CN104293741A - Respiratory syncytial virus virus-like particle vaccine as well as preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, and particularly relates to a respiratory syncytial virus (RSV) virus-like particle (VLPs) subunit vaccine as well as a preparation method and application thereof. The component of the vaccine is chimeric antigen protein which is fusion-expressed together with neutral antigenic epitope of an RSVG protein or simultaneously with the antigenic epitope of T cells of an M2 protein by taking hepatitis B virus core protein as a carrier. The high-purity antigen component is prepared by efficiently expressing antigen protein in escherichia coli and then performing in-vitro purification, degeneration and renaturation and self assembling into virus-like particles (VLPs). The RSVG protein contained in the VLPs and the antigenic epitope of the T cells of the M2 protein are simultaneously expressed, so that the capability of the vaccine for introducing specific immunity response and anti-RSV infection immunity protection can be enhanced, the balanced Th1/Th2 immunity response can be induced and the RSV vaccine is prevented from enhancing the incidence of diseases. Animals are immunized and inoculated with VLPs vaccines to induce organisms to generate high-level RSV neutral antigens, enhanced Th1 cell factor level and effective protection on RSV attack infection.

Description

A kind of respiratory syncytial virus virus sample particle vaccines and its preparation method and application

Technical field

The present invention relates to virus sample particle vaccines of a kind of chimeric expression respiratory syncytial virus multi-epitope antigen and its preparation method and application, belong to biological technical field, for the immunoprophylaxis of people and susceptible animal respiratory syncytial virus infection.

Background technology

Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) be cause infant's lower respiratory infection in global range and cause it to suffer from bronchiolitis and pneumonia, one of important pathogen body that bronchial asthma seriously can be caused even dead, nearly all children infected before two years old RSV (Black, 2003) at least one times.Age, the less state of an illness was heavier, and reinfection rate is high, occurred closely related with asthma; Immune deficiency person is easy infection more, and clinical symptom is even more serious, often lethal (Holt and Sly, 2002 of dying; Meissner et al., 2004).The elderly and immune deficiency patient are also the easy infection crowds of RSV.Can not produce the effective immunoprotection for rsv infection after people infects RSV, therefore, the easy repeated infection of RSV causes disease.In recent years, because rsv infection is in hospital and cause dead case to rise year by year.

RSV belongs to the member in Paramyxoviridae, pneumonitis virus genus, and its genome is Nonsegmented single strand RNA, is made up of 15225 Nucleotide.RSV genome encoding at least 11 kinds of protein, geneome RNA from 3 ' to 5 ' holds the albumen of coding to be followed successively by NS1, NS2, N, P, M, SH, G, F, M2 (M2-1, M2-2) and L etc., wherein surface protein Glycoprotein G and fusion protein F be mainly in and antigenic component.After organism infection RSV, G and the F antigen protein of encoding viral can stimulate immune response, produces the secretory IgA antibody of serum IgG neutralizing antibody and respiratory mucosa.Serum neutralizing antibody and respiratory tract secretory IgA are the essential substance of anti-rsv infection.In addition, M2 albumen 82-90 amino acids is an effective cytotoxic T cell epi-position (Cytotoxic T lymphocyte epitopes, CTL), significantly can activate CD8 ⁺t cell immunne response, suppresses the reaction of Th2 class, reducing vaccine enhancement disease (Vaccine Enhance Disease, VED) and occurs, having vital role at anti-rsv infection with alleviating in VED.

At present, the key agents for rsv infection disease treatment is ribavirin (Ribavirin), a class guanosine-analogue, and what can stop the synthesis of viral RNA and virus mRNA adds cap, but this medication effect is limited.Medicine for preventing spectrometry in high-risk infants to infect RSV has palivizumab (palivizumab), a kind of monoclonal antibody for RSV surface fusion protein F.This drug manufacture cost is high, needs to inject in advance and continuous injection just can reach preventive effect in 5 months, is therefore difficult to apply.The same with other viral infectious, vaccine inoculation should become prevention rsv infection available strategy.In the sixties in last century, people have prepared traditional inactivated vaccine (the Formalin inactivated RSV vaccine with Formalin inactivation RSV, FIV), clinical trial confirms, not only infant infection RSV could not be prevented after this FIV vaccine inoculation, vaccinated individuals is in RSV natural infection subsequently, the severity of bronchitis and pneumonia incidence and disease increases all greatly, number of being hospitalized for treatment obviously rises, there is vaccinating children death (Kim et al., 1969).Subsequently, RSV vaccine development is the emphasis of rsv infection diseases prevention and treatment always, but so far still without effective RSV vaccine get the Green Light listing use.What RSV vaccine was also classified as global vaccine program by the World Health Organization (WHO) first develops one of vaccine.

FIV vaccine inoculation can not be induced the effective Immune responses of the antivirus of body and be caused VED, is the major obstacle of current RSV vaccine research and development.Its reason is, FIV inoculation can not induce body to produce high-level neutralizing antibody, fails inducing anti-disease toxic effect CD8 ⁺t cell immunne response, caused by the cytokine proportional imbalance that host Th1/Th2 ratio and Th1, Th2 produce etc.And the IgE antibody that FIV inoculation induction body produces, and IL-4, IL-5 and IL-10 etc. are the CD4 of main Th2 deflection ⁺t cell response is causing playing an important role in VED.Improve vaccine-induced generation neutralizing antibody ability, the more efficient CD8 of induction ⁺t cell immunne response, and the Th1/Th2 ratio more balanced are the keys (Murata, 2009) of Development of Novel RSV vaccine.

Subunit vaccine is one of important directions of development RSV vaccine, has many bibliographical informations in recent years.Existing relevant patent report comprises: have contriver with nontoxic type heat-labile enterotoxin of E, coli LT for adjuvant, amalgamation and expression RSVG albumen aa130-230 region, and amino acid between aa182-186 is wherein replaced to the TCL epi-position (YLEKESIYY) of RSVM albumen, invent G _cTLsubunit vaccine (the patent No.: 200710066476.5).Separately there is contriver to adopt DsbA to be carrier proteins, express RSVF/M2 peptide section, for the preparation of the subunit vaccine (patent No.: 200510009119.6) of prevention rsv infection.Particularly recent; contriver is had to take adenovirus as carrier; recombinant adenovirus has been prepared by M, F of RSV and G-protein gene recombination to genome; after this recombinant adenovirus infects Mammals Vero cell; express M, F and G-protein also assembling generation virus-like particle, immune animal can induce the available protecting (number of patent application: 201310667523.7) of the anti-rsv infection of body.

Virus-like particle (Virus-like particles, VLPs) is by the protein grain of the highly structural of virus structural protein being made by manufacturers or users, lacks viral nucleic acid, without infectious.VLPs surface can be repeated high-density and be shown Surface Display of Foreign Epitopes, thus inducement efficient immunne response.Recent research shows, some VLPs effectively can pass through MHCI class and mhc class ii antigen presentation approach present antigen, induction CTL response, excites Th1 and Th2 type cell immune response, and the VLPs of some viruses can inducing dendritic shape cell and the maturation of mononuclear macrophage group and the expression of cytokine.Therefore, using chimeric virus-like particle antigen is prepared vaccine and is had feature safely and efficiently.

The cAg (HBc) of viruses of human hepatitis B (HBV) can spontaneous assembling assembly virus-like particle in virus infection, the nucleic acid of parcel virus.The virus-like particle of the spontaneous formation of HBc has two kinds of sizes, is respectively the icosahedron (Wynne et al., 1999) be made up of 180 and 240 core monomer antigen.HBc total length is 183AA, and wherein 75-82AA is antigen visualization area, is illustrated in virus like particle surface, spike district in the middle of forming.C-terminal 39 AA of HBc are rich in arginine, have the function in conjunction with packaging virus nucleic acid, do not affect HBc and form virus-like particle after removal.HBc after brachymemma can at E. coli, and be independently assembled into virus-like particle, C latter end after the middle spike district of HBc and brachymemma is all exposed to the surface of virus particle, and this region can the epitope (Birkett et al., 2002) of amalgamation and expression external source.Some HBc merging exogenous B cell epitope or T cell antigen epi-position still can be assembled into chimeric VLPs, and induction of immunity animal produces strong immunne response (Pumpens and Grens, 2001).Adopt the HBc of escherichia coli expression can form the HBc particle with natural structure, can be used for the VLPs vaccine of the different antigen fragment of construction expression.As a kind of novel epitope presentation system, HBc carrier proteins is applied to influenza virus, vaccine development (De Filette et al., 2006 of the pathogenic agent such as virus of AIDS and mycobacterium tuberculosis; Gonzalez et al., 2009; Yin et al., 2011).

The present invention is fully using for reference on productive basis, by optimization design, experiment screening, having invented based on HBc is carrier proteins, include RSVG albumen conservative antigen structure and M2 protein CTL epi-position, the subunit vaccine of virus-like particle is formed at expression in escherichia coli, this virus-like particle subunit vaccine can be used for the infection preventing humans and animals opposing RSV, has important science and using value.

Summary of the invention

Technical problem

The object of this invention is to provide a kind of novel respiratory syncytial virus virus sample particle vaccines and its preparation method and application.This vaccine safety is efficient, and not containing infectious viral nuclei acid, antigen purity is high, the natural structure of analogue antigen albumen, and immunization can induce body to produce the protective immune response of anti respiratory syncytial virus infection.For the immunoprophylaxis of the respiratory tract disease that humans and animals respiratory syncytial virus infection causes.

Technical scheme

The invention provides a kind of respiratory syncytial virus sample particle, comprise with the hepatitis B virus core protein of brachymemma (HBc1-144aa) for carrier, chimeric RSV G-protein Neutralization and crystallization wherein, the virus-like particle that can independently assemble.

The present invention further provides a kind of CTL epi-position also having merged RSV M2 albumen at above-mentioned hepatitis B virus core protein C-terminal, the virus-like particle that can independently assemble.

Wherein with the hepatitis B virus core protein of brachymemma (HBc1-144aa) for carrier, the antigen fragment (G144-204aa) (Plotnicky-Gilquin et al., the 1999) G-protein that RSV encodes being guarded B cell epi-position inserts between Asp78 and the Pro80 of HBc peptide section; Or the CTL epi-position 82-90aa (M2 of the M2 albumen of simultaneously RSV being encoded _82-90) merge at the C-terminal of truncated protein HBc1-144aa, this M2 _82-90epi-position can effectively activate body CD8 ⁺t lymphocyte immunity reaction (Srikiatkhachorn and Braciale, 1997).Through codon optimized design, screening, obtain the gene fragment (HBc-tG, SeqNO.2) merged by the 144-204aa nucleotide sequence of 1-144aa and the RSVG albumen of coding HBc and the gene fragment (HBc-tG/M2 merged by 144-204aa and the M2 protein 82-90aa nucleotide sequence of 1-144aa and the RSV G-protein of coding HBc _82-90, SeqNO.4), it is cloned into respectively in expression vector pET28, builds recombinant expression plasmid pHBc-tG and pHBc-tG/M2 _82-90.By pHBc-tG and pHBc-tG/M2 _82-90transformation of E. coli, screening is used for the engineering strain of virus-like particle (VLPs) production of vaccine, stability of characteristics; Through optimum culture condition, high expression fusion rotein HBc-tG and HBc-tG/M2 _82-90.Adopt the technological line that inclusion body separation, protein affinitive layer purification and renaturation in vitro self-assembly etc. combine, purification is used for the VLPs goods of immunization.

Respiratory syncytial virus sample particulate antigen PBS is diluted to proper concn, is prepared into respiratory syncytial virus-like particle vaccine of the present invention.

The respiratory syncytial virus virus-like particle antigen that the present invention obtains also can be used for respiratory syncytial virus infection diagnostic reagent or diagnostic kit.

Beneficial effect

The induction of respiratory syncytial virus particle vaccines Mice Inoculated produces cause of disease specificity neutralizing antibody, and after immune 2 weeks of third time, mouse obtains opposing lethal dose (3 × 10 ⁶pFU/ is only) effect of respiratory syncytial virus challenge infection, and demonstrate stronger immunological memory, the CD8 of inducement efficient ⁺t cell response, the Th1/Th2 ratio of balance and to respiratory syncytial virus strong virus attack infecting mouse 100% protective efficacy.

Accompanying drawing explanation

Fig. 1 is the RSV chimeric antigen protein gene cloning vector plasmid (for the present invention designs, entrusting commercial company's synthesis) of design and synthesis.

Fig. 2 is recombinant expression plasmid pHBc-tG and pHBc-tG/M2 _82-90build schematic diagram.

HBc-tG and HBc-tG/M2 that Fig. 3 designs for the present invention _82-90recombiant vaccine protein ingredient is identified.

A, engineering bacterium expression HBc-tG and HBc-tG/M2 _82-90recombiant vaccine protein ingredient SDS-PAGE analyzes (M: molecular weight Marker; 1,3 is express HBc-tG engineering bacteria to add the tropina that IPTG induces front and back; 2,4 for expressing HBc-tG//M2 _82-90engineering bacteria adds the tropina before and after IPTG induction); B, SDS-PAGE detect the vaccine composition (M: molecular weight Marker of purifying; 1:HBc-tG; 2:HBc-tG/M2 _82-90); C, Western Blot detects immunoreactivity (the anti-tG antibody of vaccine composition; M: molecular weight Marker; 1:HBc-tG; 2:HBc-tG/M2 _82-90).

Fig. 4 is virus-like particle (VLPs) transmission electron microscope tem observation after phospho-wolframic acid negative staining.

A, HBc-tG particle; B, HBc-tG/M2 _82-90particle;

Fig. 5 is that UV deactivation RSV and two-strain sample particle (VLPs) vaccine immune mouse humoral immunoresponse(HI) detect (A, serum total Ig G and IgG1 and IgG2a Subclass Antibodies titre; B, subclass IgG2a/IgG1 ratio; C, NAT.

Fig. 6 is UV deactivation RSV and the secretion of two-strain sample particle (VLPs) vaccine immune mouse splenic lymphocyte Th1, Th2 Cytokines Level in Patients Undergoing.

A, Th1 type cytokines; B, Th2 type cytokines;

Fig. 7 is that after UV deactivation RSV and two-strain sample particle (VLPs) vaccine immune mouse RSV attack poison infection, lung tissue secretes Th1 and Th2 Cytokines Level in Patients Undergoing.

A, Th1 type cytokines; B, Th2 type cytokines;

Fig. 8 is that after UV deactivation RSV and two-strain sample particle (VLPs) vaccine immune mouse RSV attack poison infection, lung tissue RSV titre compares.

Fig. 9 be UV deactivation RSV and two-strain sample particle (VLPs) vaccine immune mouse RSV attack poison infect after pathologic analysis (lung tissue section HE dye observation).

A, UV deactivation RSV vaccine immune mouse; B, HBc-tG particle vaccines immune mouse; C, HBc-tG/M2 _82-90particle vaccines immune mouse; D, normal mouse;

Embodiment

Now come with the following Examples to describe the present invention in further detail.There is provided the object of these embodiments to be only exemplarily the present invention to be described, can not be understood as is restriction to scope of the present invention and essence.

Not marked specific experiment condition and method in the following example, usually conveniently condition as the chief editors such as J. Pehanorm Brooker, Science Press, 1992, Molecular Cloning: A Laboratory guide (third edition); D.L. Spector etc., Science Press, 2001, the conditions described in book such as cell experiment guide, or according to the condition that manufacturer advises.

The term used in the present invention, unless otherwise specified, generally has the implication that those of ordinary skill in the art understand usually.

The structure of [embodiment 1] respiratory syncytial virus virus-like particle (VLPs) and detection

1.1, plasmid pUC57 (HBc-tG/M2 _82-90) structural representation

Plasmid pUC57-HBc-tG/M2 _82-90include the plasmid vector of the present invention through the RSVG albumen conservative antigen district of codon optimized design, screening with the chimeric coding gene sequence of CTL epi-position of M2.Protein coded by this gene order is antigen fragment (G144-204aa) (the Plotnicky-Gilquin et al. G-protein that RSV encodes being guarded B cell epi-position, 1999) between Asp78 and the Pro80 inserting HBc peptide section, the CTL epi-position 82-90aa (M2 of M2 albumen of simultaneously RSV being encoded _82-90) merge at the C-terminal of truncated protein HBc1-144aa, M2 _82-90epi-position can effectively activate body CD8 ⁺t lymphocyte immunity reaction (Srikiatkhachorn and Braciale, 1997).Entrust commercial company to synthesize by contriver, intellecture property returns contriver to own, as shown in Figure 1.

1.2, recombinant expression vector pHBc-tG and pHBc-tG/M2 _82-90build

1) recombinant expression plasmid pHBc-tG builds

(1) with plasmid pUC57-HBc-tG/M2 _82-90for template, with P1 ' and P2 ' for primer, pcr amplification goal gene fragment HBc-tG (sequence is shown in SEQIDNO.2); Primer is synthesized by Shanghai biotechnology company limited.

P1 ' (upstream primer): 5 '-ATC gAATTCaTGGATATCGACCCG-T3 ' (SEQ ID NO.5), underscore annotated sequence is EcoR I restriction enzyme site;

P2 ' (downstream primer): 5 '-TGCA cTCGAGtTACTACGGTGGTTTCC-3 ' (SEQ ID NO.6), underscore annotated sequence is Xho I restriction enzyme site.

(2) the PCR amplification system composition listed according to following table adds each component successively.

Table 1 PCR amplification system

(3) pcr amplification is carried out according to following pcr amplification program, amplifying target genes fragment:

(4), after pcr amplification completes, 2% agarose gel electrophoresis detects amplified production, and pcr amplification product size is 756bp.DNA glue is adopted to reclaim kits goal gene fragment, for construction of recombinant plasmid.(PCR primer recycling step reclaims method described in test kit specification sheets by DNA glue and completes.)

(5) goal gene fragment HBc-tG and vector plasmid pET28-a carries out double digested respectively, adds each component successively according to following system (totally 20 μ l)

Table 2 double digestion reaction system

Be mixed, be placed in 37 DEG C of water-bath digestions 5-6 hour.

(6) respectively above-mentioned digestions goal gene or carrier DNA are carried out gel electrophoresis, and reclaim DNA by above-mentioned glue recovery method of cutting.

(7) digestions goal gene is connected with carrier DNA, adds each component successively according to following linked system (totally 25 μ l).

Table 3 HBc-tG and pET28a linked system

Be mixed, be placed in 16 DEG C of ligation 6h or 4 DEG C and spend the night.

(8) above-mentioned connection product conversion bacillus coli DH 5 alpha competent cell.

1. frozen bacillus coli DH 5 alpha competent cell is taken out from-80 DEG C of refrigerators, put on room temperature or ice bath and slowly thaw.Getting 5-10 μ l connection product to join in competent cell preservation pipe (EP pipe), mixing gently, in placing 30 minutes on ice.

2. taken out from ice bath by the above-mentioned competent cell EP pipe containing connecting product, the water-bath of putting into 42 DEG C is accurately reacted 90 seconds, keeps EP to manage static in reaction process.

3. rapid being transferred to by above-mentioned EP pipe on ice bath is placed 2 minutes, and thalline is cooled rapidly.

4. aseptic technique adds 800 μ l and is preheated to 37 DEG C not containing antibiotic LB substratum in EP pipe, is placed in 37 DEG C, 80-90rpm rotating speed shaking table cultivates 45 minutes.

5. getting above-mentioned nutrient solution 50-100 μ l is added to containing on kantlex LB agar plate, and with aseptic glass rod by even for the coating of bacterium liquid, flat board is just being placed in 37 DEG C of incubators and is treating that nutrient solution is completely absorbed in 30 minutes, then flat board is inverted overnight incubation.

(9) the positive bacterium colony that bacterium colony PCR identifies, screening has transformed recombinant expression plasmid pHBc-tG.

Carry out PCR reaction by the following method, qualification, screening goal gene recombinant expression plasmid.Single bacterium colony 5-10 that picking incubated overnight grows on the LB flat board of kalamycin resistance, is inoculated in the EP pipe that 400 μ l kalamycin resistance LB substratum are housed respectively, 37 DEG C, 220rpm cultivates 4-5h.

Get 2 μ l bacterium liquid as template, with primer P1 ' and P2 ' for amplimer, add each component successively by following system.

Table 4 pHBc-tG bacterium colony PCR identification system

Pcr amplification program is:

After having increased, PCR primer being carried out agarose gel electrophoresis detection (with reference to above-mentioned electrophoresis method), identifying according to whether there being goal gene fragment amplification, screen positive recombinant expression plasmid pHBc-tG and clone.

(10) a small amount of extraction and appraisement of recombinant expression plasmid pHBc-tG

The method described by plasmid Mini Kit process specifications extracts recombinant plasmid.

1. positive colony is cultivated bacterium liquid and is inoculated in 6ml containing in the LB substratum of kantlex in 1:100 ratio, in constant-temperature table 37 DEG C, 220rpm shaking culture spends the night.

2. overnight culture gone in EP pipe, centrifugal 1 minute of 10,000rpm, incline supernatant, collects thalline.

In the EP pipe of above-mentioned collection thalline, add 250 μ l solution I (4 DEG C preservation, containing RNaseA), repeatedly blow and beat mixing with micropipet, complete resuspended thalline; In EP pipe, add 250 μ l solution II, cover tightly pipe lid, softly repeatedly put upside down EP pipe 5-6 time, room temperature leaves standstill 2 minutes; In EP pipe, add 350 μ l solution III, cover tightly pipe lid, slowly put upside down and rock several times, no longer increase to white flock precipitate.

3. centrifugal 10 minutes of 12,000rpm room temperatures, careful absorption supernatant is transferred to cover to be had in the DNA adsorption column of collection tube, centrifugal 1 minute of 10,000rpm room temperature; Liquid in collection tube is added in adsorption column again, centrifugal 1 minute of 10,000rpm room temperature; Discard the liquid in collection tube, in adsorption column, add 500 μ l Buffer HB, centrifugal 1 minute of 10,000rpm room temperature; Discard the liquid in collection tube, in adsorption column, add 700 μ l DNA Wash Buffer, centrifugal 1 minute of 10,000rpm room temperature, repeats 1 time; After discarding the liquid in collection tube, placed back in by adsorption column in collection tube, centrifugal 2 minutes of 10,000rpm is to remove the residual liquid on post film completely.

4. adsorption column is put into another aseptic EP pipe, add the aseptic ddH of Elution Buffer in 50 μ l test kits or same volume ₂o, to dissolve the DNA be adsorbed on post film, room temperature leaves standstill 2 minutes; Centrifugal 1 minute of 10,000rpm room temperature, the liquid collected in EP pipe is plasmid DNA solution.Get 1 μ l plasmid DNA solution and carry out agarose gel electrophoresis, detect DNA.

Adopt enzyme to cut to identify recombinant plasmid, in 250 μ l EP pipes, add each component successively by following system.

Table 5 recombinant expression plasmid pHBc-tG double digestion reaction system (20 μ l)

Be mixed, be placed in 37 DEG C of water-bath endonuclease reactions 5 hours, carry out DNA agarose gel electrophoresis (with reference to above-mentioned electrophoresis method), whether observation analysis band conforms to expection.

Enzyme is cut correct recombinant plasmid pHBc-tG to check order, confirm the exactness of Insert Fragment HBc-tG sequence (SeqNO.2), direction of insertion and open reading frame, as shown in Figure 2.

2) recombinant expression plasmid pHBc-tG/M2 _82-90structure

According to above-mentioned connection product conversion method and Plasmid DNA Extractions by plasmid pUC57-HBc-tG/M2 _82-90transform DH5 α competent cell, provoke cultivation in single colony inoculation to LB substratum, amplification, collect thalline, extract plasmid DNA, EcoR I and Xho I double digestion plasmid.Each component is added successively by following double digestion system.

Table 6 double digestion reaction system (20uL)

Be mixed, be placed in 37 DEG C of water-baths 6 hours, agarose gel electrophoresis detects.

Cut gluing method according to above-mentioned, reclaim goal gene fragment (HBc-tG/M2 respectively _82-90) and linearizing carrier DNA pET28-a, goal gene fragment is mixed with carrier segments 5:1 ratio, connects with T4 DNA ligase, connect product conversion to bacillus coli DH 5 alpha competent cell, bacterium colony PCR identifies positive colony, extracts recombinant expression plasmid pHBc-tG/M2 _82-90, double digestion and order-checking qualification (Seq NO.4), the operation that concrete grammar all can refer to recombinant expression plasmid pHBc-tG is carried out, as shown in Figure 2.

3) genetic engineering bacterium preparation

By recombinant plasmid pHBc-tG and pHBc-tG/M2 correct for sequencing result _82-90be transformed in e. coli bl21-CodonPlus (the DE3)-RIL competent cell for protein expression, screening obtains containing pHBc-tG or pHBc-tG/M2 _82-90genetic engineering bacterium.Concrete operations are as follows:

(1) frozen competent cell is taken out from-80 DEG C of refrigerators, room temperature or ice bath slowly thaw.Get 1 μ l recombinant expression plasmid to add in expression bacterium competence cell EP pipe, pat gently bottom EP pipe and make it mix, put ice bath 30 minutes.

(2) proceeded to fast from ice bath in the water-bath of 42 DEG C by above-mentioned EP pipe, accurately reaction 90 seconds, keeps EP to manage static in reaction process.

(3) rapid being transferred to by EP pipe on ice bath is placed 2 minutes.

(4) aseptic technique add in EP pipe 800 μ l be heated in advance 37 DEG C not containing antibiotic LB substratum, go to 37 DEG C, 80-90rpm rotating speed shaking table cultivate 45 minutes, make bacterial expression resistance marker.

(5) get 50-100 μ l and cultivate drop on kantlex, chlorampenicol resistant LB flat board, with aseptic glass rod by even for the coating of bacterium liquid, just be placed in 37 DEG C and cultivate 30 minutes after nutrient solution is absorbed, flat board is inverted overnight incubation, next day provokes 5-10 single colony inoculation from flat board and cultivates in LB substratum, the engineering bacteria that bacterium colony PCR identifies, screening contains recombinant expression plasmid.

Bacterium colony PCR adds each component successively by following system

Table 7 bacterium colony PCR identification system

Pcr amplification program

After having increased, agarose gel electrophoresis detection (with reference to above-mentioned electrophoresis method) filters out the positive colony containing object recombinant expression plasmid.And preservation genetic engineering bacterium bacterial classification as follows.

(1) the positive colony bacterium liquid identified by PCR is forwarded to by 1:100 and 5ml is housed containing in kantlex, paraxin LB substratum test tube, 37 DEG C, 220rpm rotating and culturing.

(2) when growth enters logarithmic phase, OD is detected ₆₀₀when value reaches 0.6, take out bacterium liquid.

(3) shift appropriate bacterium liquid in 1.5mlEP pipe, add 30% glycerine solution of 4 DEG C of precoolings in 1:1 ratio to bacterium liquid, piping and druming mixing.

(4) go in freezing storing box by preserving the EP pipe of engineering bacteria, frozen in-80 DEG C at least 6 hours, then engineering bacteria can be continued frozen-80 DEG C or be transferred to the medium-term and long-term preservation of liquid nitrogen.

1.3, genetic engineering bacterium expresses HBc-tG or HBc-tG/M2 albumen and qualification in a small amount

1) IPTG induces target protein to express

(1) by expressing the engineering bacteria coating of recombinant protein containing overnight incubation on kantlex, chlorampenicol resistant LB flat board, single bacterium colony is formed; Provoke single colony inoculation in containing in kantlex, paraxin LB substratum, 37 DEG C, 220rpm rotating and culturing is spent the night.

(2) be forwarded to cultivation bacterium liquid containing in kantlex, chlorampenicol resistant LB substratum in 1:100 ratio, 37 DEG C, 220rpm rotating and culturing, grows into logarithmic phase (OD to engineering bacteria ₆₀₀) value reaches 0.6.

(3) culture being in logarithmic phase is taken out from shaking table, adds IPTG and make its final concentration be 0.2mmol/L, in 37 DEG C, 220rpm continues cultivation 5 hours.

(4) be transferred to by Induced cultures in 10ml centrifuge tube, 12,000rpm, 4 DEG C centrifugal 10 minutes, abandons supernatant, collects thalline.

(5) with the resuspended thalline of 500 μ lPBS solution, 12,000rpm, 4 DEG C centrifugal 10 minutes, abandons supernatant, collects thalline, PBS repeated washing 1 time.

(6) with the resuspended thalline of 200 μ l lysate, ultrasonic disruption thalline on ice bath is put.Being set to output rating is 300W, ultrasonic 3s, interval 8s, 30-50 time altogether.After ultrasonic disruption, carry out SDS-PAGE and Western blot Analysis and Identification expression of recombinant proteins.

Cellular lysate damping fluid:

TritonX-100(v/v) 1％

Tris-Cl(pH8.0) 0.01M

NaH ₂PO ₄ 0.1M

2) target protein HBc-tG and HBc-tG/M2 _82-90qualification

sDS-PAGE analyzes

(1) gel casting

Get cleaned glass plate and adhesive tape is fixed on encapsulating support, carefully filled with by the separation gel solution of newly prepare 15% and add in glue-filling slot, avoid producing bubble, separation gel solution adds to apart from upper recess surface 2.5cm place.On separation gel solution, cover 1ml dehydrated alcohol with pipettor, horizontal rest, under room temperature, be polymerized about 20 minutes, after glue polymerization, remove tectum liquid, be inverted and remove residual liquid in 1 minute.Add on separation gel by the spacer gel solution of newly prepare 5%, insert sample comb at once, room temperature horizontal rest, treats that gel is fully polymerized.

15% separation gel solution preparation:

5% spacer gel solution preparation:

(2) sample preparation

Isopyknic 2 × sds gel sample loading buffer and thalline sample liquid are mixed, 100 DEG C are boiled 5-10 minute, immediately ice bath 5 minutes, the centrifugal 5-10 minute of room temperature 12000rpm, collect Supernatant samples and save backup.2 × sds gel sample loading buffer is prepared:

(3) electrophoresis

After gel prepares, in electrophoresis chamber, add electrophoretic buffer.Sample thief supernatant 10-30 μ l adds in sample well, carries out electrophoresis.Adjust voltage to allow sample move in spacer gel to 60V, after sample enters separation gel, voltage is adjusted to 120V and continues electrophoresis.Observe molecular weight of albumen Marker migration situation, when Marker reaches best resolution, stop electrophoresis.

5 × protein electrophoresis buffer:

(4) dye

Take off gel after electrophoresis terminates, with after tap water, gel is immersed in coomassie brilliant blue R_250 staining fluid completely, under room temperature, on horizontal shaker, shake stained over night at a slow speed.

Coomassie blue R-250 staining fluid is prepared:

Magnetic agitation makes dyestuff dissolve in 1 hour, uses Whatman filter paper filtering, room temperature preservation.

(5) decolour

After dyeing, reclaim staining fluid, gel is immersed in destainer completely, room temperature shakes at a slow speed decolouring 4-8 hour, and period changes destainer 4-5 time, treats that protein band becomes clear, discard destainer, gel tap water is cleaned and immerses in clear water, can preservation of taking pictures be carried out.

Destainer is prepared:

Glacial acetic acid 100ml

Methyl alcohol 400ml

ddH ₂O 500ml

Result shows, and genetic engineering bacterium prepared by the present invention can express HBc-tG or HBc-tG/M2 respectively _82-90albumen (as Fig. 3 A).

westernblot detects engineering bacterium expression target protein

(1) sample preparation and protein component SDS-PAGE electrophoretic separation is carried out according to aforesaid method.Concrete operations are undertaken by " molecular cloning, laboratory manual ".For ease of comparing, during electrophoresis application of sample, use each sample of same volume.

(2) transferring film and antibody response

Treat that electrophoresis terminates, take out gel and use tap water.Soak pvdf membrane and filter paper 5 minutes with transferring film damping fluid simultaneously, lay gel and pvdf membrane by negative electrode-sponge-filter paper-gel-pvdf membrane-filter paper-sponge-anode order, put into electrophoresis chamber, electrophoresis 2-3 hour under 88V voltage cold condition.Take out pvdf membrane 1 × PBST (PBS of 0.05%Tween-20) and wash 5 minutes, add the PBST Block buffer (blocking buffer) containing 5% skim-milk, hatch to close for 2 hours or 4 DEG C for 37 DEG C and spend the night.PBST washs pvdf membrane 3 times, each 10 minutes.Add anti-(RSV) G-protein polyclonal antibody of rabbit (prepared by the G-protein immunizing rabbit of this laboratory expression and purification, concrete steps participate in bavin maple master Diplomarbeit, carry out 1:1000 dilution with PBST), hatch 2 hours for 37 DEG C.PBST fully washs pvdf membrane 3-5 time, each 10 minutes.Add horseradish peroxidase (HRP) and mark goat anti-rabbit igg antibody (Abbkine Products carries out 1:5000 dilution with PBST), hatch 1 hour for 37 DEG C, PBST fully washs pvdf membrane 3-5 time, each 10 minutes.Add DAB nitrite ion (concrete formula is shown in " molecular cloning, laboratory manual "), room temperature lucifuge colour developing 5-10 minute, X-ray exposure in darkroom, record result.

Transfering buffering liquid is prepared:

Dilution buffer (TBS) is prepared:

3,3 '-diaminobenzidine (DAB) nitrite ion is prepared:

Result shows, and genetic engineering bacterium prepared by the present invention can after adding IPTG induction, high level expression HBc-tG or HBc-tG/M2 respectively _82-90albumen (Fig. 3 A).

The mass-producing preparation of [embodiment 2] respiratory syncytial virus virus-like particle (VLPs) vaccine and calibrating

2.1, the mass-producing preparation of respiratory syncytial virus sample particle (VLPs) vaccine

1) recombinant antigen protein HBc-tG and HBc-tG/M2 _82-90great expression

(1) frozen engineering bacteria is taken out, thawed from-80 DEG C of refrigerators, get 0.1ml bacterium liquid and be forwarded to (containing kantlex, paraxin) in 5mlLB substratum, 37 DEG C of 220rpm rotating and culturing are spent the night.

(2) overnight culture is seeded in the 1LLB substratum containing kantlex, paraxin in 1:100 ratio, 37 DEG C, 220rpm shaking culture.Logarithmic phase to be cultured to (bacterium liquid OD ₆₀₀value reaches 0.6), adding IPTG to final concentration is 0.2mM, 37 DEG C, 220rpm rotating and culturing 5 hours, and inducible protein is expressed.

(3) Induced cultures is transferred to 250ml centrifuge tube, 4 DEG C, 12,000rpm, centrifugal 30 minutes, abandons supernatant, collect thalline.Thalline adds 100mlPBS and suspends, and 4 DEG C, 12,000rpm, centrifugal 30 minutes, abandons supernatant, collects thalline.Repeat to add PBS and wash 2 times, collect thalline be used for protein purification or preservation-80 DEG C for subsequent use.

2) recombinant antigen protein HBc-tG and HBc-tG/M2 _82-90purifying

(1) cellular lysate process

In thalline, add lysate in 4ml/g ratio, adding proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) to final concentration is 1mM, and lysis at room temperature 30 minutes, period repeatedly stirs and is mixed.After cracking, thalline is placed in ice bath ultrasonic disruption further.Through the sample of ultrasonic disruption in 4 DEG C, centrifugal 30 minutes of 12000rpm.Collect supernatant, in precipitation, add appropriate binding buffer liquid, mixing, puts ice bath 60 minutes dissolution precipitations, 4 DEG C, centrifugal 30 minutes of 12000rpm, collects supernatant, merges for subsequent use.

Lysis buffer is prepared:

TritonX-100(v/v) 1％

Tris-Cl(pH8.0) 0.01M

NaH ₂PO ₄ 0.1M

Binding buffer liquid is prepared:

Tris-Cl(pH8.0) 0.01M

NaH ₂PO ₄ 0.1M

Urea 8M

(2) affinitive layer purification

Affinity chromatography medium Ni-NTA His-Bind resin 2 times of volume binding buffer liquid wash, add damping fluid by 50% and prepare resin suspension, add 1ml Ni-NTA His-Bind resin suspension ratio in every 4ml sample solution both are mixed, 4 DEG C, 200rpm rotates 60 minutes; Protein-bonded resin is loaded in chromatography column, use binding buffer liquid (about 5ml)-sex change wash buffer (about 20ml) washing resin successively, then denaturing elution buffer I (about 2-5ml)-denaturing elution buffer II (about 2-5ml) stepwise elution is used, Fraction collection chromatographic column effluent liquid (0.5ml/ pipe) successively, detect each component protein matter concentration, SDS-PAGE electrophoresis detection each component protein matter purity; Collect the component merging purifying target protein and be used for follow-up preparation VLPs.

Sex change wash buffer is prepared:

Tris-Cl(pH6.3) 0.01M

NaH ₂PO ₄ 0.1M

Urea 8M

Denaturing elution buffer I is prepared:

Tris-Cl(pH5.9) 0.01M

NaH ₂PO ₄ 0.1M

Urea 8M

Denaturing elution buffer II is prepared:

Tris-Cl(pH4.5) 0.01M

NaH ₂PO ₄ 0.1M

Urea 8M

3) recombinant antigen protein HBc-tG and HBc-tG/M2 _82-90renaturation and VLPs preparation

(1) dialysis tubing of the long 10-20cm of sample dialysis tubing process clip, is placed in dialysis tubing treatment solution I and boils 10 minutes, after deionized water rinsing, puts into dialysis tubing treatment solution II and boils 10 minutes, deionized water rinsing, be immersed in renaturation solution.

Dialysis tubing treatment solution I is prepared:

NaHCO ₃ 2g

EDTA(pH8.0) 0.029g

ddH ₂O 100mL

Dialysis tubing treatment solution II is prepared:

EDTA(pH8.0) 0.029g

ddH ₂O 100mL

(2) protein renaturation and VLPs assembled in vitro prick tight dialysis tubing one end handled well, the target protein component of purifying is transferred in dialysis tubing, tightens the dialysis tubing the other end, successively decrease sequentially by urea concentration, respectively in renaturation buffer, under 4 DEG C of conditions, carry out sample dialysis.Contained by renaturation buffer, urea concentration gradients is followed successively by 6M, 5M, 4M, 3M, 2M and 1M, changes renaturation buffer 1 time at interval of 3-4 hour, finally changes 500ml PBS solution into and dialyses 2 times.

Renaturation buffer is prepared:

2.2, the calibrating of respiratory syncytial virus sample particle (VLPs) vaccine

1) SDS-PAGE electrophoresis calibrating purity of protein

Concrete operations are carried out with reference to electrophoresis method in embodiment 1.

2) Western blot detects recombinant protein antigen specificity

Concrete operations are carried out with reference to authentication method in embodiment 1.

3) electron microscopic observation VLPs form

After getting above-mentioned purifying protein renaturation, assembling forms the sample 50 μ l of VLPs, protein concn is diluted to 100 μ g/ml, sample drop is added on copper mesh, static 1 minute, redundant solution is sucked along copper mesh edge with filter paper, drip 1-2 to copper mesh and drip (the distilled water preparation of 2% acetic acid uranium solution, pH6.8), dye 1 minute, suck excess stain liquid with filter paper along copper mesh edge, room temperature is static treats that copper mesh is dry, under copper mesh (negative staining sample) is put transmission electron microscope, in 200kV, × 25, particle shape is observed, record result under 000 times.

Result shows, and the technology of the present invention method can prepare high purity HBc-tG and HBc-tG/M2 _82-90vaccine component albumen (Fig. 3 B); Anti-RSV G protein antibodies detection display, prepared vaccine component albumen has the characteristic (Fig. 3 B) of reacting with specific antibody; Prepared vaccine component HBc-tG and HBc-tG/M2 _82-90after purified, renaturation, all independently can be assembled into virus-like particle (Fig. 4 A, 4B).

The Function detection of [embodiment 3] respiratory syncytial virus virus-like particle (VLPs) vaccine

Testing program

Get 5-6 female BAl BIc/c mouse 40 in age in week, be divided into A, B, C and D etc. 4 groups at random, often organize 10, mouse docking blood sampling before vaccine inoculation, separation of serum is used for antibody test as negative control sera.Respectively by following design immunization: the RSV (1 × 10 of A group injection ultraviolet inactivation ⁵tCID50); B group injection HBc-tG (VLPs), dosage 20 μ g/ is only; C group injection HBc-tG/M2 _82-90(VLPs), dosage 20 μ g/ only.Take intramuscular injection path immunity, various vaccine all immune three times, three weeks, interval; D group is control group, adopts identical approach and interval injection PBS.Latter two weeks of immunity, docking blood sampling also separation of serum.After each group of mouse inoculation vaccine, every day observes mouse state and weighs in, and compares, HBc-tG and HBc-tG/M2 with injection PBS control group mice _82-90there is not any exception in particle vaccines injection group mouse, body weight increases consistent with PBS group.

3.1, vaccinated mice humoral immunoresponse(HI) detects

1) enzyme linked immunosorbent assay (ELISA) detects mice serum antibody and subclass (IgG, IgG1 and IgG2a)

RSV after purifying is envelope antigen.Concrete steps comprise:

Bag quilt: be buffered liquid (carbonate buffer solution of 0.05MPH9.6) with bag and be diluted to 3 × 10 by purifying and through the RSV of ultraviolet inactivation ⁵tCID ₅₀as envelope antigen solution, join 96 hole ELISA enzyme plates (Corning Products, lower same) every hole 100 μ l, 4 DEG C of bags are spent the night;

Close: by bag by the ELISA enzyme plate of RSV antigen PBST (PBS is containing 0.05%Tween-20, pH7.5, lower same) washing 5 times, each 5 minutes; Add confining liquid to (100 μ l/ holes, the PBST containing 3%BSA) in each hole of enzyme plate, close 60 minutes for 37 DEG C; PBST washs 5 times;

Add serum to be checked: the PBST of mice serum containing 1%BSA makes 1:100 gradient dilution, and before mouse immune, serum is as negative control sera, is added by dilute serum in (the 100 every hole of μ l/) in each hole of ELISA enzyme plate, hatches 60 minutes for 37 DEG C; PBST washs 5 times;

Add ELIAS secondary antibody: two anti-are respectively horseradish peroxidase-labeled sheep anti-mouse igg antibody, anti-igg 1 or anti-igg 2 (Pierce Products), the PBST of sheep anti-mouse igg containing 1%BSA does 1:5000 dilution, sheep anti-mouse igg 1 and the PBST of IgG2 containing 1%BSA do 1:2000 dilution, add in (100 μ l/ hole) in each hole of elisa plate, 37 DEG C act on 60 minutes; PBST washs 5 times;

Add display substrate solution: in each hole of enzyme plate, add colour developing end solution (TMB (TMB), 100 μ l/ holes, 37 DEG C of lucifuge colour developing 15-30 minute; Every hole adds 50 μ l 2M H ₂sO ₄termination reaction;

OD ₄₅₀pH-value determination pH: wavelength 450nm place measures every hole internal optical density absorption value (OD in microplate reader ₄₅₀), serum sample OD ₄₅₀value is more than or equal to 2.1 times of negative numerical value and is judged to be the positive;

Result shows, VLPs vaccine immune mouse prepared by the present invention can induce body to produce high-level specificity RSV IgG antibody and IgG1, IgG2 Subclass Antibodies (Fig. 5 A, 5B).

Bag is buffered liquid preparation:

Na ₂CO ₃ 0.159g

NaHCO 0.293g

3

PH is regulated 9.6 to use ddH ₂o is settled to 100ml, 4 DEG C of preservations.

PBS prepares:

Use ddH ₂o is settled to 1L.

2) serum neutralizing antibody detects

Immunity plaque ethods, concrete operations are as follows:

Cell monolayer: by each hole of Vero cell suspension inoculation to 24 porocyte culture plate, treat that Vero cell grows to 95% individual layer next day for subsequent use;

Serum neutralizing antibody detects:

Viruses adsorption: serum-free DMEM nutrient solution dilution RSV is 75-100PFU/100 μ l to concentration, mixes, hatch 60 minutes for 37 DEG C with the experiment mice serum of equal-volume doubling dilution; Suck the nutrient solution in monolayer cell hole, serum-free DMEM nutrient solution washed cell individual layer 1 time, add virus-serum mixed solution (100 μ l/ hole), 37 DEG C, adsorb 2 hours in 5%CO2 incubator; Separately using RSV and nonimmune negative serum mixed solution as positive control;

Cell cultures: after absorption, sucks virus-serum mixed solution, adds the covering liquid (0.5-1ml/ hole) of 1% agarose, 37 DEG C, cultivates 3-5 days, suck covering liquid in 5%CO2 incubator;

Cell is fixed: cells rinsed with PBS 3 times, the stationary liquid (methyl alcohol: acetone-60:40) 4 DEG C adding precooling fixes 20 minutes, air drying, the PBS confining liquid (100 μ l/ hole) containing 3%BSA is added in cell hole, room temperature effect 30 minutes, washs 3 times with PBST (0.5%Tween20);

Antibody response: the mouse monoclonal antibody (primary antibodie, Millipore Products carry out 1:1000 dilution, 100 μ l/ holes with PBST) adding anti-RSVF albumen, hatches 2 hours for 37 DEG C, washs 3 times with PBST; Add the sheep anti-mouse igg (two anti-, Pierce Products, carry out 1:5000 dilution, 100 μ l/ holes with PBST) of HRP mark, hatch 1 hour for 37 DEG C;

Colour developing: cell hole adds PBST and washs 3 times, add DAB substrate colour developing (100 μ l/ hole), room temperature reaction 15-30 minute, sucks nitrite ion, count immune plaque quantity, be designated as NAT with the highest serum extent of dilution reducing by 60% plaque quantity than Positive control wells.

Result shows, VLPs vaccine immune mouse prepared by the present invention can effectively induce body to produce RSV virucidin, neutralizing antibody level identical with the RSV of UV deactivation (Fig. 5 C).

3.2, vaccinated mice cellular immunization detects

ELISA detects cytokine, and concrete operation step comprises:

Mouse spleen lymphocyte is separated:

Each group of mouse the 21st day etherization after the 3rd immunity is put to death, be soaked in 3-5 minute in 75% ethanol, aseptic technique in Bechtop, get mouse spleen, put in 35mm Tissue Culture Dish 4-5ml 1 × lymphocyte separation medium (4 DEG C of preservations, with front temperature return to room temperature, shake up), shred spleen tissue, with 5ml syringe glue Tou Xichuishi historrhexis, release cells collects splenocyte suspension, it is transferred to immediately in 15ml centrifuge tube, along tube wall slowly add 200-500 μ l RPMI-1640 substratum, keep liquid level boundary obviously; Room temperature, 1500rpm, centrifugal 30 minutes, centrifugal rear cell layering; Sucking-off buffy coat, add 10ml RPMI-1640 substratum, put upside down mixing, room temperature, 1000rpm, centrifugal 10 minutes, suck supernatant, collecting cell, with serum-free RPMI-1640 or DMEM substratum re-suspended cell, instills cell counting count board basis of microscopic observation or adopts Auto-counting of Cells instrument to carry out cell counting after doing suitably dilution.

Stimulating cytokine secretion:

The mouse boosting cell suspension of preparation is diluted to 1 × 10 with the DMEM containing 10% foetal calf serum ⁷concentration, is seeded in the 24 each holes of porocyte culture plate, 100 μ l/ holes (1 × 10 ⁶cells/well), every hole adds 10 ⁴the hot deactivation RSV of PFU, as stimulator, separately sets and does not add RSV stimulator as negative control hole, is placed in 37 DEG C, 5%CO ₂cultivate 72 hours in incubator, collecting cell culture supernatant, 4 DEG C, centrifugal 20 minutes of 2000rpm, collect supernatant, point be filled in 1.5ml EP pipe, frozen in-80 DEG C, measure for various cytokine concentration.

Cytokine concentration measures:

Enzyme plate and various detection reagent are BioLegend company of U.S. detection kit and provide component.Detect two type cytokines (Th1 class, IFN-γ, TNF-α and IL-2 respectively; Th2 class, IL-4, IL-5 and IL-10) totally 6 kinds (result illustrates see accompanying drawing and accompanying drawing).The concrete method steps introduced with reference to cytokine detection kits (BioLegend) specification sheets.

bag quilt: use1 × coating buffer dilution capture antibody (1:2000), add in 96 hole enzyme plates, 100 μ l/ holes, 4 DEG C of bags are spent the night (16-18 hour); Discarded by coating buffer, every hole adds 300 μ l washingss, and jolting enzyme plate, discards washings, firmly pats dry, repeated washing 4 times; In each hole of enzyme plate, add 1 × Assay Diluent A (200 μ l/ hole), 200rpm, rocked at room temperature hatch 1 hour; Washings detersive enzyme target 4 times;

add sample to be checked:with 1 × Assay DiluentA, standard substance cytokine is carried out 2 times of serial doubling dilutions, the standard substance of doubling dilution and sample are added in each hole of above-mentioned enzyme plate, 100 μ l/ holes, every increment product repeat 2 holes.200rpm, rocked at room temperature hatch 2 hours;

add detection antibody:enzyme plate washings washs 4 times, and add in each hole and detect antibody (1:5000 dilution), 100 μ l/ holes, 200rpm, rocked at room temperature hatch 1 hour; Enzyme plate washings washs 4 times, in each hole, add Avidin-HRP solution, 100 μ l/ holes, and 200rpm, rocked at room temperature hatch 30 minutes;

colour developing:enzyme plate washings washs 5 times, in each hole, add TMB nitrite ion, and 100 μ l/ holes, are placed in dark place, color development at room temperature 20 minutes, is positive reaction in blueness; 2M H is added in each hole ₂sO ₄stop buffer termination reaction, 100 μ l/ holes.450nm light absorption value OD is read in 30 minutes of termination reaction ₄₅₀; According to standard concentration and OD ₄₅₀value drawing standard curve; Read each sample OD ₄₅₀value, according to corresponding cytokine concentration in each cytokine standards curve calculation sample.

Result shows, VLPs vaccine immune mouse splenocyte prepared by the present invention can induce body to produce Th1 type cytokines (the FN-γ strengthened, TNF-α and IL-2) response, lower the secretion (Fig. 6 A, 6B) of Th2 type cytokines (IL-4, IL-5 and IL-10).

3.3, the lung tissue virus titer determination after VLPs vaccinated mice infects RSV and pathology damage detect

1) rsv infection of VLPs vaccine immune mouse:

VLPs vaccine immune mouse is sucked ether mode light anaesthesia about 40 ~ 50 seconds, mouse keeps dorsal position to fix, and gets the RSV virus liquid (3 × 10 that 50 μ l dilute ⁶pFU) through the inoculation of collunarium approach, be completely absorbed until virus liquid, mouse put back in mouse cage after reviving and raise.

2) mouse lung tissue virus titer measures

prepared by lung tissue lapping liquid: mouse is attacked poison with RSV and infects disconnected neck execution in latter 4th day, is soaked in 75% ethanol 5-10 minute; In Bechtop, lung tissue is taken out in aseptic technique, puts into mill, adds the plasma-free DMEM medium of 1ml precooling, fully grind, and makes lung tissue grind thoroughly; 4 DEG C, 12000rpm, centrifugal 20 minutes, it is frozen in-80 DEG C to collect supernatant, packing, detects for cytokine concentration and virus titer. vLPs vaccine immune mouse RSV attacks poison and infects cytokine concentration mensuration in rear lung tissue:get the lung tissue lapping liquid that above-mentioned preparation is frozen, detect two types totally 6 kinds of cytokines (Th1 class, IFN-γ, TNF-α and IL-2 by ELISA kit; Th2 class, IL-4, IL-5 and IL-10) concentration.Specifically with above-mentioned splenic lymphocyte factors check operation steps.

Result shows, VLPs vaccine immune mouse prepared by the present invention is after infection RSV, and lung tissue energy secreting high levels Th1 type cytokines (particularly IFN-γ) secretes; Produce low-level Th2 type cytokines (Fig. 7 A, 7B), this is conducive to body and removes rsv infection and reduce VED formation simultaneously.

lung tissue RSV titer determination:by in Vero cell suspension inoculation to 24 porocyte culture plate, treat that Vero cell grows to 95% individual layer next day, suck DMEM nutrient solution, plasma-free DMEM medium washs 2 times; Getting 50 μ l lung tissue grinding supernatant liquors is added in 150 μ l plasma-free DMEM medium, abundant mixing, mixed solution is added on 24 orifice plate Vero cell monolayers of plasma-free DMEM medium washing, in 37 DEG C, 5%CO2 incubator adsorbs 2 hours, suck mixed solution, Xiang Kongzhong adds the covering liquid (0.5-1ml/ hole) of 1% agarose, separately establishes normal mouse lung tissue lapping liquid as negative control; Cultivate 3-5 days for 37 DEG C, inhale and abandon covering liquid, PBS washs 3 times.Add precooling stationary liquid (methyl alcohol: acetone/60:40) 4 DEG C and fix 10 minutes.Dry air, adds the PBS containing 3%BSA, room temperature effect 30 minutes, washs 3 times with PBST (0.5%Tween20); Add the monoclonal antibody (primary antibodie, 1:1000) of mouse-anti RSVF albumen, hatch 2 hours for 37 DEG C, PBST washs 3 times.Add HRP mark sheep anti mouse monoclonal antibody (two resist, 1:2000), hatch 1 hour for 37 DEG C, PBST washs 3 times, add DAB substrate nitrite ion (100 μ l/ hole), color development at room temperature 15-30 minute, sucks substrate nitrite ion, counting plaque, calculates mouse lung tissue virus titer.

Result shows, VLPs vaccine immune mouse prepared by the present invention can suppress to infect RSV to be copied or accelerates the removing of RSV in Mice Body, makes immune mouse obtain the immunoprotection (Fig. 8) of anti-rsv infection.

3) mouse lung tissue HE dyes

Often group gets 5 mouse, after rsv infection the 4th day, and etherization puts to death mouse, gets full lung tissue, puts into 4% formalin solution and fix 24 hours, dehydration, paraffin embedding, section and HE dyeing, the pathological change of basis of microscopic observation analysis mouse lung tissue.

Result shows, lung tissue swelling after VLPs vaccine immune mouse prepared by the present invention can suppress rsv infection, inflammatory exudation and inflammatory cell invade the pathological changes such as profit, reduces pathologic damage (Fig. 9) that rsv infection causes.

Leading reference

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Claims (10)

1. respiratory syncytial virus (RSV) virus-like particle, it is characterized in that, with 1-144 amino acids in the hepatitis B virus core protein aminoacid sequence of brachymemma for carrier, comprise chimeric RSV G-protein Neutralization and crystallization wherein, the virus-like particle that can independently assemble.

2. respiratory syncytial virus virus-like particle according to claim 1, is characterized in that, has also merged the CTL epi-position of RSV M2 albumen at hepatitis B virus core protein C-terminal.

3. respiratory syncytial virus virus-like particle according to claim 1, is characterized in that, described RSV G-protein Neutralization and crystallization is the sequence shown in 144-204 amino acids in the aminoacid sequence of G-protein, and its aminoacid sequence is Seq NO.1.

4. respiratory syncytial virus virus-like particle according to claim 2, is characterized in that, the CTL epi-position of described RSV M2 albumen is the sequence shown in 82-90 amino acids in the aminoacid sequence of M2 albumen.

5. respiratory syncytial virus virus-like particle according to claim 3, it is characterized in that, through codon optimized design, obtain the gene fragment HBc-tG merged by HBc1-144aa and RSV G144-204aa encoding sequence, its nucleotides sequence is classified as Seq NO.2.

6. respiratory syncytial virus sample particle according to claim 4, is characterized in that, described RSV G-protein Neutralization and crystallization is the sequence shown in 144-204 amino acids in the aminoacid sequence of G-protein, and its aminoacid sequence is Seq NO.3.

7. respiratory syncytial virus virus-like particle according to claim 6, it is characterized in that, through codon optimized design, obtain the gene fragment HBc-tG/M2 merged by HBc1-144aa and RSV G144-204aa and M2 (82-90aa) encoding sequence _82-90, its nucleotides sequence is classified as Seq NO.4.

8. the recombinant expression vector of the nucleic acid containing the arbitrary described respiratory syncytial virus virus-like particle of coding claim 1-7.

9. transformed the host cell of recombinant expression vector according to claim 8.

10. the arbitrary described respiratory syncytial virus virus-like particle of claim 1-9 is for the preparation of the purposes in the medicine of prevention, Diagnosis and Treat respiratory syncytial virus infection.