CN102604898B - VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope - Google Patents

Abstract

The invention relates to VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope and application thereof, belonging to the field of biotechnology. The recombinant baculovirus is obtained by the following steps of: performing PCR (polymerase chain reaction) amplification on the rabbit hemorrhagic disease virus capsid protein VP60 (without initiator codon) gene; cloning the PCR amplification product into a pMD19-T vector and connecting into an eukaryon transfer vector; inserting the foot and mouth disease virus B cell epitope (200-213aa) to construct a baculovirus vector; and performing transfecting the Sf9 cell. The invention provides rabbit hemorrhagic disease virus capsid protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope and rabbit hemorrhagic disease virus-like particles carrying type O foot and mouth disease virus B cell epitope (chimeric protein), wherein the chimeric protein is used as antigen to immunize mice, a positive antibody against type O foot and mouth disease virus VP1 protein can be induced and generated, and therefore, the chimeric protein can be used as antigen preventing and controlling the type O foot and mouth disease vaccine.

Description

Carry the VP60 Protein reconstitution baculovirus of O type foot and mouth disease virus B cell epitope

Technical field

The present invention relates to carry rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus and the application thereof of O type foot and mouth disease virus B cell epitope, belong to biological technical field.

Background technology

Virus-like particle (virus-likeparticles, VLPs) one or more virus structural proteins by multiple copied are polymerized, mostly be shaft-like or icosahedron, diameter is greatly between 25～100nm, and its structure all keeps identical with the live virus particle with antigenicity.VLPs is formed by one or more structural protein polies, and therefore, its surface antigen is demonstrated with the mode repeated in order with a kind of, and this displaying can be induced high-caliber immunne response fast significantly.In addition, VLPs is of moderate size, and can effectively by the identification of the antigen presenting cells such as dendritic cell, be caught, thereby induce more effective immunne response.The easy purifying of VLPs, can pass through density gradient centrifugation or size chromatography purification.Based on these advantages, VLPs has become the focus of vaccine research.Rabbit haemorrhagic disease (Rabbit Hemorrhagic Disease, RHD) is the communicable disease of the rabbit that caused by rabbit hemorrhagic disease virus (RHDV), and the course of disease is short, mortality ratio is high, is the rabbit deadly infectious disease.VP60 albumen is the main structural protein of RHDV, can induce the generation neutralizing antibody in animal body, is the virus immunity protective antigen.Research finds not have under the condition of other any compositions, the VP60 capsid protein can naturally aggregate in vitro do not wrap up nucleic acid, with natural RHDV particle similar VLPs on physical aspect.This VLPs has good immunogenicity, and itself just can be used as immunological adjuvant, can enter antigen presenting cell with endocytosis or pinocytosis approach, induces respectively humoral immunoresponse(HI) and cellullar immunologic response.Research shows, but this virus-like particle chimeric expression foreign gene as vaccine carrier, this just make RHDV VP60 as other Animal diseases even the VLPs vaccine carrier of human diseases become possibility.

Foot and mouth disease (foot-and-mouth disease virus, FMD) is that the domestic animals such as the ox that caused by foot and mouth disease virus (FMDV), sheep, pig are acute, hot, the height contagious disease.Because it is propagated soon, not only to livestock industry, cause huge financial loss, and had a strong impact on international trade, OIE classifies it as one of epidemic disease that must report.FMD serotype is numerous, comprises O, A, C, Asia1, SAT1, SAT2 and SAT3 type totally 7 serotypes, between serotype, without cross protection, controls difficulty larger.Within 2003, European Union has revised FMD prevention and control strategy, has affirmed the effect of vaccine immunity when FMD breaks out, subsequently a plurality of countries of European Union by vaccine immunity as a supplement means for the control of FMD.Inactivated vaccine has been brought into play important effect in the anti-system of the FMD on the ground such as America and Europe with in eradicating, until today, inactivated vaccine remains successfully the important measures of prevention and control FMD.But weak point is also arranged: for effective immunity, the antigenic content of vaccine will guarantee, the biological safety protection rank is higher, and deactivation not exclusively may cause loose poison, and it is strong etc. that vaccine exists virus virulence to return, and in addition, the production cost of inactivated vaccine is higher.Accelerating Vaccine for hoof-and-mouth disease development research safely and effectively, is very necessary for successfully preventing, control and even finally eliminate foot and mouth disease.Along with the development of biotechnology, recombinant vaccine is following developing direction, and has obtained certain achievement.Research shows, O type FMDV VP1 has 5 B cell epitopes at least, wherein a most important epi-position is comprised of 141～160 (G-H ring) and C end 200～213 amino acids residue linear epitopes of VP1, is most important B cell epitope, is also the most important antigen site of FMDV.The scientific research personnel of Fudan University, Shanghai animal and veterinary institute has invented a kind of livestock foot-and-mouth disease viral polypeptide vaccine.This polypeptide vaccine is with the little peptide group of immunogenic 141～160 amino acids and 200～213 amino acids is arranged in chemical process composite coding VP1 albumen, by its series connection, insert again on the plasmid vector macromole and proceed to bacterial expression, obtain by fermentation the foot-and-mouth disease virus resistant vaccine, play important effect in the prevention of China's foot and mouth disease.Our seminar inserts respectively single external source B cell epitope (FMDV VP1B cell epitope 200～213aa) and two-in-series B cell epitope (FMDV VP1(141～160aa)-GS-(200～213aa at the different positions of RHDV capsid protein VP60 gene)), build 6 kinds of recombinant transfer vectors and expressed in the Sf9 cell, by electron microscopic observation and experimentation on animals, research adds these external source fragments to the assembling of VLPs and the immunogenicity of external source fragment, found that, with other insertion points, compare, insert single external source B cell epitope (FMDV VP1B cell epitope 200～213aa) at the N-of RHDV capsid protein VP60 gene end, the chimeric protein of expression of recombinant virus can form VLPs, show that capsid protein VP60 itself has vaccine carrier character, can in the situation that not change structure carry the external source fragment, chimeric protein has identical with VP60 albumen to people " O " red cell agglutination characteristic, be easy to vaccine antigen quantitatively with detect, experimentation on animals demonstration chimeric protein can be induced and be produced resisting O-type foot and mouth disease virus VP1 protein positive antibody in Mice Body.

Summary of the invention

Technical problem the objective of the invention is to build recombination bacillary viral vector by Protocols in Molecular Biology, and the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus that carries foot and mouth disease virus B cell epitope of a kind of good immune effect, simple process and the preparation method of virus like particle vaccine are provided.

Technical scheme

Carry the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus of O type foot and mouth disease virus B cell epitope, build and form by following methods: with plasmid pFastBac ^tM1-VP60 is template, pcr amplification goes out rabbit hemorrhagic disease virus capsid protein VP60 gene fragment, it is cloned into to transfer vector, again foot and mouth disease virus FMDV VP1B cell epitope 200～213aa is inserted to the correct position of VP60 gene fragment in transfer vector, then the E.coliDH10Bac competent cell that recombinant plasmid transformed is contained to shuttle vectors Bacmid, transfection Sf9 cell, obtain the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus rAcV-Bac-NF that carries O type foot and mouth disease virus B cell epitope.

The rabbit hemorrhagic disease virus like-particles chimeric protein of the O type that the carries foot and mouth disease virus B cell epitope prepared with described recombinant baculovirus rAcV-Bac-NF.Its method is: the described recombinant baculovirus of claim 1 is inoculated in to the Sf9 cell, and the harvested cell culture, be the rabbit hemorrhagic disease virus like-particles chimeric protein that carries O type foot and mouth disease virus B cell epitope.

Described recombinant baculovirus rAcV-Bac-NF or the rabbit hemorrhagic disease virus like-particles chimeric protein that carries O type foot and mouth disease virus B cell epitope are applied in preparation prevention, diagnosis or treatment foot and mouth disease medicine.

The rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope prepared with described recombinant baculovirus rAcV-Bac-NF, its preparation method is: cultivate the Sf9 cell to logarithmic phase, the described recombinant baculovirus of inoculation claim 1, there are after pathology 5 days and receive poison in cell, with PH7.4 sterilizing phosphate buffer soln, PBS is resuspended, in antigen: freund's adjuvant volume 1:1 ratio adds adjuvant, mix, be the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope.

O type foot and mouth disease virus B cell epitope inserts in rabbit hemorrhagic disease virus capsid protein VP60, do not change the biological activity of capsid protein VP60, be the red cell agglutination characteristic, chimeric protein has the red cell agglutination characteristic equally, and the titration of antigen blood clotting is carried out in available hemagglutination test (HA test) (HA).

The beneficial effect the features and advantages of the invention are as follows: the present invention chooses rabbit hemorrhagic disease virus capsid protein VP60 as the carrier of showing external source foot and mouth disease virus B cell epitope, reason is with rhabdovirus system expression VP60 albumen, recombinant protein is not having under the condition of other any compositions, can naturally aggregate in vitro and not wrap up nucleic acid, with natural RHDV virus particle similar virus-like particle (virus-likeparticles on physical aspect, VLPs), this VLPs is comprised of 180 identical subunits of structure, each subunit all shows external source foot and mouth disease virus B cell epitope, different from other display systems, a virus like particle can be showed 180 foot and mouth disease virus B cell epitopes, virtually increased the copy number of antigen effective constituent, be conducive to the raising of vaccine immunity effect, VP60 itself also can be used as immunological adjuvant simultaneously, this VLPs lacks genetic information and loses replication, and recombinant baculovirus only infects non-vertebrates simultaneously, does not infect the mankind and other animals.Therefore, with traditional aftosa vaccine, compare, utilize the virus like particle of this system expression to prepare vaccine and there is good biological safety.Eukaryotic expression system has the posttranslational modification function that is similar to mammalian cell, makes recombinant capsid protein more approach native protein on structure and function, the result demonstration of test, and the albumen of expression has the biological activity identical with rabbit hemorrhagic disease virus; The chimeric protein of expressing just can be used for the vaccine preparation without the special processing purifying, easy and simple to handle.

Evidence, the chimeric protein self assembly that the present invention expresses becomes the ability of virus like particle good, shows that capsid protein VP60 itself has vaccine carrier character, can in the situation that not change structure carry the external source fragment; Chimeric protein has identical with VP60 albumen to people " O " type red cell agglutination characteristic, be easy to vaccine antigen quantitatively with detect; The virus like particle vaccine immune mouse made from chimeric protein can be induced and be produced resisting O-type foot and mouth disease virus VP1 protein positive antibody in Mice Body.Illustrate that the present invention can provide good foot and mouth disease virus vaccine antigen, there is immune efficacy preferably, possess good biological safety simultaneously, there is important using value aspect the foot and mouth disease prevention and control.

The accompanying drawing explanation

Fig. 1 VP60 gene fragment pcr amplification

M:DNA molecular mass standard (DL2000); 1.VP60 amplified production; 2. negative control

Fig. 2 T/A clone's enzyme is cut evaluation

M1.DNA molecular mass standard (DL15000); M2.DNA molecular mass standard (DL2000); 1.pMD19-T-VP60 double digestion product (Xba I/Hind III)

Fig. 3 recombinant transfer vector pFastBac ^tMthe enzyme of HTA-VP60 is cut evaluation

M1.DNA molecular mass standard (DL15000; M2.DNA molecular mass standard (DL2000); 1.pFastBac ^tMhTA-VP60 double digestion product (Xba I/Hind III)

Fig. 4 recombinant transfer vector pFastBac ^tMthe PCR of HTA-NF identifies

M.DNA molecular mass standard (DL2000); 1.pFastBac ^tMhTA-NF; 2. negative control

The PCR of Fig. 5 recombinant shuttle vector Bacmid-NF identifies

M1.DNA molecular mass standard (DL15000); M2.DNA molecular mass standard (DL2000); 1.Bacmid (negative control) PCR identifies; 2,3,4 recombinant shuttle vector Bacmid-NF

NF gene RT-PCR result in Fig. 6 recombinant baculovirus rAcV-Bac-NF

1. blank; 2. negative control; 3. infect the cell of recombinant baculovirus rAcV-Bac-NF; M.DNA molecular weight standard (DL-2000)

Fig. 7 indirect immunofluorescene assay carrier specificity

A. infect the cell of rAcV-Bac-VP60; B. blank; C. negative control; D. infect the cell of rAcV-Bac-NF

Fig. 8 indirect immunofluorescene assay epitope specificity

A. infect the cell of rAcV-Bac-VP60; B. blank; C. negative control; D. infect the cell of rAcV-Bac-NF

Fig. 9 immunoblotting detects carrier specificity

A:SDS-PAGE，1.AcV-Bac；2.rAcV-Bac-VP60；3.rAcV-Bac-NF

B:Western blot，1.AcV-Bac；2.rAcV-Bac-VP60；3.rAcV-Bac-NF

Figure 10 immunoblotting detects epitope specificity

A:SDS-PAGE，1.AcV-Bac；2.rAcV-Bac-VP60；3.rAcV-Bac-NF

B:Western blot，1.AcV-Bac；2.rAcV-Bac-VP60；3.rAcV-Bac-NF

Figure 11 electron microscopic observation result

A.RHDV-VP60-VLPs，B.NF-VLPs

Figure 12 blood clotting result

A.RHDV-VP60-VLPs，B.NF-VLPs

Epi-position ELISA antibody horizontal in Figure 13 mice serum

FMDV.O type foot and mouth disease virus inactivated vaccine (positive control); PBS. negative control; NF. carry the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine of foot and mouth disease virus B cell epitope

Embodiment

The present invention is without initiator codon by pcr amplification rabbit hemorrhagic disease virus capsid protein VP60() gene, pcr amplification product is cloned into to the donor plasmid pFastBac of baculovirus expression system ^tMin HTA, obtain recombinant transfer vector pFastBac ^tMhTA-VP60, and then foot and mouth disease virus B cell epitope (200～213aa) is inserted to pFastBac ^tMthe appropriate location of HTA-VP60, obtain recombinant transfer vector pFastBac ^tMhTA-NF, transform the E.coli DH10Bac that contains shuttle plasmid Bacmid with this carrier, through screening, obtain recombinant baculovirus plasmid Bacmid-NF, and transfection Sf9 cell, obtain recombinant baculovirus rAcV-Bac-NF.With recombinate shape virus infection Sf9 cell, mosaic gene to be expressed, then using the chimeric protein of recombinant baculovirus expression as antigen, make the rabbit hemorrhagic disease virus like-particles vaccine that carries foot and mouth disease virus B cell epitope.

The step of the concrete technological line that the present invention adopts:

(1) structure of recombinant baculovirus plasmid Bacmid-NF

1. following primer is synthesized in design:

Underscore mark place is restriction enzyme site

2. synthesize FMDV B cell epitope:

FMDV VP1B cell epitope (200～213aa:RHKQEIVAPVKQKL, the gene order that it is corresponding:

5 '-CGTCACAAACAGGAAATCGTAGCTCCAGTAAAACAGAAGTTG-3 ') synthetic by invitrogen company.

3.VP60 the amplification of gene:

With plasmid pFastBac ^tM1-VP60(Wang Fang etc.; the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit; journal of animal science and veterinary medicine; 2008; 39 (10); 1382～1387) be template, take VP60-F/VP60-R as primer, pcr amplification goes out VP60 gene (without initiator codon) fragment.The PCR25ul reaction system of pcr amplification VP60 is as follows: pre-mixed PCR damping fluid 2 * (Mix) 12.5 μ l, template 0.5 μ l, upstream primer VP60-F0.5 μ l, downstream primer VP60-R, ddH ₂o11 μ l, cumulative volume 25 μ l, moment centrifugal mixing, adopt the heat lid to carry out pcr amplification reaction: 94 ℃ of sex change 5min, 94 ℃ of sex change 1min, 52.6 ℃ (58.1 ℃, 52.6 ℃ and 59.4 ℃) annealing 1min, 72 ℃ are extended 2min, after 30 circulations, 72 ℃ are extended 10min, finish reaction; The PCR product is carried out to agarose gel electrophoresis and identify that the capsid protein VP60 gene amplification segment size of acquisition is 1737bp(Fig. 1).

4. the structure of recombinant baculovirus plasmid Bacmid-NF:

4.1 recombinant transfer vector pFastBac ^tMthe structure of HTA-VP60 first is cloned into cloning vector pMD19-T(purchased from Dalian precious biotechnology company limited by the VP60 gene of amplification), cut evaluation (Fig. 2) with XbaI/Hind III enzyme, then be cloned into transfer vector pFastBac ^tMhTA(is purchased from Invitrogen company) in, cut evaluation with XbaI/Hind III enzyme, obtain recombinant transfer vector pFastBac ^tMhTA-VP60(Fig. 3).

4.2 the preparation FMDV-F/FMDV-R of the FMDV VP1B cell epitope double-stranded DNA of phosphorylation band cohesive terminus forms double-stranded after anneal, concrete operations are 95 ℃ of insulation 10min in the PCR instrument; 80 ℃ of 10min, 65 ℃ of 10min, 50 ℃ of 10min, 37 ℃ of 10min, 25 ℃ of 10min.

FMDV VP1B cell epitope double-stranded DNA phosphorylation, 50 μ l systems are as follows:

4.3 recombinant transfer vector pFastBac ^tMthe structure of HTA-NF is by recombinant transfer vector pFastBac ^tMhTA-VP60 cuts digestion with the EcoRI/XbaI enzyme, reclaim carrier segments, then with T4DNA Ligase, with the FMDV VP1B cell epitope double-stranded DNA of annealing phosphatizing treatment, be connected, transform DH5 α competent cell, carry out PCR evaluation order-checking with the Pn1/Pn2 primer, obtain recombinant transfer vector pFastBac ^tMhTA-NF(Fig. 4).Recombinant transfer vector pFastBac ^tMthe FMDV VP1B cell epitope double-stranded DNA linked system of HTA-VP60 and annealing phosphatizing treatment is as follows:

The PCR evaluation, PCR25 μ l reaction system is as follows:

Low-speed centrifugal 10s mixes, and adopts heat lid PCR to carry out cyclic amplification.

PCR identification reaction parameter is as follows:

4.4 the structure of recombinant baculovirus plasmid Bacmid-NF is by recombinant transfer vector pFastBac ^tMhTA-NF transforms the E.coliDH10Bac competent cell (purchased from Invitrogen company) that contains shuttle vectors Bacmid, converted product is applied to and contains three kinds of microbiotic (50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins), X-gal(100 μ g/ml) and IPTG(20 μ g/ml) the LB flat board, after 37 ℃ of cultivation 48h, select white colony and cultivate and extract plasmid, as template, with general pUC/M13 upstream primer and specificity downstream primer VP60-R(Fig. 5 the 2nd swimming lane), specificity upstream primer VP60-F and pUC/M13 downstream primer (Fig. 5 the 3rd swimming lane), specificity upstream primer VP60-F and downstream primer VP60-R(Fig. 5 the 4th swimming lane) carry out the PCR evaluation, simultaneously with on pUC/M13, downstream primer is about 300bp(Fig. 5 the 1st swimming lane to the amplified production size of the empty Bacmid plasmid that do not insert exogenous genetic fragment), the success of proof swivel base, positive recombinant plasmid called after Bacmid-NF.

(20 μ l) is as follows for the PCR reaction system:

The PCR cycling condition is: 95 ℃ of denaturation 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min.

(2) transfectional cell, obtain recombinant baculovirus

1. transfectional cell and purifying

Take LipofectaminTM2000(purchased from Invitrogen company) as cotransfection reagent, according to explanation, by recombinant shuttle vector plasmid Bacmid-NF transfection Sf9 monolayer cell.After transfection, every 12h observes once, until cytopathy is when obvious, collecting cell and supernatant liquor, as recombinant baculovirus stoste-4 ℃ preservation.Will be containing the recombinant baculovirus called after rAcV-Bac-NF of goal gene.By going down to posterity with 1% volume ratio inoculation Sf9 cell (purchased from Invitrogen company) after twice of f1 disease venom freeze thawing, obtain the 2nd generation recombinant virus.

2. identify

2.1RT-PCR identify

Collect the cell that infects recombinant virus, extract cell RNA, with VP60-F/VP60-R, carry out the RT-PCR evaluation, result shows that electrophoretic band is 1788bp(Fig. 6 the 3rd swimming lane), with the predicted gene in the same size.The cell that the viral AcV-Bac that the empty Bacmid transfection Sf9 cell that extracts from E.coliDH10Bac of simultaneously usining obtains infects, as negative control (Fig. 6 the 2nd swimming lane), is usingd normal Sf9 cell as blank (Fig. 6 the 1st swimming lane), band do not occur.

Reverse transcription concrete steps reference molecule cloning experimentation guide carries out, and the RT reaction system is as follows:

Then carry out following reaction conditions: 30 ℃ of reaction 10min, 42 ℃ of reaction 30min, 99 ℃ of reaction 5min, finish reaction.-20 ℃ of storages are standby.

Then carry out the PCR program, reaction system is as follows:

Then carry out following reaction conditions: 94 ℃ of denaturation 3min; 94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min, finish reaction.

2.2 the evaluation of expression product

2.2.1 indirect immunofluorescene assay carrier specificity recombinant virus rAcV-Bac-NF infects the Sf9 cell, the cell that the viral AcV-Bac that the empty Bacmid transfection Sf9 cell that extracts from E.coli DH10Bac of simultaneously usining obtains infects, as negative control, is usingd normal Sf9 cell as blank.After infecting Sf9 cell 24h, the dilution RHDV monoclonal antibody of the 1:40 of take A3C is primary antibodie, and the dilution fluorescent mark goat anti-rabbit igg of 1:100 is two anti-, carries out indirect IF staining.Result shows, infect the Sf9 cell (Fig. 7 D) of recombinant baculovirus rAcV-Bac-NF, recombinant baculovirus rAcV-Bac-VP60 (the Wang Fang etc. that infection contains the RHDVVP60 gene, the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit, journal of animal science and veterinary medicine, 2008, 39 (10), 1382～1387) cell (Fig. 7 A) has very strong specificity fluorescent, and blank (Fig. 7 B), negative control (Fig. 7 C) is interior without special fluorescence, illustrate that in recombinant virus rAcV-Bac-VP60, carrier VP60 has obtained expression, and expression product is present in insect cell.

2.2.2 the indirect immunofluorescene assay epitope specificity infects the Sf9 cell with recombinant virus rAcV-Bac-NF, the cell that the viral AcV-Bac that the empty Bacmid transfection Sf9 cell that extracts from E.coli DH10Bac of simultaneously usining obtains infects, as negative control, is usingd normal Sf9 cell as blank.Infecting the Sf9 cell after 24 hours, the dilution ox of the 1:40 of take " O " type FMDV polyvalent antibody is primary antibodie, and the dilution fluorescent mark goat-anti of 1:5000 ox IgG is two anti-, carries out indirect IF staining.Result shows, the Sf9 cell (Fig. 8 D) that infects recombinant baculovirus rAcV-Bac-NF has very strong specificity fluorescent, and blank (Fig. 8 B), negative control (Fig. 8 C), recombinant baculovirus rAcV-Bac-VP60 (the Wang Fang etc. that infection contains RHDV VP60 gene, the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit, journal of animal science and veterinary medicine, 2008, 39 (10), 1382～1387) cell (Fig. 8 A) is interior without special fluorescence, illustrate that in recombinant virus rAcV-Bac-NF, FMDV VP1B cell epitope has obtained expression, and expression product is present in insect cell.

2.2.3 immunoblotting detects carrier specificity and collects the cell culture that infects recombinant virus, adds sample-loading buffer to carry out protein electrophorese (SDS-PAGE) after freeze thawing treatment; With VP60 monoclonal antibody A3C(number of patent application: 200910029627.9; Publication number: CN101519447; Title: anti-rabbit hemorrhagic disease virus VP 60 albumen monoclonal antibody) be primary antibodie, 1:40 dilution, the goat anti-rabbit igg of HRP mark (biological brilliant U.S. company) is two anti-, immunoblotting (Westernblot) is carried out in the 1:5000 dilution.The SDS-PAGE result shows, infect the Sf9 cell (Fig. 9 A:3) of recombinant baculovirus rAcV-Bac-NF, recombinant baculovirus rAcV-Bac-VP60 (the Wang Fang etc. that infection contains RHDV VP60 gene, the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit, journal of animal science and veterinary medicine, 2008, 39 (10), 1382～1387) cell (Fig. 9 A:2) specific band that is about 60kDa all occurs after expressing, with the expection in the same size, Sf9(Fig. 9 A:1 of infection AcV-Bac) specific band of 60kDa does not appear being about after expression, Western blot result shows, infect the Sf9 cell (Fig. 9 B:3) of recombinant baculovirus rAcV-Bac-NF, recombinant baculovirus rAcV-Bac-VP60 (the Wang Fang etc. that infection contains RHDV VP60 gene, the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit, journal of animal science and veterinary medicine, 2008, 39 (10), 1382～1387) cell (Fig. 9 B:2) specific band occurs in the 60kDa left and right, with the expection in the same size, infect Sf9(Fig. 9 B:1 of AcV-Bac) in corresponding position, band does not appear, illustrate that in recombinant virus rAcV-Bac-NF, carrier VP60 has obtained expression.

2.2.4 immunoblotting detects epitope specificity and collects the cell culture that infects recombinant virus, adds sample-loading buffer to carry out protein electrophorese (SDS-PAGE) after freeze thawing treatment; Niu Yuan " O " the type FMDV polyvalent antibody of take is primary antibodie, 1:40 dilution, and the goat-anti ox IgG of HPR mark is two anti-, immunoblotting (Western blot) is carried out in the 1:5000 dilution.The SDS-PAGE result is identical with Fig. 9 A, and Western blot result shows, infects the Sf9 cell (Figure 10 B:3) of recombinant baculovirus rAcV-Bac-NF at a specific band of 60kDa left and right appearance, with the expection in the same size; Recombinant baculovirus rAcV-Bac-VP60 (the Wang Fang etc. that infection contains RHDV VP60 gene; the expression of rabbit hemorrhagic disease virus capsid protein in insect cell reaches the immune protective effect to rabbit; journal of animal science and veterinary medicine; 2008; 39 (10); 1382～1387) Sf9(Figure 10 B:1 of cell (Figure 10 B:2) and infection AcV-Bac) in corresponding position, band do not occur, illustrate that in recombinant virus rAcV-Bac-NF, FMDV VP1B cell epitope has obtained expression.

(3) electron microscopic observation of chimeric protein

Collect the cell culture that infects recombinant baculovirus rAcV-Bac-NF, multigelation, centrifugal removal cell debris, supernatant is standby as chimeric protein, by chimeric protein sample drop to be checked on the load sample copper mesh, absorption 2min, suck unnecessary sample with filter paper, then drips 2% phospho-wolframic acid dye liquor on copper mesh, fixing 2min, finally remove unnecessary phospho-wolframic acid dye liquor, drying at room temperature 5min is observed on H-7650 type transmission electron microscope.Result shows, the chimeric protein that recombinant baculovirus rAcV-Bac-NF expresses can form virus like particle (Figure 11 B), and size is about 30～40nm, be similar to RHDV VLPs(Figure 11 A of the recombinant baculovirus rAcV-Bac-VP60 expression that contains RHDV VP60 gene), illustrate that its self assembly ability is good.

(4) the blood clotting characteristic of chimeric protein

Collect the cell culture that infects recombinant baculovirus rAcV-Bac-NF, multigelation, centrifugal removal cell debris, supernatant is standby as chimeric protein, U-shaped blood-coagulation-board 1st～10 holes respectively add PBS liquid 50 μ l, then draw chimeric protein 50 μ l and add the 1st hole to mix, and draw mixing liquid 50 μ l from the 1st hole and add the 2nd hole, doubling dilution to the 10 holes successively, the 10th hole is drawn 50 μ l and is discarded.Every hole adds 50 μ l1% people " O " type red corpuscle, and after vibration mixes, the RHDV VLPs simultaneously expressed with the recombinant baculovirus rAcV-Bac-VP60 that contains RHDV VP60 gene compares, observations after 4 ℃ of standing 40min.The result demonstration, RHDV VLPs hemagglutinative titer is 2 ⁹(Figure 12 A), the chimeric protein hemagglutinative titer is 2 ⁶(Figure 12 B), illustrate that O type foot and mouth disease virus B cell epitope inserts in rabbit hemorrhagic disease virus capsid protein VP60, do not change the biological activity of capsid protein VP60, it is the red cell agglutination characteristic, chimeric protein has the red cell agglutination characteristic equally, the titration of antigen blood clotting is carried out in available hemagglutination test (HA test) (HA), is quantitatively laying a good foundation of chimeric protein antigen.

(5) in vaccine preparation and Mice Body, immunogenicity detects

Cultivate the Sf9 cell to logarithmic phase, inoculate corresponding recombinant baculovirus rAcV-Bac-NF, there are after pathology 5 days and receive poison in cell, with sterilizing phosphate buffer soln (PBS, PH7.4) resuspended, in antigen: freund's adjuvant volume 1:1 ratio adds adjuvant, mixes, and is the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope.

By above-mentioned virus like particle vaccine subcutaneous inoculation ICR mouse (immunity 3 times), and establish control group, exempt from the rear the 0th respectively at head, 1, 2, 3, 4, within 5 and 6 weeks, gather mice serum, indirect ELISA (O type FMDV ELISA detection kit purchased from Wuhan section before biological products limited liability company) detects the epitope antibodies level, result shows, carry the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine of foot and mouth disease virus B cell epitope and can induce generation resisting O-type foot and mouth disease virus VP1 protein positive antibody (Figure 13, NF), positive control O type foot and mouth disease virus inactivated vaccine can be induced the higher resisting O-type foot and mouth disease virus VP1 protein positive antibody (Figure 13 of generation, FMDV), negative control PBS does not produce resisting O-type foot and mouth disease virus VP1 protein positive antibody (Figure 13, PBS).Illustrate that the present invention has immune efficacy preferably in actual applications, thering is important using value aspect the foot and mouth disease prevention and control, possess good biological safety simultaneously.

SEQUENCE LISTING

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<400> 3

atgcgtcaca aacaggaa 18

<210> 4

<211> 18

<212> DNA

<213> 1

<220>

<221> Pn2

<222> (1)..(18)

<223>

<400> 4

ctacaccagg atccatgc 18

<210> 5

<211> 41

<212> DNA

<213>

<220>

<221 > FMDV VP1 B cell epitope (200～213 aa)

<222> (1)..(41)

<223>

<400> 5

cgtcacaaac aggaaatcgt agctccagta aaacagaagt t 41

<210> 6

<211> 51

<212> DNA

<213>

<220>

<221> FMDV-F：

<222> (1)..(51)

<223>

<400> 6

ctagacgtca caaacaggaa atcgtagctc cagtaaaaca gaagttgtga a 51

<210> 7

<211> 51

<212> DNA

<213>

<220>

<221> FMDV-R：

<222> (1)..(51)

<223>

<400> 7

tgcagtgttt gtcctttagc atcgaggtca ttttgtcttc aacactttcg a 51

Claims (2)

1. carry the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus of O type foot and mouth disease virus B cell epitope, build and form by following methods: with plasmid pFastBac ^tM1-VP60 is template, and pcr amplification goes out rabbit hemorrhagic disease virus capsid protein VP60 gene fragment, and it is cloned into to transfer vector, at RHDV capsid protein VP60 gene the N-endinsert the encoding gene of single foot and mouth disease virus FMDV VP1 B cell epitope 200～213aa, FMDV VP1 B cell epitope 200～213 aa:RHKQEIVAPVKQKL, the gene order that it is corresponding: 5 '-cgtcacaaacaggaaatcgtagctccagtaaaacagaagttG-3 '; Then recombinant plasmid transformed is contained to shuttle vectors Bacmid's e. colidH10Bac competent cell, then transfection Sf9 cell, obtain the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus rAcV-Bac-NF that carries O type foot and mouth disease virus B cell epitope.

2. the rabbit hemorrhagic disease virus like-particles chimeric protein of the O type that the carries foot and mouth disease virus B cell epitope prepared with the described recombinant baculovirus of claim 1.

The method of 3, with the described recombinant baculovirus preparation of claim 1, carrying the rabbit hemorrhagic disease virus like-particles chimeric protein of O type foot and mouth disease virus B cell epitope, be characterised in that: the described recombinant baculovirus of claim 1 is inoculated in to the Sf9 cell, the harvested cell culture, be the rabbit hemorrhagic disease virus like-particles chimeric protein that carries O type foot and mouth disease virus B cell epitope.

4, the purposes of the described recombinant baculovirus of claim 1 in preparation prevention, diagnosis or treatment foot and mouth disease medicine.

5, the described purposes of rabbit hemorrhagic disease virus like-particles chimeric protein in preparation prevention, diagnosis or treatment foot and mouth disease medicine of carrying O type foot and mouth disease virus B cell epitope of claim 2 or 3.

6, the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope prepared with the described recombinant baculovirus of claim 1.

7, the method for preparing vaccine with the described recombinant baculovirus of claim 1, be characterised in that: cultivate the Sf9 cell to logarithmic phase, the described recombinant baculovirus of inoculation claim 1, there are after pathology 5 days and receive poison in cell, resuspended with PH 7.4 sterilizing phosphate buffer soln PBS, in antigen: freund's adjuvant volume 1:1 ratio adds adjuvant, mixes, and is the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope.

8, measure the described method of carrying the rabbit hemorrhagic disease virus like-particles chimeric protein antigen hemagglutinative titer of O type foot and mouth disease virus B cell epitope of claim 2 or 3 with hemagglutination test (HA test).

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