggcaggtgga?caggatgagg?attaa 3045
<210>61
<211>2583
<212>DNA
<213〉human immunodeficiency virus
<220>
<221>CDS
<222>(1)..(2583)
<223>
<400>61
atgagagtga?gggggatgca?gaggaattgg?caggacttgg?ggaagtgggg?ccttttattc 60
ctgggaatat?taataatctg?taatgctgaa?caaaatttgt?gggtcacagt?ctattatggg 120
gtacctgtgt?ggaaagaagc?atccactact?ctattctgtg?catcagatgc?taaatcatat 180
gaaagagaag?tgcataatgt?ctgggctaca?catgcctgtg?tacccacaga?tcccaatcca 240
caagaactag?ttctgggaaa?tgtaacagaa?aattttgata?tgtggaaaaa?tgatatggta 300
gaacaaatgc?atgaagatat?aattagctta?tgggatcaaa?gcctaaaacc?atgtgtgaag 360
ttaaccccac?tctgtgttac?tttaaactgt?actaatgcca?aggccattaa?ctcctctgac 420
aatgccacca?attctactcc?cttgcctata?accagccccc?tgaaggaaga?ggcaggggca 480
atacaaaact?gttctttcaa?tgtgaccaca?gagttaagag?ataagaagaa?gaaagaatat 540
gcaatttttt?ataaacttga?tatagtacca?atagaacaaa?tcagcaataa?aagttacaat 600
acatacaggc?taataaattg?taacacctca?accactacac?aggcttgtcc?aaaggtatct 660
tgggatccaa?ttcccataca?ttattgtgct?ccagctggtt?ttgcgattct?aaagtgtaat 720
aataaaacgt?tcaatgggac?agggccatgc?aagaatgtca?gtacagtaca?atgtacacat 780
ggaattaaac?cagtggtatc?aactcaattg?ttgttaaatg?gtagcctagc?agaagaagat 840
atagtaatca?gatttcaaaa?tatctcagat?aatgcaaaaa?ccataatagt?acactttaat 900
gaatctgtac?agattaattg?tacaagaccc?aacaacaata?caagaaaagg?tatacatatg 960
ggaccaggac?aagcatttta?tgcagcagga?gaaataatag?gagacatcag?aaaagcatat 1020
tgtactatta?atggaacaca?atggaatgac?actttagaac?aggtaaaggc?aaagttgcag 1080
gagcatttcc?ctaatagaac?aataatgttt?aactcatctg?caggagggga?cctagaaatt 1140
acaacacata?tttttaattg?tagaggagaa?tttttttact?gcaatacatc?agaactgttt 1200
aataacacaa?aaaacaatgg?caccatcatt?ctcccatgta?aaataaaaca?aattgtaaac 1260
atgtggcaga?gagtaggacg?agcaatgtat?gccaatccca?ttgcaggaaa?aattaactgt 1320
aactcaaata?ttacaggtct?attattgaca?agagatggtg?gtaatcaggc?taatgagact 1380
agtggtattc?agactaatgg?gactgagatc?ttcagacctg?gaggaggaaa?tatgagagac 1440
aattggagaa?gtgaattata?taaatataaa?gtagtacaaa?ttaaaccact?aggaatagca 1500
cccaccaggg?caaaaagacg?agtggtgcag?agagcaagaa?gagcagtggg?aataggagct 1560
cttttcattg?gattcttagg?agcagcagga?agcactatgg?gcgcggcgtc?aataacgctg 1620
acggtacagg?ccagacaatt?attgtctgga?atagtgcaac?agcaaaacaa?tttactgcag 1680
gctattgaag?cgcaacagca?tctgttgcag?ctcacagtct?ggggcattaa?acagctccag 1740
gcaagactcc?tggctgtgga?aagattccta?catgatcaac?agctcctagg?gctttggggc 1800
tgctctggaa?aactcatctg?caccactagt?gtgccctgga?actctagttg?gagcaataaa 1860
tcttatgaca?agatttggga?taacatgacc?tggatggagt?gggaaaaaga?gattagcaat 1920
tactcagatg?aaatatacag?gttaattgaa?aaatcgcaga?accagcagga?aataaatgaa 1980
caagaattat?tagcattgga?taaatgggca?agtctgtgga?attggtttga?cataacaagc 2040
tggctgtggt?atataaaaat?attcataatg?atagtaggag?gcttgatagg?cttaagaata 2100
gtttttactg?tgctttctat?agtaaataga?gttaggaagg?gatactcacc?tttgtcattt 2160
cagacccata?tcccaagctc?gagggaacaa?cccgacaggc?ccgaaggaat?cgaagaagga 2220
ggtggagagc?aagacaaaga?cagatccgtg?agattagtga?gcggattctt?agctcttgtc 2280
tggaacgacc?tgagggacct?gtgcctcttc?agctaccacc?tcttgagaga?cttcacatta 2340
cttgcagcga?ggattgtgga?cagagggctg?aggagggggt?gggaagccct?caaatatctg 2400
tggaatctcg?cgcagtattg?gagtcgggaa?ctaaagaata?gtgctattag?cttgtttaat 2460
accacagcaa?tagtagtagc?tgaagggaca?gatagagttc?tagaagcttt?gcaaagagct 2520
ggtagagctg?ttctccatat?ccctagaaga?ataagacagg?gctttgaaag?agctttgcta 2580
taa 2583
<210>62
<211>19
<212>DNA
<213〉artificial sequence
<400>62
gaggactctt?ggactctca 19
<210>63
<211>25
<212>DNA
<213〉artificial sequence
<400>63
ccgtgtgcac?ttcgcttcac?ctctg 25
<210>64
<211>19
<212>DNA
<213〉artificial sequence
<400>64
catcacatca?ggattccta 19
<210>65
<211>25
<212>DNA
<213〉artificial sequence
<400>65
ctcagtttac?tagtgccatt?tgttc 25
<210>66
<211>21
<212>DNA
<213〉artificial sequence
<400>66
catcacatca?ggattcctaa?a 21
<210>67
<211>25
<212>DNA
<213〉artificial sequence
<400>67
agaagatctc?aatctcggga?atctc 25
<210>68
<211>18
<212>DNA
<213〉artificial sequence
<400>68
cccgctcaat?gcctggag 18
<210>69
<211>111
<212>DNA
<213〉hepatitis C virus
<220>
<221>CDS
<222>(1)..(111)
<223>
<400>69
atggttttcg?gcggccattg?gggtgtggtg?tttggcttgg?cctatttttc?catgcaagga 60
gcgtgggcca?aagtcattgc?catcctcctc?cttgtcgcag?gagtggacgc?g 111
<210>70
<211>1098
<212>DNA
<213〉hepatitis C virus
<220>
<221>CDS
<222>(1)..(1098)
<223>
<400>70
ggcacctata?ccaccggtgc?agtcacgggt?cgaaccacca?atgtgtttac?tagcctcttt 60
tcatctgggt?cccagcagaa?gctcagttta?atcaacacca?atggcagctg?gcacataaac 120
cggaccgccc?tcaattgcaa?tgacagcctg?cagacggggt?ttatcgcctc?cttgttttac 180
acgcacaggt?ttaacagctc?cggctgcccc?gagcgcttgt?cttcctgccg?caggctggaa 240
gatttccgca?tcgggtgggg?aaccttggaa?tacgagacta?atgtcaccaa?tgatgaggac 300
atgaggccgt?actgctggca?ttaccctcca?agaccctgcg?gtatcgtcca?ggctaagacg 360
gtttgcgggc?cggtctattg?tttcaccccc?agccctgtcg?tcgtgggtac?cactgacagg 420
cagggcgtgc?ccacttacag?ctggggggaa?aatgagaccg?atgttttctt?gttaaatagt 480
acaagacccc?cgcaaggagc?ctggttcggc?tgcacttgga?tgaatgggac?tgggttcact 540
aagacatgcg?gtgcaccacc?ttgccgcatt?aggagggact?acaacggaac?cctcgaccta 600
ttgtgcccca?cagactgttt?cagaaagcac?ccagatacta?cctaccttaa?gtgtggagcg 660
gggccttggt?tgacccccaa?atgcatggta?gactatccct?atagattgtg?gcattatccg 720
tgcactgtga?attttaccat?cttcaaggtg?cggatgtatg?tgggaggagt?ggagcatcgg 780
ttccatgcag?cgtgcaatta?cacgcgcggg?gaccgctgca?gtttggagga?cagggacagg 840
ggtcagcaga?gtccactact?gcattccacc?accgagtggg?cggtgttgcc?atgctctttc 900
tccgacctac?cggcactatc?cactggtcta?ctgcacctcc?accaaaacat?cgtggacgtg 960
cagtacctct?atggactctc?tccggctatc?acaaaataca?tcgtgaagtg?ggaatgggtg 1020
atccttcttt?tcctgttgct?ggcagacgcc?agggtctgtg?cttgcctctg?gatgctcatt 1080
atattgggcc?aggccgaa 1098
<210>71
<211>28
<212>DNA
<213〉artificial sequence
<400>71
aagtgttgct?ttcattgcca?agtttgtt 28
<210>72
<211>10
<212>PRT
<213〉artificial sequence
<400>72
Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val
1 5 10
<210>73
<211>9
<212>PRT
<213〉artificial sequence
<400>73
Trp?Leu?Ser?Leu?Leu?Val?Pro?Phe?Val
1 5
<210>74
<211>9
<212>PRT
<213〉artificial sequence
<400>74
Phe?Leu?Leu?Thr?Arg?Ile?Leu?Thr?Ile
1 5
<210>75
<211>10
<212>PRT
<213〉artificial sequence
<400>75
Tyr?Val?Asn?Val?Asn?Met?Gly?Leu?Lys?Leu
1 5 10
<210>76
<211>9
<212>PRT
<213〉artificial sequence
<400>76
Glu?Tyr?Leu?Val?Ser?Phe?Gly?Val?Trp
1 5
<210>77
<211>14
<212>PRT
<213〉artificial sequence
<400>77
Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr?Glu
1 5 10

Claims (25)

1. the cocktail vaccine of anti-immunologic tolerance virus and/or togavirus, it comprises two or more element and optional virus antigen recombiant protein and pharmaceutically acceptable excipient, described element exists with the form of recombinant DNA and/or recombinant virus, described element is selected from (A) neutralizing antibody, receptor blocking agent, membrane fusion inhibitor, cytokine coded sequence, and/or RNA interferes sequence; (B) immunological adjuvant coded sequence; (C) antigen gene, virus antigen and immunological adjuvant fusion gene, virus antigen epitope coded sequence, multi-epitope fusion gene and/or virus antigen epitope-MHC-I SCT gene; (D) coded sequence of two of the togavirus fusion rotein seven peptide repetitive sequence HR1, HR2 and/or its polymer.
2. according to the cocktail vaccine of claim 1, wherein said immunologic tolerance virus is hepatitis B virus (HBV) or hepatitis C virus (HCV).
3. according to the cocktail vaccine of claim 1, wherein said togavirus is human immunodeficiency virus (HIV), respiratory syncytial virus (RSV) or sars coronavirus (SARS-Cov).
4. according to the cocktail vaccine of claim 1, wherein said immunological adjuvant is heat shock protein gp96 or its n terminal fragment.
5. according to the cocktail vaccine of claim 1, wherein said virus antigen recombiant protein can be selected from, but be not limited to following sequence expressed proteins: the comparison conserved sequence of virus antigen, main epidemic strain sequence or its partial sequence, or the fusion gene sequence of virus antigen and adjuvant fusion.
6. according to the cocktail vaccine of claim 1, described RNA interferes its target sequence of sequence to be selected from, but is not limited to encoding viral sequence or non-coding sequence.
7. according to the cocktail vaccine of claim 1, wherein said membrane fusion inhibitor can be selected from, but is not limited to seven peptide repetitive sequence HR1, HR2 or its polymer that the HIV film merges.
8. according to the cocktail vaccine of claim 1, wherein said cytokine can be selected from, but is not limited to cytokine IFN α (1 and 2) or γ, TNF α or IL-2.
9. according to the cocktail vaccine of claim 1, wherein said antigen gene can be selected from, but is not limited to the comparison conserved sequence of virus antigen, main epidemic strain sequence or its partial sequence, or the fusion gene sequence of virus antigen and adjuvant fusion.
10. according to the cocktail vaccine of claim 1, wherein said virus antigen epitope coded sequence can be selected from, but is not limited to viral structural gene or reconciles the gene epitope sequences.
11. according to the cocktail vaccine of claim 1, wherein said multi-epitope fusion gene can be selected from, but is not limited to viral Th epi-position and MHC-I restriction epi-position and adjuvant or/and the ubiquitin fusion sequence.
12. according to the cocktail vaccine of claim 1, wherein said virus antigen epitope-MHC-I SCT gene can be selected from but be not limited to, the fusion sequence of HLA-A2, HLA-A11, HLA-A24, HLA-30 and virus antigen and β 2m.
13. according to the cocktail vaccine of claim 1, wherein said recombinant DNA is a recombiant plasmid.
14. according to the cocktail vaccine of claim 13, wherein said plasmid is a carrier for expression of eukaryon.
15. according to the cocktail vaccine of claim 1, wherein said recombinant virus is recombinant adenovirus or vaccinia virus recombinant.
16. according to the cocktail vaccine of claim 1, it is the multichip carrier vaccine that comprises recombinant DNA, adenovirus and/or poxvirus that makes up by described element is randomly made up.
17. according to the cocktail vaccine of claim 1, it is by described element being covered the single carrier bacterin that makes up in same DNA or adenovirus or the poxvirus vector.
18. cocktail vaccine according to claim 1 or 2, wherein said cocktail vaccine is a HBV therapeutic cocktail vaccine, it comprises two or more element, described element exists with the form of recombinant DNA and/or recombinant virus, and described element is selected from: (1) effectively reduces RNA interfering (siRNA) and/or interferon (IFN) α or the β coded sequence that HBV-DNA duplicates; (2) adjuvant molecule gp96 or its n terminal fragment coded sequence; (3) fusion gene that merges of the structural antigens HBsAg of HBV, HBcAg or itself and adjuvant and/or its epitope coded sequence, multi-epitope fusion gene, epitope-MHC-I SCT gene.
19. cocktail vaccine according to claim 1 or 2, wherein said cocktail vaccine is a HCV therapeutic cocktail vaccine, it comprises two or more element, described element exists with the form of recombinant DNA and/or recombinant virus, and described element is selected from: (1) effectively reduces RNA interfering (siRNA) and/or IFN α or the β coded sequence that HCV duplicates; (2) adjuvant molecule gp96 or its n terminal fragment coded sequence; (3) fusion gene that merges of the structural antigens Core of HCV, E1, E2 coded sequence or itself and adjuvant and/or epitope coded sequence, multi-epitope fusion gene, the epitope-MHC-I SCT gene of HCV.
20. cocktail vaccine according to claim 1 or 3, wherein said cocktail vaccine is the preventative cocktail vaccine of HIV, it comprises two or more element, described element exists with the form of recombinant DNA and/or recombinant virus, and described element is selected from: seven peptide repetitive sequence HR1, the HR2 of (1) HIV capable of blocking and cell fusion or the coded sequence of its polymer; (2) adjuvant molecule gp96 or its n terminal fragment coded sequence; (3) fusion gene that merges of the structural antigens env of HIV, gag, pol coded sequence and/or itself and adjuvant and/or epitope coded sequence, multi-epitope fusion gene, the epitope-MHC-I SCT gene of HIV.
21. cocktail vaccine according to claim 1 or 3, wherein said cocktail vaccine is a HIV therapeutic cocktail vaccine, it comprises two or more element, described element exists with the form of recombinant DNA and/or recombinant virus, and described element is selected from: seven peptide repetitive sequence HR1, the HR2 of (1) HIV capable of blocking and cell fusion or the coded sequence of its polymer; (2) can effectively suppress RNA interfering (siRNA) and/or IFN α or the β coded sequence that HIV duplicates; (3) adjuvant molecule gp96 or its n terminal fragment coded sequence; (4) fusion gene that merges of the structural antigens env of HIV, gag, pol and/or itself and adjuvant and/or epitope coded sequence, multi-epitope fusion gene, the epitope-MHC-I SCT gene of HIV.
22. according to the cocktail vaccine of claim 1,20 or 21, wherein said polymer can be HR212.
23. prevent and/or treat purposes in the medicine of the disease that causes by immunologic tolerance virus and immunodeficiency virus in preparation according to each cocktail vaccine among the claim 1-22.
24. comprising, a method for preparing the described medicine of claim 23, described method will be selected from the element of following two or more: (A) neutralizing antibody, receptor blocking agent, membrane fusion inhibitor, cytokine coded sequence, and/or RNA interferes sequence; (B) immunological adjuvant coded sequence; (C) antigen gene, virus antigen and immunological adjuvant fusion gene, virus antigen epitope coded sequence, multi-epitope fusion gene and/or virus antigen epitope-MHC-I SCT gene; (D) coded sequence of two of the togavirus fusion rotein seven peptide repetitive sequence HR1, HR2 and/or its polymer HR212, be built into active carrier with recombinant DNA, recombinant virus respectively, described recombinant DNA and/or recombinant virus active carrier are mixed with optional virus antigen recombiant protein and the pharmaceutical excipient of choosing wantonly.
25. the method for the disease that a treatment is caused by immunologic tolerance virus and immunodeficiency virus is wherein to the described medicine of patient's administration claim 23 of the described disease of trouble.
CN200610165104A 2006-12-13 2006-12-13 Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application Expired - Fee Related CN101057975B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN200610165104A CN101057975B (en) 2006-12-13 2006-12-13 Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN200610165104A CN101057975B (en) 2006-12-13 2006-12-13 Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application

Publications (2)

Publication Number Publication Date
CN101057975A true CN101057975A (en) 2007-10-24
CN101057975B CN101057975B (en) 2012-10-10

Family

ID=38864378

Family Applications (1)

Application Number Title Priority Date Filing Date
CN200610165104A Expired - Fee Related CN101057975B (en) 2006-12-13 2006-12-13 Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application

Country Status (1)

Country Link
CN (1) CN101057975B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103501807A (en) * 2011-02-23 2014-01-08 迈阿密大学 Combined cell based gp96-ig-siv/hiv, recombinant gp120 protein vaccination for protection from siv/hiv
US8968720B2 (en) 2008-03-20 2015-03-03 University Of Miami Heat shock protein GP96 vaccination and methods of using same
CN110904138A (en) * 2018-09-17 2020-03-24 杭州博茵生物技术有限公司 Escherichia coli soluble protein expression vector of Msyb fusion tag, construction method and application
WO2021155846A1 (en) * 2020-02-05 2021-08-12 翁炳焕 Preparation method for novel coronavirus pneumonia bivalent vaccine
WO2024175111A3 (en) * 2023-02-24 2024-10-03 Suzhou Sanegene Bio Inc. Small interfering rna targeting hbv and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011513399A (en) 2008-03-03 2011-04-28 ザ ユニバーシティー オブ マイアミ Immunotherapy with allogeneic cancer cells

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8968720B2 (en) 2008-03-20 2015-03-03 University Of Miami Heat shock protein GP96 vaccination and methods of using same
CN103501807A (en) * 2011-02-23 2014-01-08 迈阿密大学 Combined cell based gp96-ig-siv/hiv, recombinant gp120 protein vaccination for protection from siv/hiv
CN110904138A (en) * 2018-09-17 2020-03-24 杭州博茵生物技术有限公司 Escherichia coli soluble protein expression vector of Msyb fusion tag, construction method and application
WO2021155846A1 (en) * 2020-02-05 2021-08-12 翁炳焕 Preparation method for novel coronavirus pneumonia bivalent vaccine
WO2024175111A3 (en) * 2023-02-24 2024-10-03 Suzhou Sanegene Bio Inc. Small interfering rna targeting hbv and uses thereof

Also Published As

Publication number Publication date
CN101057975B (en) 2012-10-10

Similar Documents

Publication Publication Date Title
CN1282656C (en) HIV peptides from conserved regions in GAGP 17 and 924 and their application in e.g. vaccines
CN1188519C (en) Fusion proteins comprising HIV-1 Tat and/or Nef proteins
CN1636063A (en) Genetic vaccine against human immunodeficiency virus
CN1179973C (en) HBV core antigen particles with multiple immunogenic components attached via peptide ligands
CN1213068C (en) Improvments in or relating to immune responses to HIV
CN1446102A (en) Genetic vaccine that mimics natural viral infection and induces long-lasting immunity to pathogens
CN1533284A (en) Use of biologically active HIV-1 Tat, fragments or derivatives thereof for preventing or therapeutic vaccination and/or treating other diseases
CN1122613A (en) Process for the preparation of immunogens or diagnostic reagents, and immunogens or diagnostic reagents thereby obtainable
CN1659187A (en) Ferritin fusion proteins for use in vaccines and other applications
CN1230549A (en) Escape mutant of surface antigen of hepatitis B virus
CN101057975A (en) Cocktail vaccine for anti immune tolerance and immunodeficiency virus and its application
CN1639324A (en) Viral variants with altered susceptibility to nucleoside analogs and uses thereof
CN1602202A (en) A method for identification and development of therapeutic agents
CN1483736A (en) Immunogen for producing vaccine or medicine to treat hepatitis B, its preparation process and use
CN101036784A (en) Toxicity T cell position vaccine of the cell for treating Hepatitis B and the preparing method
CN1255540C (en) Vaccine-induced hepatitis B viral strain and uses thereof
CN1874787A (en) Composition for the prophylaxis/treatment of HBV infections and HBV-mediated diseases
CN1934248A (en) HBV variants detection and application
CN1812810A (en) Materials and methods for immunizing against fiv infection.
CN1570115A (en) Optimized SARS coronavirus spike protein gene
CN1249241C (en) HIV-like particles and use thereof
CN1612942A (en) Viral capsid assembly intermediates
CN1809381A (en) HIV-1 envelope glycoproteins having unusual disulfide structure
CN1720261A (en) Vaccine
CN1268392C (en) Vaccine of recombined albumen for preventing and treating infection of human C type hepatitis virus and its usage

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20121010

Termination date: 20211213