CN103864902B - A kind of bivalent DNA vaccine connection peptides and application thereof - Google Patents

Abstract

The invention discloses a kind of bivalent DNA vaccine connection peptides and application thereof.Pcr amplification HSV-2 glycoprotein gene and adjuvant genes also introduce restriction enzyme site, introduce connection peptides (G by Overlap extension PCR method ₄s) ₂iZ (G ₄s) ₂, obtain a kind of bivalent DNA vaccine.Adopt prime-boost principle immune mouse, detect humoral immunization, cellular immunization and the mucosal immune response reaction that this vaccine causes; By vaginal infection 150LD ₅₀hSV-2 virus, detect serotype antibody and evaluate the disease severity of each group in 15 days.Find that the bivalent DNA vaccine of restructuring can produce stronger humoral immunization, cellular immunization and mucosal immune response reaction than unit price DNA vaccination, also detect the ability of stronger neutralization virus; The Immunoreactivity of the bivalent vaccine of restructuring and memory immune reaction can produce the antibody of high titre more rapidly, have better protected effect to mouse.

Description

A kind of bivalent DNA vaccine connection peptides and application thereof

Technical field

The present invention relates to genetically engineered and field of immunology, more specifically relate to a kind of bivalent DNA vaccine connection peptides, also relate to this connection peptides and preparing the application in bivalent DNA vaccine.

Background technology

Bivalent DNA vaccine forms the DNA molecular merged after referring to and adopting gene splicing technology and DNA recombinant technology to be connected by two kinds of genes, bifunctional protein molecular can be translated with separately or merging by after goal gene transfered cell, body can be made to produce the biotechnological formulation of specific immunity.

Two genes are with connexon (linker) or directly connect, use same open reading frame, translation produces a chimeric bifunctional fusion proteins molecule, general method is deleted by the termination codon of first gene, and the initiation codon of its reading frame and second gene or 5 ' UTR are connected and also can correctly read over.Suitable connexon is conducive to the correct folding of two albumen and produces independently function separately, thus the equivalent of translation skill on realization theory is expressed.The method of design of connexon is varied, multiselect is selected as neutral hydrophobic amino acid, as being rich in the polypeptide chain of glycine and Serine, its length to the folding of protein and stability extremely important, the subject matter of this method is: 1. probably because the change of conformation makes the function of wherein one or two gene be affected; If 2. two gene products have different cellular localizations, also can affect the normal function of gene, namely the function of gene is also uneven.

Chemokine is the molecule of normal presence in animal body, and side reaction is little, can induce and change immunoreactive intensity and type, found that multiple chemokine has adjuvant effect at present, but its detailed mechanism of action is not still fully aware of as gene adjuvant.CCL19 and CCL28 is regulated by different effect links or strengthens the immunological effect of DNA vaccination and play adjuvant effect.The Main Function of CCL19 recruits the T cell region of T cell to secondary lymphoid organ, thus promote activation, the propagation of T cell.Its ligand molecular CCR7 is also expressed in B cell and the DC cell of activation, and thus CCL19-CCR7 signal can promote B cell, DC cell and the interaction of T cell in secondary lymphoid tissue.In addition bibliographical information CCL19 is also had can to strengthen antitumor response and the specific immunne response of HSV-1.The ligand molecular of CCL28 has CCR3 and CCR10, its wide expression is in the epithelial cell of many mucosa, the plasmocyte can recruiting secretion IgA antibody migrates to multiple mucosa from Lymphoid tissue, CCL28 as gene adjuvant in the research and development of influenza vaccines, and there is the anti-microbial activity of wide spectrum, research finds that CCL28 also can strengthen the immunne response ability of HIV albumen, and its potential mechanism still needs to be illustrated further.

HSV-2 does not also have desirable vaccine at present; the method of the most frequently used production vaccine is exactly the vaccine made as immunogen with the envelope glycoprotein of virus; conventional has gD and gB; but the speed of its neutralizing antibody produced is slow, the ability of neutralization virus is lower, the transformation period is shorter, has and only can protect seronegative crowd and can not protect the deficiencies such as the male sex.The bivalent vaccine prepared by connection peptides can improve the expression efficiency of adjuvant and antigen, forms tripolymer during two kinds of genetic expression, can realize turn improving immunogenicity while albumen equivalent is expressed; Simultaneously according to the characteristic distributions of the ligand molecular of adjuvant can be directed antigen molecule is offered to T or the DC cell recognition in secondary lymphoid tissue, produce efficient cell immune response.The research of the people such as Mark Melchers finds CD40L and the HIV-1Env albumen connected by several linker, IZ motif (GCN4) can make that the trimeric configuration of Env albumen is more stable to be facilitated the maturation of dendritic cell simultaneously and add the secretion of cytokine, thus improves the neutralising capacity of antibody.

In view of above research, more firm space conformation is formed in order to make the fusion rotein of expression envelope glycoprotein gene and adjuvant genes, and give play to respective independently function, the present invention extends IZ motif, adds the peptide chain (G4S) that a section has hydrophobicity and certain extensibility before and after it ₂, two kinds of mutual interference effects of albumen can be alleviated to greatest extent, normally can translate in somatocyte after connection, fold, and albumen tripolymer can be formed, after immunity, also obtain satisfied result.The bivalent DNA vaccine of this restructuring has genetic stability and repeatable and more promptly can produce the antibody of high titre, stronger virus neutralizing cpaacity good to the immune protective effect of mouse.

Summary of the invention

The object of the invention is to there are provided a kind of bivalent DNA vaccine connection peptides, its nucleotides sequence is classified as shown in SEQ ID NO.1, and corresponding aminoacid sequence is for shown in SEQ ID NO.2.Viral envelope glycoprotein gene and adjuvant genes are by this connection peptides ((G4S) ₂-IZ-(G4S) ₂) can normally translate in somatocyte after connection, folding, and albumen tripolymer can be formed.

Another object of the present invention there are provided a kind of bivalent DNA vaccine connection peptides and is preparing the application in bivalent DNA vaccine.The vaccine quality utilizing this DNA vaccination connection peptides to prepare is easy to control, and easy scale operation, easy storage and transport, cost is low.The protein molecular of the bifunctional fusion formed not only enhances the immunogenicity of DNA vaccination; also achieve viral envelope glycoprotein gene and adjuvant genes high expression in same cell simultaneously; the antibody of high titre more promptly can be produced after immune mouse; and there is stronger antibody neutralising capacity, and good to the immune protective effect of mouse.

In application as above, by (G4S) ₂-IZ-(G4S) ₂cCL19 and HSV-2 glycoprotein gd connects by linker, obtain a kind of fusion rotein, its nucleotides sequence is classified as shown in SEQ ID NO.3, it is connected with carrier for expression of eukaryon, obtained a kind of bivalent DNA vaccine p-gDIZCCL19 of restructuring, its sequence is for shown in SEQ ID NO.7.

In application as above, by (G4S) ₂-IZ-(G4S) ₂cCL28 and HSV-2 glycoprotein gd connects by linker, and obtain a kind of fusion rotein, its nucleotides sequence is classified as shown in SEQ ID NO.4, it is connected with carrier for expression of eukaryon, has obtained a kind of bivalent DNA vaccine p-CCL28IZgD of restructuring.

In application as above, by (G4S) ₂-IZ-(G4S) ₂cCL19 and HSV-2 glycoprotein gB connects by linker, obtains a kind of fusion rotein, and its nucleotides sequence is classified as shown in SEQ ID NO.5, it is connected with carrier for expression of eukaryon, has obtained a kind of bivalent DNA vaccine p-CCL19IZgB of restructuring.

In application as above, by (G4S) ₂-IZ-(G4S) ₂cCL19 and HSV-2 glycoprotein gB connects by linker, obtains a kind of fusion rotein, and its nucleotides sequence is classified as shown in SEQ ID NO.4, it is connected with carrier for expression of eukaryon, has obtained a kind of bivalent DNA vaccine p-gBIZCCL19 of restructuring.

In order to realize above-mentioned object, the present invention adopts following technical measures:

Bivalent DNA vaccine connection peptides is preparing the application in bivalent DNA vaccine, and its application process is as follows:

(1) PCR method amplicon virus envelope glycoprotein gene and adjuvant genes, and introduce restriction enzyme site.At (G4S) during design primer ₂-IZ-(G4S) ₂remove terminator codon in the upstream gene of linker sequence, in downstream gene, remove signal peptide sequence.

(2) enzyme cuts viral envelope glycoprotein gene and adjuvant genes PCR primer, is first connected into carrier for expression of eukaryon in order, then is amplified (G4S) in the middle of viral envelope glycoprotein gene and adjuvant genes by over-lap PCR method ₂-IZ-(G4S) ₂linker sequence.

(3) connect product conversion to E.coli DH5 α competence bacterial strain, double digestion checking after picking list bacterium colony enlarged culturing, extraction recombinant plasmid, is the bivalent DNA vaccine of restructuring of the present invention through the recombinant plasmid that sequence verification sequence is correct.

Preferably, the envelope glycoprotein gene of described HSV-2 is gD or gB;

Preferably, described adjuvant genes is CCL19 or CCL28;

Preferably, described carrier for expression of eukaryon is pcDNA3.1 (+).

According to aforesaid method, four kinds of obtained bivalent DNA vaccines are as follows:

P-gDIZCCL19 is the pcDNA3.1 (+) containing nucleotide sequence shown in SEQ ID NO.3; This nucleotide sequence is positioned at the downstream of pcDNA3.1 (+) promotor Pcmv;

P-CCL28IZgD is the pcDNA3.1 (+) containing nucleotide sequence shown in SEQ ID NO.4; This nucleotide sequence is positioned at the downstream of pcDNA3.1 (+) promotor Pcmv;

P-CCL19IZgB is the pcDNA3.1 (+) containing nucleotide sequence shown in SEQ ID NO.5; This nucleotide sequence is positioned at the downstream of pcDNA3.1 (+) promotor Pcmv;

P-gBIZCCL19 is the pcDNA3.1 (+) containing nucleotide sequence shown in SEQ ID NO.6.This nucleotide sequence is positioned at the downstream of pcDNA3.1 (+) promotor Pcmv;

The application of bivalent DNA vaccine in intramuscular injection type vaccine of restructuring, its process is as follows:

To inject after two weeks again booster immunization once with the principle initial immunity of prime-boost, twice immunization method and dosage identical.Within after last immunity the 14th day, gather blood, vagina sample, spleen cell, the antagonism of machines body acupuncture originates in the reaction of raw humoral immunization, cellular immunization and mucosal immune response respectively, and the protectiveness effect of vaccine on mouse.

The present invention compared with prior art, has the following advantages and effect:

The present invention adds the joint (G4S) that two are rich in glycine and Serine ₂linker---(G4S) ₂iZ (G4S) ₂, envelope glycoprotein gene and adjuvant genes can be helped correctly to translate, fold, define the normal recombinant protein playing respective function, the space conformation of this recombinant protein has stronger immunogenicity than the DNA vaccination of unit price.Simultaneously under gene adjuvant CCL19 and CCL28 effect; achieve and directionally HSV-2 envelope glycoprotein is offered to the B cell of maturation, T cell or dendritic cell (DC); enhance vaccinal immune response and memory immune reaction, the DNA vaccinations demonstrating four kinds of restructuring of the present invention first can more promptly produce the antibody of high titre, stronger virus neutralizing cpaacity have better immune protective effect to mouse than the DNA vaccination of unit price.

Accompanying drawing explanation

Fig. 1 is the vector construction schematic flow sheet of restructuring bivalent DNA vaccine p-gDIZCCL19.

Fig. 2 is the titre schematic diagram of antigen-specific IgG in the serum of p-gDIZCCL19 group.

Fig. 3 is the titre schematic diagram of antigen-specific IgA in p-gDIZCCL19 group vaginal douche.

Fig. 4 is the neutralising capacity schematic diagram of p-gDIZCCL19 group Serum Antibody.

Fig. 5 is the Th1/Th2 cellullar immunologic response reaction schematic diagram of p-gDIZCCL19 group splenocyte antigen-specific.

Fig. 6 is that after p-gDIZCCL19 group attacks poison, the end point titres of the 5th day mice serum somatotype antibody analyzes schematic diagram.

Fig. 7 is that p-gDIZCCL19 group attacks mouse disease severity scale schematic diagram in malicious latter 15 days.

Fig. 8 is the qualification figure of restructuring vector in vitro expressed fusion protein.

Wherein, scheming A is SDS-PAGE (upper); Figure B is native-PAGE (under).

Embodiment

Technical scheme of the present invention, if not otherwise specified, to be in molecular biology conventional technical scheme, described reagent if not otherwise specified, all purchased from biochemical shop.

Embodiment 1:

Bivalent DNA vaccine connection peptides is preparing the application in bivalent DNA vaccine, and its application process is as follows:

1. the amplification of gene:

A. on pcDNA3.1 (+) (Invitrogen company) plasmid vector, successively building HSV-2gD or gB, (it is HG52 strain that HSV-2 tests strain, from British royal laboratory, LGC impurity of the drug standard substance company obtains) (GenBank:Z86099.2) envelope glycoprotein gene and adjuvant genes CCL19 (Kai Hu.JOURNAL OF IMMUNOLOGY, Aug.2013, p.1935-1947), its molecular formula is: pcDNA3.1 (+)-Hind III-gD (EcoR I)/gB (BamH I) (removing termination codon)-(G4S) ₂-IZ-(G4S) ₂-CCL19 (removing signal peptide)-XhoI, called after p-gDIZCCL19 (pcDNA3.1 (+) recombinant plasmid for containing nucleotide sequence shown in SEQ IDNO.3) and p-gBIZCCL19 (pcDNA3.1 (+) recombinant plasmid for containing nucleotide sequence shown in SEQ ID NO.6) respectively, primer sequence is in table 1.

B. on pcDNA3.1 (+) (Invitrogen company) plasmid vector, adjuvant genes CCL19 or CCL28 (Kai Hu.JOURNAL OF IMMUNOLOGY is successively built, Aug.2013, p.1935-1947) and HSV-2gD or gB envelope glycoprotein gene, its molecular formula is: pcDNA3.1 (+)-Hind III-CCL28/CCL19 (removing termination codon)-KpnI-(G4S) ₂-BamHI-IZ-(G4S) 2-EcoRI-gD/gB (removing signal peptide)-Xba I, called after p-CCL28IZgD (pcDNA3.1 (+) recombinant plasmid for containing nucleotide sequence shown in SEQ ID NO.4) and p-CCL19IZgB (pcDNA3.1 (+) recombinant plasmid for containing nucleotide sequence shown in SEQ ID NO.5) respectively, primer sequence is in table 2.

The primer sequence table of table 1p-gDIZCCL19 and p-gBIZCCL19

Table 2 builds the primer sequence table of p-CCL28IZgD and p-CCL19IZgB

Be described for the goal gene built in p-gDIZCCL19 below:

(1) first, pcDNA3.1 (+) (commercially available, Invitrogen company) plasmid vector builds the genome of gene adjuvant CCL19.To gene adjuvant CCL19 genome (Kai Hu.JOURNAL OF IMMUNOLOGY, Aug.2013, p.1935-1947) carries out introducing BamH I-(G4S) at 5 ' end and 3 ' end respectively by primer when pcr amplification obtains goal gene fragment and the amplification of the part of the 252bp after removing signal peptide ₂with Xho I two restriction enzyme sites and (G4S) ₂, primer sequence is BamHI-(G4S) ₂-CCL19:5 '-TAT gGATCCgGTGGCGGTGGCTCCGGCGGTGGTGGGTCCGGTGCTAATGATGCGGAAGA-3 ' and CCL19-XhoI:5 '-CCG cTCGAGtCAAGACACAGGGCTCCTTC-3 '.

(2) then, linker is built in the genomic upstream of gene adjuvant CCL19, the gene of linker adopts the synthesis of montage Overlap extension PCR (splicing overlap extention PCR, SOE PCR) method order, and IZ two ends are respectively containing one (G4S) ₂, (G4S) ₂sequence is introduced by primer when increasing by linker upstream and downstream goal gene gD and CCL19 respectively, and the upstream primer sequence of synthesis IZ is respectively: IZ1:5 '- aTCGCCAGAATCAAGAAGCTgATCGGCGAGAGAGGTGGCGGTGGCTCCGGCGG-3 ', IZ2:5 '- cATCGAGAACGAG aTCGCC aGAATCAAGAAGCT-3 ', BamH I-IZ3:5 '-TAT gGATCCAGAATGAAGCAGATCGAGGAcAAGATCGAGGAG -3 ' and IZ4-(G4S) 2 (EcoR I): 5 '-CCG gAAT tCAGAATGAAGCAGATCGAGGA-3 ', in upstream primer, the oligonucleotide sequence of single straight line mark is restriction enzyme site, and the oligonucleotide sequence of other same line mark is the part of overlapping amplification, and the downstream primer of synthesis IZ all adopts the reverse primer of IZ1-4: CCG cTCGAGtCAAGACACAGGGCTCCTTC.

(3) last, the genome of HSV-2gD (HG52 strain) is built in the upstream of linker, gD is the 1254bp part after genome ends removes termination codon, introduces Hind III and (G4S) respectively during amplification by primer at 5 ' end and 3 ' end ₂-EcoR I two restriction enzyme sites and (G4S) ₂, primer sequence is: upstream 5 '-CCC aAGCTTaTGGGGCGTTTGACCTCCGG-3 ' and downstream 5 '-CCG gAATTCgGTGGCGGTGGCTCCGGCGGTGGTGGGTCCGTAAAACAATGGCTGGTGCG-3 '.

P-gBIZCCL19; P-CCL28IZgD; The building process of the object fragment in p-CCL19IZgB is identical with p-gDIZCCL19.

Building p-gBIZCCL19 goal gene primer used is BamHI-(G4S) 2-CCL19, CCL19-XhoI and Hind in table 1 III-gB and gB-(G4S) ₂-BamH I;

Building p-CCL28IZgD goal gene primer used is Hind III-CCL28, CCL28-Kpn I, EcoRI-gD and gD-XbaI in table 2;

Building p-CCL19IZgB goal gene primer used is Hind III-CCL19, CCL19-Kpn I, EcoRI-gB and gB-XbaI in table 2;

The primer sequence that above IZ gene is used is identical, is IZ1, IZ2, BamH I-IZ3, IZ4-(G4S) ₂the reverse primer of (EcoR I) and IZ1-4.

2. the structure of recombinant plasmid:

(1) endonuclease reaction: by the above object gene amplification, cut glue after agarose electrophoresis and reclaim PCR primer, the each DNA fragmentation reclaimed and pcDNA3.1 (+) plasmid vector carry out double digestion (NEB company) with corresponding restriction enzyme respectively, object fragment (plasmid vector) endonuclease reaction system is: object fragment 36 μ L (plasmid vector 2 μ L), restriction enzyme 1 and each 2 μ L of restriction enzyme 2,10 × restriction endonuclease damping fluid 5 μ L, BSA0.5 μ L, adds water to cumulative volume 50 μ L.Reaction conditions: 37 DEG C of water-baths 3 hours, digestion products is separated through the agarose gel electrophoresis of 1%.

(2) ligation: glue reclaims the DNA fragmentation that enzyme is cut, then successively object fragment is carried out ligation according to above molecular formula and carrier pcDNA3.1 (+), reaction system: object fragment 15 μ L, carrier 2 μ L, 10 × ligase enzyme damping fluid 2 μ L, T4 ligase enzyme 1 μ L (NEB company), reaction conditions: 16 DEG C of water-baths 16 hours.

(3) transform and checking: last, connect product conversion to E.coli DH5 α competence bacterial strain, conversion condition: ddH2O45 μ L, 1M CaCl ₂2.5 μ L, 0.6M MgCl ₂2.5 μ L, connection product 10 μ L.Reaction conditions: ice bath 40 minutes, 42 DEG C thermal shocks 1.5 minutes, ice bath 2 minutes.Converted product is coated on the LB flat board of amicillin resistance, double digestion checking after picking list bacterium colony Zengjing Granule, extraction recombinant plasmid, the bivalent DNA vaccine of restructuring of the present invention is, i.e. p-gDIZCCL19 (sequence is shown in SEQ ID NO.7) through the recombinant plasmid that sequence verification sequence is correct; P-CCL28IZgD(contains the nucleotide sequence shown in SEQ ID NO.4); P-CCL19IZgB(contains the nucleotide sequence shown in SEQ ID NO.5); P-gBIZCCL19(contains the nucleotide sequence shown in SEQ ID NO.6).Wherein for p-gDIZCCL19 group, the structure flow process of recombinant plasmid is shown in Fig. 1.

Embodiment 2:

The application of bivalent DNA vaccine in preparation intramuscular injection type vaccine, the steps include:

1. the enlarged culturing of plasmid: by the bivalent DNA vaccine p-gDIZCCL19 prepared above, p-CCL28IZgD, p-gBIZCCL19, p-CCL19IZgB and pcDNA3.1 (+) empty carrier, above antigen gD or gB (called after p-gD or p-gB) is built separately with at pcDNA3.1 (+), independent structure gene adjuvant CCL19 or CCL28 (called after p-CCL19 or p-CCL28) plasmid (for univalent vaccine) Transformed E .coli DH5 α all again, line is coated on the agarose plate containing amicillin resistance, cultivate 16 hours for 37 DEG C, picking list bacterium colony adds the LB liquid medium of 5mL, 37 DEG C of 200rmp jolt cultivation 16 hours, then add 500mL by 1:100 and prepare the sterilized 2 × YT liquid nutrient medium having penbritin in advance, 37 DEG C of 200rmp jolt cultivation 20 hours, test kit extraction plasmid is extracted in a large number with the endotoxic plasmid that goes of MACHEREY-NAGEL company, finally be dissolved in not containing in endotoxic deionized water, DNA concentration is measured with ultraviolet spectrophotometer, the plasmid amount needed for 1mol antigen is calculated respectively according to the molecular weight of recombinant plasmid.This plasmid stroke-physiological saline solution diluted before immunity, vortex mixes, and faces used time preparation.

2. immunization strategy: be injected in by the dosage of 1moL/40 μ L in the quadriceps muscle of thigh of SPF level BALB/c mouse in a 6 week age back leg, and with

100V, 50ms shock by electricity 3 times, shock by electricity 3 times after electrode is reverse again, promote that the DNA vaccination of plasmid form is transfected into muscle cell.To inject after two weeks again booster immunization once with the principle initial immunity of prime-boost, twice immunization method and dosage identical.

3. test echelon design:

(1) experimental group: p-gDIZCCL19; P-CCL28IZgD; P-CCL19IZgB; P-gBIZCCL19 (each 1mol)

(2) positive controls: p-gD or p-gB+pcDNA3.1 (+) (each 1mol); P-gD or p-gB+pCCL19 (each 1mol); P-gD or p-gB+pCCL28 (each 1mol)

(3) negative control group: pcDNA3.1 (+) (1mol)

4. sample collecting: gather blood, vagina sample, spleen in after last immunity the 14th day, originates in the reaction of raw humoral immunization, cellular immunization and mucosal immune response with the antagonism of following experimental technique machines body acupuncture respectively.

(1) serum and vagina sample: gather about 250 μ L blood from mouse ectocanthion, little of blood coagulation in 37 DEG C of placements 1, centrifugal 15 minutes of 6000rpm, packing serum in 6 hours; Gather vaginal douche (100 μ L/ only) with the aseptic PBS containing proteinase inhibitor (Roche company), centrifugal 5 minutes of 13000rpm, draws supernatant and retains, be all stored in-80 DEG C

Refrigerator, for subsequent use, for ELISA experiment and external Neutralizing test.

(2) splenocyte: after mouse the 2nd immunity 14 days cervical dislocation are put to death, be soaked in the alcohol of 75%, after about 5 minutes, the spleen of aseptic collection mouse, organize collecting cell after Falcon70 μm of filter screen (BD Biosciences company) grinding, be separated with mouse lymphocyte parting liquid (it is biotech firm that Beijing reaches section) and reclaim lymphocyte (method is see specification sheets), finally with 0.8mL containing 10%FBS with dual anti-complete RPMI-1640 substratum is resuspended, counting, for follow-up Chemotaxis test and flow cytometry.

5. indirect ELISA experiment

(1) envelope antigen: with PBS by prokaryotic expression and HSV-2gD or the gB proteantigen of purifying be diluted to concentration be 2.0 μ g/mL wrap by ELISA96 orifice plate, every hole 50 μ L, 4 DEG C place 14-16 hour.

(2) wash plate: get rid of antigen protein, clean elisa plate 3 times with PBST, pat dry on thieving paper at every turn.

(3) close: every hole adds 300 μ L1%BSA confining liquids, close 1 hour for 37 DEG C.

(4) wash plate: abandon confining liquid, wash plate same (2).

(5) sample to be tested (antibody) is added: (the initial dilution ratio of serum is 1:5000 to add the serum of 3 times of gradient doubling dilutions or vaginal douche, the initial dilution ratio of vaginal douche is 1:10), every hole 50 μ L, hatches 1 hour for 37 DEG C.

(6) wash plate: get rid of testing sample, wash plate same (2).

(7) incubate primary antibodie: add goat anti-mouse IgG (H+L) antibody of HRP mark or biotin labeled goat anti-mouse IgA, antibody antibody diluent dilution (1:5000), every hole 50 μ L, hatches 1 hour for 37 DEG C.Every plate arranges the negative control not adding sample.

(8) wash plate: get rid of primary antibodie, clean elisa plate 5 times with PBST, pat dry on thieving paper at every turn.

(incubate the Streptomycin sulphate of HRP mark again: with this during survey IgA, press 1:5000 dilution with antibody diluent, every hole 50 μ L, hatches 0.5 hour for 37 DEG C.Wash plate same (8).)

(9) colour developing and detection: add TMB chromogenic substrate, every hole 50 μ L, room temperature lucifuge develops the color 5 minutes, adds stop buffer (2M H ₂sO ₄), every hole 50 μ L.Microplate reader is used to read OD immediately ₄₅₀and OD ₅₇₀the value of (reference wavelength).

(10) analytical data: the end point titres (End-point titer) calculating antibody with Excel and GraphPad Software.

(11) detection of Subclass of antibody: except detecting the IgA antibody of IgG in serum and vaginal douche, the antibody typing test kit specification sheets provided according to Southern Biotechnology company and Invitrogen detects IgM, IgG1, IgG2a, IgG2b and IgG3 antibody in serum.

6. external Neutralizing test

(1) cell is spread: in detecting first 1 day by Vero cell with every milliliter of 2-3 × 10 ⁵individual cell is inoculated in 96 porocyte culture plates, 37 DEG C, 5%CO2 hatches 12-14h.

(2) sample process: serum sample needs to be placed in 56 DEG C of water-baths, and 60 minutes with deactivation complement.The experimental group serum starting point concentration of gD antigen dilutes according to 1:20 (gB dilutes according to the 1:10) DMEM of serum-free, follow-up with 3 times of gradient doubling dilutions, totally 6 gradients, and each gradient does 4 repetitions, every hole 50 μ L.

(3) virus dilution: the DMEM of HSV-2 virus serum-free to be neutralized is diluted to 100TCID ₅₀/ 50 μ L, for subsequent use.

(4) neutralizing effect: the HSV-2 virus of dilution mixes by 1:1 with the diluent of serum/vaginal douche, 37 DEG C, 5%CO ₂hatch 1 hour.

(5) add cell: sample-viral mixed solution 100 μ L is added Vero cell monolayer, add the DMEM of 100 μ L containing 2%FBS, 37 DEG C, 5%CO ₂hatch and measure TCID ₅₀the identical time, terminate to observe.

(6) contrast is set up: the positive control and 0.1,1,10, the 100 times of TCID that do not add virus with batch Setup Experiments ₅₀the negative control (each extent of dilution 4 hole) of HSV-2.As Positive control wells and 0.1,1 times of TCID at the end of experiment ₅₀control wells should there is not pathology, and 100TCID ₅₀must there is cytopathy in hole, experiment is set up.Often criticize the immune serum contrast of Setup Experiments pcDNA3.1 (+) experimental group simultaneously.

(7) result judges: the neutralising capacity of sample is expressed as and suppresses virus replication active.

7. the Th1/Th2 cellullar immunologic response reaction of splenocyte antigen-specific

(1) cell counting: the spleen cell 1 × 10 getting fresh separated ⁷individually add 24 orifice plates, every increment product do 1 repetition, complement to 1mL with complete RPMI-1640 substratum.

(2) stimulator: gD or the gB antigen protein adding HSV-2 reaches 1 μ g/ hole, does the positive control adding canavaline lectin A (Con-A) and the negative control only adding substratum (not adding any stimulator) with criticizing.

(3) cultivate: culture plate is placed in 37 DEG C, 5%CO ₂cultivate 5 days in incubator.

(4) supernatant is received: after filtering with 0.22 μm of disposable filter, by supernatant packing, frozen in-80 DEG C, for subsequent use.

(5) operation steps: detect (article No.: 551287) with the CBA test kit of BD Biosciences company, detect the Th1/Th2 cytokine (containing IFN-γ, IL-2, IL-4, IL-5 and TNF) that murine antigen is special, working method refers to test kit specification sheets.

(6) upper machine testing and analysis: the standard substance using test kit to carry, sets up negative control simultaneously.First complete the detection of standard substance and negative control, then carry out the detection of sample successively, detect data and use the FCAP Arrayv1.0 analysis software that provides of BD company to process, automatic drawing standard curve and calculate the content of each cytokine.

8. Chemotaxis test: the splenocyte of fresh separated after counting, with 2 × 10 ⁶/ 500 μ L are added on Transwell12 orifice plate (the Corning company in 0.3 μm of aperture, article No.: upper room 3402), the lower room of Transwell is added on the 1.5mL RPMI-1640 perfect medium contained and do not contain the recombined small-mouse CCL19 albumen of 30ng/mL and the CCL28 albumen (R & D Systems company) of 500ng/mL, every increment product do 2 repetitions, culture plate are placed in 37 DEG C and containing 5%CO ₂incubator hatch 2 hours.Take out the upper room of transwell, the cell in the lower room of piping and druming mixing, counts with the cell of cell counting count board to migration.(experimental group is for containing CCL19 or CCL28 albumen for the cell of the cell/control group average mobility of mobility=experimental group average mobility; Control group is not containing CCL19 or CCL28 albumen).

9. attack poison checking: mouse immune carries out vaginal infection HSV-2 (HG52 strain) to mouse after 49 days and attacks poison (strain is purchased from British royal laboratory LGC impurity of the drug standard substance company), attack poison first 5 days subcutaneous multi-point injection Progesterone (2mg/ only), 1% vetanarcol (according to appointment the mouse 1mg of 20g body weight) are first used to anaesthetize when attacking poison, then vaginal infection 150LD ₅₀hSV-2 test strain 10 μ L (PFU/mL=2.2 × 10 ⁷).After attacking poison, blood sampling in the 5th day, detects serum antibody response situation (working method is shown in the antibody typing analysis in 5).In other 15 days to the disease symptoms of mouse mark (Natalie V, et al.JOURNAL OF VIROLOGY, May2011, p.5036-5047): 1 grade, non-evident sympton; 1 grade, slightly rubescent; 2 grades, red and swollen as seen; 3 grades, seriously red and swollen; 4 grades, fester and/or lose hair or feathers; 5 grades, serious morbidity (any mouse disease severity reaches 5 grades will carry out euthanasia), comparative experiments group and other disease severity difference organized.

10. the qualification of recombinant vectors vivoexpression fusion rotein: by 3 μ g recombinant plasmid vector transfections to 293T cell, with Lipofectamine2000 (purchased from American Invitrogen) reagent transfection, transfection 48 h before harvest supernatant, within 13000rpm/ minute centrifugal 10 minutes, draw supernatants for 4 DEG C, by the oligomeric state of sex change PAGE electrophoresis (SDS-PAGE) and Native PAGE electrophoresis (native-PAGE) and Western blotting qualification albumen.

11. statistical study

This research is according to repeating, contrasting and random experiment principle, data represent with mean ± standard deviation, by SPSS13.0 software analysis data, draw with GraphPad Prism5.0 software, adopt one-way analysis of variance and Spearman Rank correlation, comparing between two with LSD-t inspection, take p=0.05 as notable level.In figure, * represents that p<0.05, * * represents p<0.01, represents that between two groups, significant difference is remarkable; NS represents that between two groups, significant difference is not remarkable.

12. results and conclusion

Following result if not otherwise specified, is the detected result of the sample that last immunity gathers for latter 14th day

(1) detection of antibody: in serum and vaginal douche sample gD and gB specific antibody end point titres the results are shown in Table 3, IgG and IgA of bivalent DNA vaccine p-gDIZCCL19, p-CCL19IZgB, p-gBIZCCL19 of restructuring is all high than positive controls; Although the IgG of p-CCL28IZgD does not have the height of positive controls, IgA higher (p<0.01).Therefore, the bivalent DNA vaccine of restructuring can stimulate body to produce high efficiency humoral immunization (IgG and IgM) and mucosa-immune (IgA).Wherein for p-gDIZCCL19 group, in its serum, the titre of antigen-specific IgG and IgA compares and sees Fig. 2,3, in figure, * represents p<0.01, * * and represents p<0.001, think that experimental group p-gDIZCCL19 (1mol) and the antibody titers between positive controls p-gD+pCCL19 (1mol) and p-gD+p-cDNA3.1 (+) group have significant difference and pole significant difference respectively, and have dose-dependant.

The titre of IgG and IgM that the mouse also finding immune p-gDIZCCL19 and p-CCL19IZgB in detecting in addition produces after immunity the last time on the 5th day is higher than other groups, and antibody titer can maintain the longer time, the 5th day time immunity p-gDIZCCL19 and p-CCL19IZgB mouse produce IgG be respectively 294410.21 ± 61.57 and 57873.66101.21, IgM be respectively: 334.01 ± 21.17 and 231.52 ± 51.66; IgG213779.52 ± 251.33/39441.14 ± 45.63 of its end point titres control group p-gD+pCCL19/p-gB+pCCL19 separately and IgM218.32 ± 11.77/161.28 ± 15.85 height.

The end point titres of antigen-specific antibodies IgG and IgA in table 3 immune mouse antiserum(antisera)

(2) detection of NAT: the neutralising capacity of the serum antibody of the bivalent DNA vaccine of restructuring is apparently higher than other groups (p<0.01), can learn that the antiserum(antisera) that experimental group vaccine produces has stronger neutralising capacity, the results are shown in Table 4,5.Wherein for p-gDIZCCL19 group, the neutralising capacity of its serum antibody relatively see Fig. 4, and have dose-dependant.

The neutralising capacity (%) of table 4HSV-2gD group serum antibody

The neutralising capacity (%) of table 5HSV-2gB group serum antibody

(3) the Th1/Th2 cellullar immunologic response reaction of splenocyte antigen-specific: IFN-γ, TNF, IL-2 are the cytokines of Th1 emiocytosis, IL-4 and IL5 is the cytokine of Th2 emiocytosis, the former scale has showed the function of Th1 cell, and the scale of the latter has showed the function of Th2 cell; Th1 cell mainly regulates cell immune response, and Th2 cell mainly regulates humoral immune reaction.The splenocyte of experimental mice can produce to have than the cytokine of control group must be increased (see table 6) in various degree, TNF and IL-2 as wherein p-gDIZCCL19 and p-CCL28IZgD generation is higher than corresponding control group, therefore can produce stronger cellular immunization.Be 35.80% by the percentage of cells of the positive dendritic cell of CCL19 part (CCR7) in the splenocyte of Flow cytometry p-gDIZCCL19 group mouse in addition, more than control group p-gD+pCCL1916.83%, dendritic cell is the cell that HLA-II antigen is the strongest, and the bivalent DNA vaccine p-gDIZCCL19 of therefore this restructuring is better than univalent vaccine in the ability regulating cellular immunization; The dendritic cell that in the splenocyte of p-CCL19IZgB group mouse, CCL19 part (CCR7) is positive and B cell percentage ratio are respectively 43.12% and 10.48%, and (dendritic cell and B cell percentage ratio are respectively 28.93% and 6.04%) that p-gBIZCCL19 is only dendritic cell (42.79%) the control group p-gB+pCCL19 more common than them is many; Visible bivalent vaccine inducing mouse creates stronger cellular immunization.Wherein for p-gDIZCCL19 group, the Th1/Th2 cellullar immunologic response of splenocyte antigen-specific reacts to compare sees Fig. 5.

The antigen specific cytokine of table 6 splenocyte secretion

(4) Chemotaxis test: this experiment is the detection by quantitative to the immunocyte (T, B, DC) of expressing CCR7 (part of CCL19 molecule) and CCR10 (part of CCL28 molecule) molecule, the results are shown in Table 7, the immunocyte of expressing CCR7 molecule in the spleen of known experimental group p-gDIZCCL19 and pCCL19IzgB mouse is more than control group p-gD+p-cDNA3.1 (+) or p-gD+pCCL19.Chemokines CC CL19 protein molecular can be expressed in somatocyte after immunization p-gDIZCCL19 and p-CCL19IzgB, it can attract the immune cell migration of expressing corresponding acceptor to corresponding site by chemotactic as gene adjuvant, attested function is: attract the T cell district that inmature and central memory T cell and maturation Dendritic Cells Migration to secondary lymphoid is tied, may be relevant to the effect of its immunostimulant.Known by result, after p-gDIZCCL19 and pCCL19IzgB immunity, CCL19 chemotactic has attracted relatively many immunocytes to enter in spleen, has stronger immune protective relevant after attacking poison to this group mouse.

Table 7 splenocyte antigen-specific mobility (%)

(5) attack poison after antibody typing analysis: attack poison after the 5th day gather blood, in serum somatotype antibody end point titres the results are shown in Table 8.Experimental group p-gDIZCCL19, p-CCL28IZgD and p-CCL19IZgB produce the amount all high than control group (p<0.01) of IgG1, IgG2a, IgG2b and IgM, show that the reaction of its memory immune is rapider.Wherein for p-gDIZCCL19, after attacking poison, Fig. 6 is shown in the end point titres analysis of the 5th day mice serum somatotype antibody, this result is consistent with the serum antibody situation of mouse vaccine Immunoreactivity, no matter experimental group p-gDIZCCL19 is in vaccine immunity reaction or is the group responded the earliest in memory immune response after attacking poison.

The end point titres of somatotype antibody in table 8HSV-2gD group serum

(6) disease severity analysis after attacking poison: observe through attacking poison, experimental group p-gDIZCCL19, p-CCL28IZgD and p-CCL19IZgB mouse there will not be red and swollen reaction in early days attacking poison, only p-gDIZCCL19 group mouse has the phenomenon of depilation late, and the early stage red and swollen rate of control group mice is all higher, late period plucking rate also higher (the results are shown in Table 9).Experimental group p-gDIZCCL19, p-CCL28IZgD and p-CCL19IZgB mouse are under this high virus challenge dose, and comparatively other organize healthy state.Comprehensively the detected result of (5) and (6) can be known: memory immune reacts more early, has better protected effect to mouse.Wherein for p-gDIZCCL19, attack mouse disease severity scale in malicious latter 15 days and see Fig. 7, experimental group p-gDIZCCL19 mouse is under this high virus challenge dose, and healthy state comparatively control group is good.

After poison attacked by table 9,1-15 days mouse disease severity are graded

(7) qualification of recombinant vectors vivoexpression fusion rotein: Western blotting the results are shown in SDS-PAGE (figure A) and native-PAGE (figure B) result, fusion rotein can be gone out by normal expression by the visible recombinant vectors of SDS-PAGE, be respectively the albumen that molecular weight ratio gD and gB is high.But the applied sample amount of p-CCL28IZgD albumen is lower in detecting, its result (loading Buffer is 2 × Buffer) is not measured in native-PAGE electrophoresis, in SDS-PAGE, visible its has fusion rotein (loading Buffer is 5 × Buffer), and all the other are respectively organized has trimer protein to express (figure B) all as seen.

Conclusion: the bivalent DNA vaccine that this connection peptides obtains can bring out body and produce humoral immunization, cellular immunization and the mucosal immunoreaction stronger than univalent vaccine, and the antiserum(antisera) of generation has stronger neutralising capacity, has better protected effect to mouse.

SEQUENCE LISTING

<110> Wuhan Virology Institute,Chinan academy of Sciences

<120> bivalent DNA vaccine connection peptides and application thereof

<130> bivalent DNA vaccine connection peptides and application thereof

<160> 7

<170> PatentIn version 3.1

<210> 1

<211> 159

<212> DNA

<213> artificial sequence

<400> 1

ggtggcggtg gctccggcgg tggtgggtcc agaatgaagc agatcgagga caagatcgag 60

gagatcctga gcaagatcta ccacatcgag aacgagatcg ccagaatcaa gaagctgatc 120

ggcgagagag gtggcggtgg ctccggcggt ggtgggtcc 159

<210> 2

<211> 53

<212> PRT

<213> artificial sequence

<400> 2

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Met Lys Gln Ile Glu

1 5 10 15

Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu

20 25 30

Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Arg Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser

50

<210> 3

<211> 1614

<212> DNA

<213> artificial sequence

<400> 3

atggggcgtt tgacctccgg cgtcgggacg gcggccctgc tagttgtcgc ggtgggactc 60

cgcgtcgtct gcgccaaata cgccttagca gacccctcgc ttaagatggc cgatcccaat 120

cgatttcgcg ggaagaacct tccggttttg gaccagctga ccgacccccc cggggtgaag 180

cgtgtttacc acattcagcc gagcctggag gacccgttcc agccccccag catcccgatc 240

actgtgtact acgcagtgct ggaacgtgcc tgccgcagcg tgctcctaca tgccccatcg 300

gaggcccccc agatcgtgcg cggggcttcg gacgaggccc gaaagcacac gtacaacctg 360

accatcgcct ggtatcgcat gggagacaat tgcgctatcc ccatcacggt tatggaatac 420

accgagtgcc cctacaacaa gtcgttgggg gtctgcccca tccgaacgca gccccgctgg 480

agctactatg acagctttag cgccgtcagc gaggataacc tgggattcct gatgcacgcc 540

cccgccttcg agaccgcggg tacgtacctg cggctagtga agataaacga ctggacggag 600

atcacacaat ttatcctgga gcaccgggcc cgcgcctcct gcaagtacgc tctccccctg 660

cgcatccccc cggcagcgtg cctcacctcg aaggcctacc aacagggcgt gacggtcgac 720

agcatcggga tgttaccccg ctttatcccc gaaaaccagc gcaccgtcgc cctatacagc 780

ttaaaaatcg ccgggtggca cggccccaag cccccgtaca ccagcaccct gctgccgccg 840

gagctgtccg acaccaccaa cgccacgcaa cccgaactcg ttccggaaga ccccgaggac 900

tcggccctct tagaggatcc cgccgggacg gtgtcttcgc agatcccccc aaactggcac 960

atcccgtcga tccaggacgt cgcgccgcac cacgcccccg ccgcccccag caacccgggc 1020

ctgatcatcg gcgcgctggc cggcagtacc ctggcggcgc tggtcatcgg cggtattgcg 1080

ttttgggtac gccgccgcgc tcagatggcc cccaagcgcc tacgtctccc ccacatccgg 1140

gatgacgacg cgcccccctc gcaccagcca ttgttttacg gatccactag tccagtgtgg 1200

tggggtggcg gtggctccgg cggtggtggg tccagaatga agcagatcga ggacaagatc 1260

gaggagatcc tgagcaagat ctaccacatc gagaacgaga tcgccagaat caagaagctg 1320

atcggcgaga gaggtggcgg tggctccggc ggtggtgggt ccggtgctaa tgatgcggaa 1380

gactgctgcc tgtctgtgac ccagcgcccc atccctggga acatcgtgaa agccttccgc 1440

taccttctta atgaagatgg ctgcagggtg cctgctgttg tgttcaccac actaaggggc 1500

tatcagctct gtgcacctcc agaccagccc tgggtggatc gcatcatccg aagactgaag 1560

aagtcttctg ccaagaacaa aggcaacagc accagaagga gccctgtgtc ttga 1614

<210> 4

<211> 1659

<212> DNA

<213> artificial sequence

<400> 4

atgcagcaag cagggctcac actcatggct gtggctgtgt gtgtggcttt tcaaacctca 60

gaagccatac ttcccatggc ctccagctgt tgcactgagg tgtctcatca tgtttccgga 120

agacttctgg aaagagtgag ttcatgcagc atccagagag ctgacgggga ctgcgacctg 180

gctgctgtca tccttcatgt taaacgtaga agaatctgca tcagcccgca caatcgtact 240

ttgaagcagt ggatgagagc ctcagaggta aagaagaatg gcagagaaaa cgtatgttct 300

gggaaaaaac aacccagcag gaaggacaga aaagggcaca ctacgagaaa gcacagaaca 360

cgtggaacac acaggcacga agcctctcgt ggtggcggtg gctccggcgg tggtgggtcc 420

agaatgaagc agatcgagga caagatcgag gagatcctga gcaagatcta ccacatcgag 480

aacgagatcg ccagaatcaa gaagctgatc ggcgagagag gtggcggtgg ctccggcggt 540

ggtgggtccg ccaaatacgc cttagcagac ccctcgctta agatggccga tcccaatcga 600

tttcgcggga agaaccttcc ggttttggac cagctgaccg acccccccgg ggtgaagcgt 660

gtttaccaca ttcagccgag cctggaggac ccgttccagc cccccagcat cccgatcact 720

gtgtactacg cagtgctgga acgtgcctgc cgcagcgtgc tcctacatgc cccatcggag 780

gccccccaga tcgtgcgcgg ggcttcggac gaggcccgaa agcacacgta caacctgacc 840

atcgcctggt atcgcatggg agacaattgc gctatcccca tcacggttat ggaatacacc 900

gagtgcccct acaacaagtc gttgggggtc tgccccatcc gaacgcagcc ccgctggagc 960

tactatgaca gctttagcgc cgtcagcgag gataacctgg gattcctgat gcacgccccc 1020

gccttcgaga ccgcgggtac gtacctgcgg ctagtgaaga taaacgactg gacggagatc 1080

acacaattta tcctggagca ccgggcccgc gcctcctgca agtacgctct ccccctgcgc 1140

atccccccgg cagcgtgcct cacctcgaag gcctaccaac agggcgtgac ggtcgacagc 1200

atcgggatgt taccccgctt tatccccgaa aaccagcgca ccgtcgccct atacagctta 1260

aaaatcgccg ggtggcacgg ccccaagccc ccgtacacca gcaccctgct gccgccggag 1320

ctgtccgaca ccaccaacgc cacgcaaccc gaactcgttc cggaagaccc cgaggactcg 1380

gccctcttag aggatcccgc cgggacggtg tcttcgcaga tccccccaaa ctggcacatc 1440

ccgtcgatcc aggacgtcgc gccgcaccac gcccccgccg cccccagcaa cccgggcctg 1500

atcatcggcg cgctggccgg cagtaccctg gcggcgctgg tcatcggcgg tattgcgttt 1560

tgggtacgcc gccgcgctca gatggccccc aagcgcctac gtctccccca catccgggat 1620

gacgacgcgc ccccctcgca ccagccattg ttttactag 1659

<210> 5

<211> 2730

<212> DNA

<213> artificial sequence

<400> 5

atggcccccc gtgtgacccc actcctggcc ttcagcctgc tggttctctg gaccttccca 60

gccccaactc tggggggtgc taatgatgcg gaagactgct gcctgtctgt gacccagcgc 120

cccatccctg ggaacatcgt gaaagccttc cgctaccttc ttaatgaaga tggctgcagg 180

gtgcctgctg ttgtgttcac cacactaagg ggctatcagc tctgtgcacc tccagaccag 240

ccctgggtgg atcgcatcat ccgaagactg aagaagtctt ctgccaagaa caaaggcaac 300

agcaccagaa ggagccctgt gtctggtggc ggtggctccg gcggtggtgg gtccagaatg 360

aagcagatcg aggacaagat cgaggagatc ctgagcaaga tctaccacat cgagaacgag 420

atcgccagaa tcaagaagct gatcggcgag agaggtggcg gtggctccgg cggtggtggg 480

tccgccccgg cggccccccg cgcctcgggc ggcgtggccg cgaccgtcgc ggcgaacggg 540

ggtcccgcct cccggccgcc ccccgtcccg agccccgcga ccaccagggc ccggaagcgg 600

aaaaccaaaa agccgcccga gcggcccgag gcgaccccgc cccccgacgc caacgcgacc 660

gtcgccgccg gccacgccac gctgcgcgcg cacctgcggg aaatcaaggt cgagaacgcc 720

gatgcccagt tttacgtgtg cccgcccccg acgggcgcca cggtggtgca gtttgagcag 780

ccgcgccgct gcccgacgcg cccggagggg cagaactaca cggagggcat cgcggtggtc 840

ttcaaggaga acatcgcccc gtacaaattc aaggccacca tgtactacaa agacgtgacc 900

gtgtcgcagg tgtggttcgg ccaccgctac tcccagttta tggggatatt cgaggaccgc 960

gcccccgttc ccttcgagga ggtgatcgac aagattaacg ccaagggggt ctgccgctcc 1020

acggccaagt acgtgcggaa caacatggag accaccgcgt ttcaccggga cgaccacgag 1080

accgacatgg agctcaagcc ggcgaaggtc gccacgcgca cgagccgggg gtggcacacc 1140

accgacctca agtacaaccc ctcgcgggtg gaggcgttcc atcggtacgg cacgacggtc 1200

aactgcatcg tcgaggaggt ggacgcgcgg tcggtgtacc cgtacgatga gtttgtgctg 1260

gcgacgggcg actttgtgta catgtccccg ttttacggct accgggaggg gtcgcacacc 1320

gagcacacca gctacgccgc cgaccgcttc aagcaggtcg acggcttcta cgcgcgcgac 1380

ctcaccacga aggcccgggc cacgtcgccg acgacccgca acttgctgac gacccccaag 1440

tttaccgtgg cctgggactg ggtgccgaag cgaccggcgg tctgcaccat gaccaagtgg 1500

caggaggtgg acgagatgct ccgcgccgag tacggcggct ccttccgctt ctcctccgac 1560

gccatctcga ccaccttcac caccaacctg acccagtact cgctctcgcg cgtcgacctg 1620

ggcgactgca tcggccggga tgcccgcgag gccatcgacc gcatgtttgc gcgcaagtac 1680

aacgccacgc acatcaaggt gggccagccg cagtactacc tggccacggg gggcttcctc 1740

atcgcgtacc agcccctcct cagcaacacg ctcgccgagc tgtacgtgcg ggagtacatg 1800

cgggagcagg accgcaagcc ccggaatgcc acgcccgcgc cactgcggga ggcgcccagc 1860

gccaacgcgt ccgtggagcg catcaagacc acctcctcga tcgagttcgc ccggctgcag 1920

tttacgtata accacataca gcgccacgtg aacgacatgc tggggcgcat cgccgtcgcg 1980

tggtgcgagc tgcagaacca cgagctgact ctctggaacg aggcccgcaa gctcaacccc 2040

aacgccatcg cctccgccac cgtcggccgg cgggtgagcg cgcgcatgct cggagacgtc 2100

atggccgtct ccacgtgcgt gcccgtcgcc ccggacaacg tgatcgtgca gaactcgatg 2160

cgcgtcagct cgcggccggg gacgtgctac agccgccccc tggtcagctt tcggtacgaa 2220

gaccagggcc cgctgatcga ggggcagctg ggcgagaaca acgagctgcg cctcacccgc 2280

gacgcgctcg agccgtgcac cgtgggccac cggcgctact tcatcttcgg cgggggctac 2340

gtgtacttcg aggagtacgc gtactctcac cagctgagtc gcgccgacgt caccaccgtc 2400

agcaccttca tcgacctgaa catcaccatg ctggaggacc acgagtttgt gcccctggag 2460

gtctacacgc gccacgagat caaggacagc ggcctgctgg actacacgga ggtccagcgc 2520

cgcaaccagc tgcacgacct gcgctttgcc gacatcgaca cggtcatccg cgccgacgcc 2580

aacgccgcca tgttcgcggg gctgtgcgcg ttcttcgagg ggatggggga cttggggcgc 2640

gcggtcggca aggtcgtcat gggagtagtg gggggcgtgg tgtcggccgt ctcgggcgtg 2700

tcctccttta tgtccaaccc cttcggggcg 2730

<210> 6

<211> 3114

<212> DNA

<213> artificial sequence

<400> 6

atgcgcgggg ggggcttgat ttgcgcgctg gtcgtggggg cgctggtggc cgcggtggcg 60

tcggcggccc cggcggcccc ccgcgcctcg ggcggcgtgg ccgcgaccgt cgcggcgaac 120

gggggtcccg cctcccggcc gccccccgtc ccgagccccg cgaccaccag ggcccggaag 180

cggaaaacca aaaagccgcc cgagcggccc gaggcgaccc cgccccccga cgccaacgcg 240

accgtcgccg ccggccacgc cacgctgcgc gcgcacctgc gggaaatcaa ggtcgagaac 300

gccgatgccc agttttacgt gtgcccgccc ccgacgggcg ccacggtggt gcagtttgag 360

cagccgcgcc gctgcccgac gcgcccggag gggcagaact acacggaggg catcgcggtg 420

gtcttcaagg agaacatcgc cccgtacaaa ttcaaggcca ccatgtacta caaagacgtg 480

accgtgtcgc aggtgtggtt cggccaccgc tactcccagt ttatggggat attcgaggac 540

cgcgcccccg ttcccttcga ggaggtgatc gacaagatta acgccaaggg ggtctgccgc 600

tccacggcca agtacgtgcg gaacaacatg gagaccaccg cgtttcaccg ggacgaccac 660

gagaccgaca tggagctcaa gccggcgaag gtcgccacgc gcacgagccg ggggtggcac 720

accaccgacc tcaagtacaa cccctcgcgg gtggaggcgt tccatcggta cggcacgacg 780

gtcaactgca tcgtcgagga ggtggacgcg cggtcggtgt acccgtacga tgagtttgtg 840

ctggcgacgg gcgactttgt gtacatgtcc ccgttttacg gctaccggga ggggtcgcac 900

accgagcaca ccagctacgc cgccgaccgc ttcaagcagg tcgacggctt ctacgcgcgc 960

gacctcacca cgaaggcccg ggccacgtcg ccgacgaccc gcaacttgct gacgaccccc 1020

aagtttaccg tggcctggga ctgggtgccg aagcgaccgg cggtctgcac catgaccaag 1080

tggcaggagg tggacgagat gctccgcgcc gagtacggcg gctccttccg cttctcctcc 1140

gacgccatct cgaccacctt caccaccaac ctgacccagt actcgctctc gcgcgtcgac 1200

ctgggcgact gcatcggccg ggatgcccgc gaggccatcg accgcatgtt tgcgcgcaag 1260

tacaacgcca cgcacatcaa ggtgggccag ccgcagtact acctggccac ggggggcttc 1320

ctcatcgcgt accagcccct cctcagcaac acgctcgccg agctgtacgt gcgggagtac 1380

atgcgggagc aggaccgcaa gccccggaat gccacgcccg cgccactgcg ggaggcgccc 1440

agcgccaacg cgtccgtgga gcgcatcaag accacctcct cgatcgagtt cgcccggctg 1500

cagtttacgt ataaccacat acagcgccac gtgaacgaca tgctggggcg catcgccgtc 1560

gcgtggtgcg agctgcagaa ccacgagctg actctctgga acgaggcccg caagctcaac 1620

cccaacgcca tcgcctccgc caccgtcggc cggcgggtga gcgcgcgcat gctcggagac 1680

gtcatggccg tctccacgtg cgtgcccgtc gccccggaca acgtgatcgt gcagaactcg 1740

atgcgcgtca gctcgcggcc ggggacgtgc tacagccgcc ccctggtcag ctttcggtac 1800

gaagaccagg gcccgctgat cgaggggcag ctgggcgaga acaacgagct gcgcctcacc 1860

cgcgacgcgc tcgagccgtg caccgtgggc caccggcgct acttcatctt cggcgggggc 1920

tacgtgtact tcgaggagta cgcgtactct caccagctga gtcgcgccga cgtcaccacc 1980

gtcagcacct tcatcgacct gaacatcacc atgctggagg accacgagtt tgtgcccctg 2040

gaggtctaca cgcgccacga gatcaaggac agcggcctgc tggactacac ggaggtccag 2100

cgccgcaacc agctgcacga cctgcgcttt gccgacatcg acacggtcat ccgcgccgac 2160

gccaacgccg ccatgttcgc ggggctgtgc gcgttcttcg aggggatggg ggacttgggg 2220

cgcgcggtcg gcaaggtcgt catgggagta gtggggggcg tggtgtcggc cgtctcgggc 2280

gtgtcctcct ttatgtccaa ccccttcggg gcgcttgccg tggggctgct ggtcctggcc 2340

ggcctggtcg cggccttctt cgccttccgc tacgtcctgc aactgcaacg caatcccatg 2400

aaggccctgt atccgctcac caccaaggaa ctcaagactt ccgaccccgg gggcgtgggc 2460

ggggaggggg aggaaggcgc ggaggggggc gggtttgacg aggccaagtt ggccgaggcc 2520

cgagaaatga tccgatatat ggctttggtg tcggccatgg agcgcacgga acacaaggcc 2580

agaaagaagg gcacgagcgc cctgctcagc tccaaggtca ccaacatggt tctgcgcaag 2640

cgcaacaaag ccaggtactc tccgctccac aacgaggacg aggccggaga cgaagacgag 2700

ctcggtggcg gtggctccgg cggtggtggg tccagaatga agcagatcga ggacaagatc 2760

gaggagatcc tgagcaagat ctaccacatc gagaacgaga tcgccagaat caagaagctg 2820

atcggcgaga gaggtggcgg tggctccggc ggtggtgggt ccggtgctaa tgatgcggaa 2880

gactgctgcc tgtctgtgac ccagcgcccc atccctggga acatcgtgaa agccttccgc 2940

taccttctta atgaagatgg ctgcagggtg cctgctgttg tgttcaccac actaaggggc 3000

tatcagctct gtgcacctcc agaccagccc tgggtggatc gcatcatccg aagactgaag 3060

aagtcttctg ccaagaacaa aggcaacagc accagaagga gccctgtgtc ttga 3114

<210> 7

<211> 7039

<212> DNA

<213> artificial sequence

<400> 7

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt 900

taagcttatg gggcgtttga cctccggcgt cgggacggcg gccctgctag ttgtcgcggt 960

gggactccgc gtcgtctgcg ccaaatacgc cttagcagac ccctcgctta agatggccga 1020

tcccaatcga tttcgcggga agaaccttcc ggttttggac cagctgaccg acccccccgg 1080

ggtgaagcgt gtttaccaca ttcagccgag cctggaggac ccgttccagc cccccagcat 1140

cccgatcact gtgtactacg cagtgctgga acgtgcctgc cgcagcgtgc tcctacatgc 1200

cccatcggag gccccccaga tcgtgcgcgg ggcttcggac gaggcccgaa agcacacgta 1260

caacctgacc atcgcctggt atcgcatggg agacaattgc gctatcccca tcacggttat 1320

ggaatacacc gagtgcccct acaacaagtc gttgggggtc tgccccatcc gaacgcagcc 1380

ccgctggagc tactatgaca gctttagcgc cgtcagcgag gataacctgg gattcctgat 1440

gcacgccccc gccttcgaga ccgcgggtac gtacctgcgg ctagtgaaga taaacgactg 1500

gacggagatc acacaattta tcctggagca ccgggcccgc gcctcctgca agtacgctct 1560

ccccctgcgc atccccccgg cagcgtgcct cacctcgaag gcctaccaac agggcgtgac 1620

ggtcgacagc atcgggatgt taccccgctt tatccccgaa aaccagcgca ccgtcgccct 1680

atacagctta aaaatcgccg ggtggcacgg ccccaagccc ccgtacacca gcaccctgct 1740

gccgccggag ctgtccgaca ccaccaacgc cacgcaaccc gaactcgttc cggaagaccc 1800

cgaggactcg gccctcttag aggatcccgc cgggacggtg tcttcgcaga tccccccaaa 1860

ctggcacatc ccgtcgatcc aggacgtcgc gccgcaccac gcccccgccg cccccagcaa 1920

cccgggcctg atcatcggcg cgctggccgg cagtaccctg gcggcgctgg tcatcggcgg 1980

tattgcgttt tgggtacgcc gccgcgctca gatggccccc aagcgcctac gtctccccca 2040

catccgggat gacgacgcgc ccccctcgca ccagccattg ttttacggat ccactagtcc 2100

agtgtggtgg ggtggcggtg gctccggcgg tggtgggtcc agaatgaagc agatcgagga 2160

caagatcgag gagatcctga gcaagatcta ccacatcgag aacgagatcg ccagaatcaa 2220

gaagctgatc ggcgagagag gtggcggtgg ctccggcggt ggtgggtccg gtgctaatga 2280

tgcggaagac tgctgcctgt ctgtgaccca gcgccccatc cctgggaaca tcgtgaaagc 2340

cttccgctac cttcttaatg aagatggctg cagggtgcct gctgttgtgt tcaccacact 2400

aaggggctat cagctctgtg cacctccaga ccagccctgg gtggatcgca tcatccgaag 2460

actgaagaag tcttctgcca agaacaaagg caacagcacc agaaggagcc ctgtgtcttg 2520

actcgagtct agagggccct tcgaacaaaa actcatctca gaagaggatc tgaatatgca 2580

taccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc 2640

ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 2700

tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag 2760

gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga 2820

caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag 2880

ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2940

ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 3000

tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 3060

gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 3120

gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 3180

ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 3240

ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 3300

tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg 3360

tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag 3420

tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 3480

catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 3540

ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 3600

gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 3660

ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag 3720

acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 3780

gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 3840

gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 3900

tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 3960

ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 4020

ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 4080

tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 4140

gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 4200

gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 4260

ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 4320

ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 4380

gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 4440

ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 4500

atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgcgaaat 4560

gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta 4620

tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 4680

ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta 4740

caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag 4800

ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag 4860

ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 4920

aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 4980

gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5040

gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 5100

ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 5160

atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 5220

gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 5280

gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 5340

gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 5400

gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 5460

aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 5520

ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 5580

taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 5640

tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 5700

gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 5760

taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 5820

tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 5880

tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 5940

ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 6000

taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 6060

tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 6120

cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 6180

gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 6240

cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 6300

ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 6360

aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 6420

atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 6480

tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 6540

gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 6600

aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 6660

acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 6720

ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 6780

tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 6840

aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 6900

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 6960

 

atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 7020

 

aaaagtgcca cctgacgtc 7039

 

 

Claims (10)

1. a bivalent DNA vaccine connection peptides, its sequence is for shown in SEQ ID NO.2.

2. connection peptides according to claim 1 is preparing the application in bivalent DNA vaccine, and described connection peptides is for connecting HSV-2gD or gB and adjuvant CCL19 or CCL28.

3. application according to claim 2, its application process is as follows:

(1) PCR method amplicon virus envelope glycoprotein gene and adjuvant genes, and introduce restriction enzyme site;

(2) enzyme cuts viral envelope glycoprotein gene and adjuvant genes PCR primer, is connected into carrier for expression of eukaryon in order, then is amplified (G4S) in the middle of viral envelope glycoprotein gene and adjuvant genes by over-lap PCR method ₂-IZ-(G4S) ₂sequence;

(3) connect product conversion to E.coli DH5 α competence bacterial strain, picking list bacterium colony enlarged culturing, extract double digestion checking after recombinant plasmid, the recombinant plasmid correct through sequence verification sequence and get final product.

4. a bivalent DNA vaccine for restructuring, is the recombinant plasmid containing nucleotide sequence shown in SEQ ID NO.3.

5. bivalent DNA vaccine according to claim 4, its nucleotides sequence is classified as shown in SEQ ID NO.7.

6. a bivalent DNA vaccine for restructuring, is the recombinant plasmid containing nucleotide sequence shown in SEQ ID NO.4.

7. a bivalent DNA vaccine for restructuring, is the recombinant plasmid containing nucleotide sequence shown in SEQ ID NO.5.

8. a bivalent DNA vaccine for restructuring, is the recombinant plasmid containing nucleotide sequence shown in SEQ ID NO.6.

9., according to the bivalent DNA vaccine of any one restructuring described in claim 4 or 6 or 7 or 8, it is characterized in that, described plasmid is pcDNA3.1 (+).

10. the application of bivalent DNA vaccine in preparation intramuscular injection type vaccine of the restructuring according to any one of claim 4-8.