CN102604898A - VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope - Google Patents
VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope Download PDFInfo
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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
Technical field
The present invention relates to carry the 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-like particles; VLPs) one or more virus structural proteins by multiple copied are polymerized; Mostly be shaft-like or icosahedron, between 25～100nm, its structure all keeps identical with the live virus particle with antigenicity to diameter greatly.VLPs is formed by one or more structural protein polies, therefore, its surface antigen with a kind of in order and the multiple mode showed that this displaying can be induced high-caliber immunne response fast significantly.In addition, VLPs is of moderate size, and can effectively be caught by the identification of antigen presenting cells such as BMDC, thereby induce more effective immunne response.VLPs is prone to purifying, can pass through density gradient centrifugation or size chromatography purification.Based on these advantages, VLPs has become the focus of vaccine research.(Rabbit Hemorrhagic Disease is the communicable disease of the rabbit that caused by rabbit hemorrhagic disease virus (RHDV) RHD) to rabbit haemorrhagic disease, 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.Discover under the condition that does not have other any compositions to exist, the VP60 capsid protein external can aggregate into naturally 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 get into antigen presenting cell with endocytosis or pinocytosis approach, induces HI and cellullar immunologic response respectively.Research shows, but this virus-like particle chimeric expression foreign gene and as vaccine carrier, and this just makes RHDV VP60 become possibility as the VLPs vaccine carrier of other Animal diseases even human diseases.
(foot-and-mouth disease virus FMD) is acute, hot, the height contagious diseases of domestic animal such as the ox that caused by foot and mouth disease virus (FMDV), sheep, pig to foot and mouth disease.Because it propagates fast, has not only caused enormous economic loss to livestock industry, and has had a strong impact on international trade, OIE classifies it as one of eqpidemic disease that must report.FMD serotype is numerous, comprises O, A, C, Asia1, SAT1, SAT2 and SAT 3 types totally 7 serotypes, does not have cross protection between serotype, and the control difficulty is bigger.European Union had revised FMD prevention and control strategy in 2003, had affirmed the effect of vaccine immunity when FMD breaks out, and a plurality of countries of European Union are used for vaccine immunity the control of FMD as the means of supplementing out economy subsequently.Inactivated vaccine has been brought into play important effect in the anti-system of the FMD on ground such as America and Europe with in eradicating, and up to today, inactivated vaccine remains successfully the important measures of prevention and control FMD.But weak point is arranged also: for effective immunity, the antigenic content of vaccine will guarantee that the Biosafety protection level is higher, and it is strong etc. that the deactivation poison that not exclusively possibly cause loosing, vaccine exist virus virulence to return, and in addition, the production cost of inactivated vaccine is higher.Accelerating novel safely and effectively aftosa vaccine development research, is very necessary for successfully preventing, control and even finally eliminate foot and mouth disease.Along with development of biology, recombinant vaccine is the development in future direction, and has obtained certain achievement.Research shows; O type FMDV VP1 has 5 B cell epitopes at least; A wherein most important epi-position is made up of 141～160 (G-H ring) and C end 200～213 amino acids residue linear epitopes of VP1, is most important B cell epitope, also is 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 that immunogenic 141～160 amino acids and 200～213 amino acids are arranged in the chemical process composite coding VP1 albumen; With its series connection; Insert again on the plasmid vector macromole and change bacterial expression over to; Promptly get the foot-and-mouth disease virus resistant vaccine by fermentation, in the prevention of China's foot and mouth disease, play important effect.Our seminar is at RHDV capsid protein VP60 gene
Different positionsInsert respectively single external source B cell epitope (FMDV VP1 B cell epitope 200～213aa) and two-in-series B cell epitope (FMDV VP1 (141～160aa)-GS-(200～213aa)),
Make up 6 kindsRecombinant transfer vector is also expressed in the Sf9 cell, and through electron microscopic observation and experimentation on animals, research adds these exogenous segment to the assembling of VLPs and the immunogenicity of exogenous segment,
The result finds, inserts the site with other and compares,At RHDV capsid protein VP60 gene
The N-endInsert single external source B cell epitope (FMDV VP1 B cell epitope 200～213aa), the chimeric protein of expression of recombinant virus can form VLPs,
Show that capsid protein VP60 itself has vaccine carrier character, can under the situation that does not change structure, carry exogenous segment; Chimeric protein has identical with VP60 albumen to the people
" O " RCA characteristic is easy to the quantitative of vaccine antigen and detectsExperimentation on animals demonstration chimeric protein can be induced in the mouse body and produced resisting O-type foot and mouth disease virus VP1 protein positive antibody.
Summary of the invention
Technical problemThe objective of the invention is to make up recombination bacillary viral vector, the easy 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, technology and the preparation method of virus like particle vaccine are provided through Protocols in Molecular Biology.
Technical scheme
Carry the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus of O type foot and mouth disease virus B cell epitope, form by following method structure: with plasmid pFastBac
TM1-VP60 is a template; Pcr amplification goes out rabbit hemorrhagic disease virus capsid protein VP60 gene fragment; It is cloned into transfer vector; Again foot and mouth disease virus FMDV VP1 B cell epitope 200～213aa is inserted the correct position of VP60 gene fragment in the transfer vector, then recombinant plasmid transformed is contained shuttle vectors Bacmid's
E. coliThe DH10Bac competent cell, transfection Sf9 cell, the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus rAcV-Bac-NF of O type foot and mouth disease virus B cell epitope is carried in acquisition.
Rabbit hemorrhagic disease virus like-particles chimeric protein with the O type that the carries foot and mouth disease virus B cell epitope of said recombinant baculovirus rAcV-Bac-NF preparation.Its method is: the said recombinant baculovirus of claim 1 is inoculated in the Sf9 cell, and the harvested cell culture is the rabbit hemorrhagic disease virus like-particles chimeric protein that carries O type foot and mouth disease virus B cell epitope.
Said 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 with said recombinant baculovirus rAcV-Bac-NF preparation; Its preparation method is: cultivate the Sf9 cell to logarithmic phase; The said recombinant baculovirus of inoculation claim 1; There were after the pathology 5 days to receive poison in cell, resuspended with PH 7.4 sterilization phosphate buffer soln PBS, in antigen: freund's adjuvant volume 1:1 ratio adding adjuvant; Mixing is 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 among the rabbit hemorrhagic disease virus capsid protein VP60; Do not change the biological activity of capsid protein VP60; Be the RCA characteristic, chimeric protein has the RCA characteristic equally, and the titration of antigen blood clotting is carried out in available hemagglutination test (HA test) (HA).
Beneficial effectCharacteristics of the present invention and advantage are following: 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 under the condition that does not have other any compositions to exist; External can aggregate into naturally do not wrap up nucleic acid, with natural RHDV virus particle on physical aspect similarly virus-like particle (virus-like particles, VLPs), this VLPs is made up of 180 identical subunits of structure; Each subunit all shows external source foot and mouth disease virus B cell epitope; Different with other display systems, a virus like particle can be showed 180 foot and mouth disease virus B cell epitopes, has increased the copy number of antigen effective constituent virtually; Help the raising that vaccine immunity is renderd a service, 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, compare, utilize the virus like particle of this system expression to prepare vaccine and have good biological safety with traditional aftosa vaccine.Eukaryotic expression system has the posttranslational modification function that is similar to mammalian cell, make recombinant capsid protein on 26S Proteasome Structure and Function more near native protein, the result of test shows that expressed proteins has the biological activity identical with rabbit hemorrhagic disease virus; The chimeric protein of expressing need not the special processing purifying and just can be used for the vaccine preparation, and is easy and simple to handle.
It is good that the chimeric protein oneself that evidence, the present invention are expressed is assembled into the ability of virus like particle, shows that capsid protein VP60 itself has vaccine carrier character, can carry exogenous segment under the situation that does not change structure; Chimeric protein has identical with VP60 albumen to people " O " type RCA characteristic, be easy to vaccine antigen quantitatively with detect; With the virus like particle vaccine immune mouse that chimeric protein is processed, in the mouse body, can induce to produce resisting O-type foot and mouth disease virus VP1 protein positive antibody.Explain that the present invention can provide good foot and mouth disease virus vaccine antigen, have immune efficacy preferably, possess good biological safety simultaneously, have important use value aspect the foot and mouth disease prevention and control.
Description of drawings
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
TM
The 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
TM
The PCR of HTA-NF identifies
M. dna molecular quality 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 among 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 immunofluorescence detects 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 immunofluorescence detects 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 through pcr amplification rabbit hemorrhagic disease virus capsid protein VP60 (no initiator codon) gene, pcr amplification product is cloned into the donor plasmid pFastBac of baculovirus expression system
TMAmong the HTA, obtain recombinant transfer vector pFastBac
TMHTA-VP60, and then with foot and mouth disease virus B cell epitope (200～213aa) insertion pFastBac
TMThe appropriate location of HTA-VP60 obtains recombinant transfer vector pFastBac
TMHTA-NF contains shuttle plasmid Bacmid's with this carrier conversion
E. coliDH10Bac obtains recombinant baculovirus plasmid Bacmid-NF through screening, and transfection Sf9 cell obtains recombinant baculovirus rAcV-Bac-NF.With recombinate shape virus infection Sf9 cell, mosaic gene to be expressed, then with the chimeric protein of recombinant baculovirus expression as antigen, process 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 a restriction enzyme site
2. synthetic FMDV B cell epitope:
FMDV VP1 B cell epitope (200～213 aa:RHKQEIVAPVKQKL, the gene order that it is corresponding: 5 '-cgtcacaaacaggaaatcgtagctccagtaaaacagaagtt-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) being template, is primer with VP60-F/VP60-R, and pcr amplification goes out VP60 gene (no initiator codon) fragment.The PCR 25ul reaction system of pcr amplification VP60 is following: pre-mixed PCR damping fluid 2 * (Mix) 12.5 μ l, template 0.5 μ l, upstream primer VP60-F 0.5 μ l, downstream primer VP60-R, ddH
2O 11 μ l, TV 25 μ l, moment centrifugal mixing, adopt the heat lid to carry out pcr amplification reaction: 94 ℃ of sex change 5 min; 94 ℃ of sex change 1 min, 52.6 ℃ (58.1 ℃, 52.6 ℃ and 59.4 ℃) annealing, 1 min, 72 ℃ are extended 2min; After 30 circulations, 72 ℃ are extended 10min, finish reaction; The PCR product is carried out agarose gel electrophoresis 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 is cloned into cloning vector pMD19-T (available from the precious biotechnology in Dalian ltd) earlier with the VP60 gene of amplification, cuts evaluation (Fig. 2) with Xba I/ Hind III enzyme, is cloned into transfer vector pFastBac then
TMAmong the HTA (available from Invitrogen company), cut evaluation, obtain recombinant transfer vector pFastBac with Xba I/ Hind III enzyme
TMHTA-VP60 (Fig. 3).
4.2 the preparation FMDV-F/FMDV-R of the FMDV VP1 B cell epitope double-stranded DNA of phosphorylation band cohesive terminus forms double-stranded after anneal, concrete operations are 95 ℃ of insulation 10 min in the PCR appearance; 80 ℃ of 10 min, 65 ℃ of 10 min, 50 ℃ of 10 min, 37 ℃ of 10 min, 25 ℃ of 10 min.
FMDV VP1 B cell epitope double-stranded DNA phosphorylation, 50 μ l systems are following:
4.3 recombinant transfer vector pFastBac
TMThe structure of HTA-NF is with recombinant transfer vector pFastBac
TMHTA-VP60 cuts digestion with EcoR I/Xba I enzyme, reclaims carrier segments, is connected with the FMDV VP1 B cell epitope double-stranded DNA that the annealing phosphorylation is handled with T4 DNA Ligase then, transforms DH5
Competent cell carries out PCR evaluation and order-checking with Pn1/Pn2 primer, obtains recombinant transfer vector pFastBac
TMHTA-NF (Fig. 4).Recombinant transfer vector pFastBac
TMHTA-VP60 is following with the FMDV VP1 B cell epitope double-stranded DNA linked system that the annealing phosphorylation is handled:
PCR identifies that PCR 25 μ l reaction systems are following:
Low-speed centrifugal 10 s mixings adopt heat lid PCR to carry out cyclic amplification.
PCR identification reaction parameter is following:
4.4 the structure of recombinant baculovirus plasmid Bacmid-NF is with recombinant transfer vector pFastBac
TMHTA-NF transforms and contains shuttle vectors Bacmid's
E. coliDH10Bac competent cell (available from Invitrogen company); It is dull and stereotyped that converted product is applied to the LB that contains three kinds of microbiotic (50 μ g/ml kantlex, 7 μ g/ml qingfengmeisu qiongs, 10 μ g/ml tsiklomitsins), X-gal (100 μ g/ ml) and IPTG (20 μ g/ ml); Behind 37 ℃ of cultivation 48h; Select white colony and cultivate and extract plasmid; As template; Carrying out PCR 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) identifies; Simultaneously the amplified production size of the empty Bacmid plasmid that do not insert exogenous genetic fragment is about 300 bp (Fig. 5 the 1st swimming lane), prove swivel base successfully, male recombinant plasmid called after Bacmid-NF with pUC/M13 upstream and downstream primer.
PCR reaction system (20 μ l) is as follows:
The PCR cycling condition is: 95 ℃ of preparatory sex change 5 min; 94 ℃ of sex change 1 min, 55 ℃ of annealing 1 min, 72 ℃ are extended 2 min, 30 circulations; 72 ℃ are extended 10 min.
(2) transfectional cell obtains recombinant baculovirus
1. transfectional cell and purifying
With LipofectaminTM2000 (available from Invitrogen company) is cotransfection reagent, according to explanation, with recombinant shuttle vector plasmid Bacmid-NF transfection Sf9 monolayer.Every 12h observes once after the transfection, and when cytopathy was obvious, collecting cell and supernatant were as recombinant baculovirus stoste-4 ℃ preservation.The recombinant baculovirus called after rAcV-Bac-NF that will contain goal gene.Gone down to posterity with 1% volume ratio inoculation Sf9 cell (available from Invitrogen company) in twice of f1 disease venom freeze thawing back, obtain the 2nd generation recombinant virus.
2. identify
2.1 RT-PCR identifies
Collect the cell that infects recombinant virus, extract cell RNA, carry out RT-PCR with VP60-F/VP60-R and identify that the result shows that electrophoretic band is 1788 bp (Fig. 6 the 3rd swimming lanes), consistent with the predicted gene size.Simultaneously with from
E. coliThe cell that the virus of A cV-Bac that the empty Bacmid transfection Sf9 cell that extracts among the DH10Bac obtains infects as blank (Fig. 6 the 1st swimming lane), does not occur band with normal Sf9 cell as negative control (Fig. 6 the 2nd swimming lane).
Reverse transcription concrete steps reference molecule cloning experimentation guide carries out, and the RT reaction system is following:
Carry out following reaction conditions then: 30 ℃ of reaction 10 min, 42 ℃ of reaction 30 min, 99 ℃ of reaction 5 min finish reaction.-20 ℃ of storages are subsequent use.
Carry out the PCR program then, reaction system is following:
Carry out following reaction conditions then: 94 ℃ of preparatory sex change 3 min; 94 ℃ of sex change 1 min, 57 ℃ of annealing 1min, 72 ℃ are extended 2 min, 30 circulations; 72 ℃ are extended 10 min, finish reaction.
2.2 the evaluation of expression product
Infect the Sf9 cell 2.2.1 indirect immunofluorescence detects carrier specificity recombinant virus rAcV-Bac-NF, simultaneously with from
E. coliThe cell that the virus of A cV-Bac that the empty Bacmid transfection Sf9 cell that extracts among the DH10Bac obtains infects is as negative control, with normal Sf9 cell as blank.After infecting Sf9 cell 24 h, be one anti-with the dilution RHDV monoclonal antibody of 1:40 A3C, the dilution fluorescent mark goat anti-rabbit igg of 1:100 is two anti-, carries out indirect IF staining.The result shows; The recombinant baculovirus rAcV-Bac-VP60 that the Sf9 cell (Fig. 7 D) of infection recombinant baculovirus rAcV-Bac-NF, infection contain RHDV VP60 gene (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) cell (Fig. 7 A) has very strong specificity fluorescent, and no special fluorescence in the blank (Fig. 7 B), negative control (Fig. 7 C); Explain that carrier VP60 has obtained expression among the recombinant virus rAcV-Bac-VP60, and expression product is present in the insect cell.
Infect the Sf9 cell 2.2.2 indirect immunofluorescence detects epitope specificity with recombinant virus rAcV-Bac-NF, the while with from
E. coliThe cell that the virus of A cV-Bac that the empty Bacmid transfection Sf9 cell that extracts among the DH10Bac obtains infects is as negative control, with normal Sf9 cell as blank.Infecting the Sf9 cell after 24 hours, is one anti-with the dilution ox of 1:40 " O " type FMDV polyvalent antibody, and the dilution fluorescent mark goat-anti of 1:5000 ox IgG is two anti-, carries out indirect IF staining.The 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), infects the recombinant baculovirus rAcV-Bac-VP60 that contains RHDV VP60 gene (Wang Fang etc., the expression of rabbit hemorrhagic disease virus capsid protein in insect cell and to the immune protective effect of rabbit; Journal of animal science and veterinary medicine; 2008,39 (10), 1382～1387) no special fluorescence in the cell (Fig. 8 A); Explain that FMDV VP1 B cell epitope has obtained expression among the recombinant virus rAcV-Bac-NF, and expression product is present in the insect cell.
2.2.3 immunoblotting detects carrier specificity and collects the cell culture that infects recombinant virus, adds sample-loading buffer after the freeze thawing treatment and carries out protein electrophorese (SDS-PAGE); With VP60 monoclonal antibody A3C (number of patent application: 200910029627.9; Publication number: CN101519447; Title: be one anti-anti-rabbit hemorrhagic disease virus VP 60 albumen monoclonal antibody), the 1:40 dilution, the goat anti-rabbit igg of HRP mark (biological brilliant U.S. company) is two anti-, 1:5000 dilution carrying out immunoblotting (Western blot).SDS-PAGE result shows; Infect the Sf9 cell (Fig. 9 A:3) of recombinant baculovirus rAcV-Bac-NF, the recombinant baculovirus rAcV-Bac-VP60 (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, and is consistent with the expection size, do not occur being about the specific band of 60kDa after the Sf9 (Fig. 9 A:1) of infection AcV-Bac expresses; Western blot result shows; The recombinant baculovirus rAcV-Bac-VP60 that the Sf9 cell (Fig. 9 B:3) of infection recombinant baculovirus rAcV-Bac-NF, infection contain RHDV VP60 gene (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; A specific band appears in cell 39 (10), 1382～1387) (Fig. 9 B:2) about 60kDa, consistent with the expection size; The Sf9 (Fig. 9 B:1) that infects AcV-Bac band do not occur in corresponding position, explains that carrier VP60 has obtained expression among the recombinant virus rAcV-Bac-NF.
2.2.4 immunoblotting detects epitope specificity and collects the cell culture that infects recombinant virus, adds sample-loading buffer after the freeze thawing treatment and carries out protein electrophorese (SDS-PAGE); With Niu Yuan " O " type FMDV polyvalent antibody is one anti-, the 1:40 dilution, and the goat-anti ox IgG of HPR mark is two anti-, 1:5000 dilution carrying out immunoblotting (Western blot).SDS-PAGE result is identical with Fig. 9 A, and Western blot result shows that a specific band appears in the Sf9 cell (Figure 10 B:3) that infects recombinant baculovirus rAcV-Bac-NF about 60kDa, and is consistent with the expection size; Infection contains the recombinant baculovirus rAcV-Bac-VP60 (Wang Fang etc. of 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 band do not occur in corresponding position, explains that FMDV VP1 B cell epitope has obtained expression among the recombinant virus rAcV-Bac-NF.
(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 subsequent use as chimeric protein, and chimeric protein sample drop to be checked in carrying on the appearance copper mesh, is adsorbed 2 min; Inhale with filter paper and to remove unnecessary sample, drip 2% phospho-wolframic acid dye liquor then on copper mesh, fixedly 2min; Remove unnecessary phospho-wolframic acid dye liquor at last, drying at room temperature 5min observes on H-7650 type transmission electron microscope.The 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 the RHDV VLPs (Figure 11 A) of the recombinant baculovirus rAcV-Bac-VP60 expression that contains RHDV VP60 gene, explain that its self-assembling 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 subsequent use as chimeric protein; U type blood-coagulation-board the 1st～10 hole respectively adds PBS liquid 50
l; Draw chimeric protein 50
l then and add the 1st hole mixing; Draw mixing liquid 50
l from the 1st hole and add the 2nd hole; Doubling dilution to the 10 holes successively, the 10th hole draw 50
l discards.Every hole adding 50
l 1% people " O " type red corpuscle; Behind the vibration mixing; The RHDV VLPs that expresses with the recombinant baculovirus rAcV-Bac-VP60 that contains RHDV VP60 gene simultaneously compares, and 4 ℃ leave standstill observations behind the 40min.The result shows that RHDV VLPs hemagglutinative titer is 2
9(Figure 12 A), chimeric protein hemagglutinative titer are 2
6(Figure 12 B); Explain that O type foot and mouth disease virus B cell epitope inserts among the rabbit hemorrhagic disease virus capsid protein VP60; Do not change the biological activity of capsid protein VP60, i.e. RCA characteristic, chimeric protein has the RCA characteristic equally; The titration of antigen blood clotting is carried out in available hemagglutination test (HA test) (HA), quantitatively lays a good foundation for chimeric protein is antigenic.
(5) immunogenicity detects in vaccine production and the mouse body
Cultivate the Sf9 cell to logarithmic phase; Inoculate corresponding recombinant baculovirus rAcV-Bac-NF, wait cell to have after the pathology 5 days to receive poison, with sterilization phosphate buffer soln (PBS; PH 7.4) resuspended; In antigen: freund's adjuvant volume 1:1 ratio adds adjuvant, and mixing is the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope.
With the subcutaneous immune ICR mouse of above-mentioned virus like particle vaccine (immunity 3 times), and establish control group, exempt from back the 0th respectively at head; 1,2,3; 4,5 and 6 weeks were gathered mice serums, and indirect ELISA (O type FMDV ELISA detection kit available from Wuhan section before biological products Ltd) detects the epitope antibodies level; The result shows, the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope can induce produce 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 and produced higher resisting O-type foot and mouth disease virus VP1 protein positive antibody (Figure 13; FMDV), negative control PBS do not produce resisting O-type foot and mouth disease virus VP1 protein positive antibody (Figure 13, PBS).Explain that the present invention has immune efficacy preferably in practical application,, possess good biological safety simultaneously having important use value aspect the foot and mouth disease prevention and control.
SEQUENCE?LISTING
< 110>Jiangsu Province Agriculture Science Institute
< 120>carry the VP60 albumen recombinant baculovirus of O type foot and mouth disease virus B cell epitope
<130> 0
<160> 7
<170> PatentIn?version?3.1
<210> 1
<211> 26
<212> DNA
< 213>manual work
<220>
<221> VP60-F
<222> (1)..(26)
<223>
<400> 1
ttttctagag?agggcaaagc?ccgcac 26
<210> 2
<211> 27
<212> DNA
< 213>manual work
<220>
<221> VP60-R
<222> (1)..(27)
<223>
<400> 2
gccaagcttt?cagacataag?aaaagcc 27
<210> 3
<211> 18
<212> DNA
<213> 1
<220>
<221> Pn1
<222> (1)..(18)
<223>
<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 (8)
1. carry the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus of O type foot and mouth disease virus B cell epitope, form by following method structure: with plasmid pFastBac
TM1-VP60 is a template; Pcr amplification goes out rabbit hemorrhagic disease virus capsid protein VP60 gene fragment; It is cloned into transfer vector, again foot and mouth disease virus FMDV VP1 B cell epitope 200～213aa is inserted in the VP60 gene fragment, then recombinant plasmid transformed is contained shuttle vectors Bacmid's
E. coliThe DH10Bac competent cell, transfection Sf9 cell again obtains to carry the rabbit hemorrhagic disease virus capsid protein VP60 recombinant baculovirus rAcV-Bac-NF of O type foot and mouth disease virus B cell epitope.
2. with the rabbit hemorrhagic disease virus like-particles chimeric protein of the O type that the carries foot and mouth disease virus B cell epitope of the said recombinant baculovirus of claim 1 preparation.
3. with the method for the rabbit hemorrhagic disease virus like-particles chimeric protein of the O type that the carries foot and mouth disease virus B cell epitope of the said recombinant baculovirus of claim 1 preparation; Be characterised in that: the said recombinant baculovirus of claim 1 is inoculated in the Sf9 cell; The harvested cell culture is the rabbit hemorrhagic disease virus like-particles chimeric protein that carries O type foot and mouth disease virus B cell epitope.
4. the said recombinant baculovirus of claim 1 prevents, diagnoses or treat the purposes in the foot and mouth disease medicine in preparation.
5. claim 2 or the 3 said rabbit hemorrhagic disease virus like-particles chimeric proteins that carry O type foot and mouth disease virus B cell epitope prevent, diagnose or treat the purposes in the foot and mouth disease medicine in preparation.
6. the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope for preparing with the said recombinant baculovirus of claim 1.
7. the method for preparing vaccine with the said recombinant baculovirus of claim 1; Be characterised in that: cultivate the Sf9 cell to logarithmic phase, the said recombinant baculovirus of inoculation claim 1 waits cell to have after the pathology 5 days and receives poison; Resuspended with PH 7.4 sterilization phosphate buffer soln PBS; In antigen: freund's adjuvant volume 1:1 ratio adds adjuvant, and mixing is the rabbit hemorrhagic disease virus capsid protein virus like particle vaccine that carries foot and mouth disease virus B cell epitope.
8. measure claim 2 or 3 said methods 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 with hemagglutination test (HA test).
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| CN111793650A (en) * | 2020-07-08 | 2020-10-20 | 中国农业科学院兰州兽医研究所 | Preparation method and application of chimeric south Africa type 2 foot-and-mouth disease virus multi-epitope gene porcine parvovirus H-like particle |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105056227A (en) * | 2015-07-21 | 2015-11-18 | 复旦大学 | Anti-FMDV (foot and mouth disease virus) VLP (virus-like particles) vaccine and preparation method thereof |
| CN105056227B (en) * | 2015-07-21 | 2017-12-29 | 复旦大学 | A kind of viroid particle VLP vaccines of foot-and-mouth disease virus resistant and preparation method thereof |
| CN111793650A (en) * | 2020-07-08 | 2020-10-20 | 中国农业科学院兰州兽医研究所 | Preparation method and application of chimeric south Africa type 2 foot-and-mouth disease virus multi-epitope gene porcine parvovirus H-like particle |
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