CN103122353B - Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof - Google Patents

Porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as preparation method and application thereof Download PDF

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CN103122353B
CN103122353B CN201210364265.0A CN201210364265A CN103122353B CN 103122353 B CN103122353 B CN 103122353B CN 201210364265 A CN201210364265 A CN 201210364265A CN 103122353 B CN103122353 B CN 103122353B
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baculovirus
cell
recombinant baculovirus
virus
mouth disease
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CN103122353A (en
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李祥敏
钱平
杨应立
陈焕春
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Huazhong Agricultural University
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Abstract

The invention discloses porcine O-type foot-and-mouth disease virus recombinant baculovirus as well as a preparation method and application thereof. Sequences of VP0, VP1 and VP3 genes are artificially synthesized by referring to an FMDV (Foot And Mouth Disease Virus) O-type epidemic strain gene sequence; the VP0, VP1 and VP3 genes are connected to pFBDPHmHNM1P10eGFP plasmid by adopting the plasmid as a framework vector, so that a baculovirus transfer vector pFBDPHmVP013 is obtained. The baculovirus transfer vector pFBDPHmVP013 is mixed with DH10Bac escherichia coli competent cells, and the positive bacterial colony is selected to obtain a recombinant shuttle vector Bacmid; the shuttle vetcor Bacmid is transferred with a sf9 cell, and the recombinant baculovirus QP-Ac-FVLP is obtained by collecting the cell supernatant. The recombinant baculovirus can be used for efficiently expressing FMDVVP0, Vp1 and Vp3 proteins and forming virus-like particles. And the virus-like particles are used for preparing subunit vaccine, so that the organism is induced to generate specific immunity response after the mouse is immunized.

Description

One boar O type foot and mouth disease virus recombinant baculovirus and preparation method and application
Technical field
The present invention relates to biotechnology and field of virology.More specifically, the present invention relates to the recombinant baculovirus QP-Ac-FVLP of a boar O type foot and mouth disease virus, the preparation method who also relates to the recombinant baculovirus QP-Ac-FVLP of a boar O type foot and mouth disease virus, the recombinant baculovirus QP-Ac-FVLP that also relates to a boar O type foot and mouth disease virus prevents the application in pig O type mouth disease virus infection subunit vaccine in preparation.Utilize expression of recombinant virus of the present invention manually modified swine foot-and-mouth disease virus O type VP0, VP1 and VP3 gene and be assembled into swine foot-and-mouth disease virus sample virion VLP, can be for the preparation of O type foot and mouth disease virus safe vaccine.
Background technology
Foot and mouth disease (Foot and Mouth Disease, FMD) is a kind of transmissible disease that is caused acute, hot, height contact infecting both domestic animals and human artiodactylous by foot and mouth disease virus (Foot and Mouth Dis ease Virus, FMDV).The popular history of this disease is more of a specified duration; host range is wide; infectivity is extremely strong; route of transmission is many, the fast (Chen Puyan of speed; 2006), once thereby outburst very easily causes serious morbidity animal production function and the livestock product quality of reducing that be very popular, to livestock industry, cause tremendous economic loss; so OIE (OIE) is classified as the strong zoonosis of category-A (Pereira HG, 1981; Mark et al., 1999), China Ministry of Agriculture is classified as one-level zoonosis, once there is epidemic situation, must report.Its host is mainly artiodactyls as other wildlifes (Doel, 2005 such as bovine livestock, pig, sheep and camel, deer; Dus Santos et al., 2005).And showing cat, dog, birds, rodents, bat, insect etc., correlative study all can propagate this disease.O type foot and mouth disease is deep-rooted, long-standing in China.FMDV strain separated before 2009 mostly is Chinese topological type, and new variation has occurred the fashion trend of 2010 Nian Hou China foot and mouth disease, and the O type FMDV of mainly take is honest and just, and horse strain is the Burma 98(MYA98 of South East Asia topological type) as main.FMDV belongs to Picornaviridae (Picornaviridae), Hostis (Aphthovirus), this virus is sub-thread normal chain picornavirus, and genome is about 8500 Nucleotide, mainly by formations such as structure gene (P1 district) and nonstructural genes (P2, P3 district).4 kinds of structural protein of P1 district coding FMDV are respectively VP4, VP0, and VP3 and VP1, wherein only have VP0, and VP3 and VP1 albumen can form virus-like particle.The subunit that these four kinds of structural protein form pentamer forms viral capsid, avoids viral RNA by nuclease degradation, determines viral host range and antigenicity and energy identification specificity cell receptor.
Aftosa vaccine inoculation is reliable tools and the effective means (Casas et al., 1988) as specificity prevention foot and mouth disease.Current anti-FMDV vaccine mainly contains inactivated vaccine and these two kinds of conventional vaccines of attenuated vaccine, although these two kinds of vaccines are brought into play certain effect in the anti-system of foot and mouth disease, has some shortcomings.Weak malicious seedling is because easily cause immune animal to produce viremia and long-term band poison, also potential virulence is returned strong risk, so the world only has a few countries and area studies to use, and inactivated vaccine has the safe and reliable and good feature of immune effect, most countries prevents foot and mouth disease with inactivated vaccine.Yet because of the reason of himself, also there are many defects in inactivated vaccine: 1. immune animal is without cross protection, and therefore a kind of inactivated vaccine cannot provide comprehensive protection; 2. in preparation process, there is the risk of viral escape; Because of containing in a organized way or cellular constituent immunity there is side reaction.
Except above-mentioned conventional vaccine, people are seeking foot-and-mouth disease gene engineering vaccine as subunit vaccine, polypeptide vaccine, the live vector vaccines such as poxvirus, adenovirus.But these vaccines still exist some deficiency, are not accepted by people, as subunit vaccine and polypeptide vaccine involve great expense, immune effect is not good; Although the vaccine of the live vector such as poxvirus, adenovirus has certain immune effect, restricted due to vector virus, people would rather select conventional vaccine also not select this recombinant vaccine to prevent foot and mouth disease.Therefore, this area has efficient, safe, the cheap recombinant vaccine for foot and mouth disease in the urgent need to developing.
Baculovirus is the large-scale shaft-like togavirus of a class, and genome is circular double stranded DNA, and size is about 80-180kbp.Baculovirus parasitizes arthropods as pathogenic micro-organism, has the host specificity of height, and its host mainly contains lepidopteran Diptera and hymenopteran, not yet finds arthropods baculovirus host in addition.In numerous members of baculovirus, studying at present and utilizing maximum is many embedding nuclear polyhedrosis virus of autographa california (Autographa califonica mult iple nuclear polyhedrosis virus, AcMNPV).AcMNPV viral genome is the supercoiled double-stranded DNA of virus covalently closed circular, about 130kb, and its genome sequence is measured at present.In recent years, rhabdovirus expression vector, with the advantage of self, has accounted for leading status on expression vector.Baculovirus expression system is compared with other expression system, it is simple to operate, safe that baculovirus expression system has, can hold large goal gene, express foreign protein effect high, there is the effect of posttranslational modification, the immunogenicity of expressing protein, biological activity and the natural advantages such as protein similar (Anderson et al., 1995; Wang et al., 2001; Ribeiro et al., 2001).The virus sample particle vaccines that development in recent years is got up (Virus like particles; VLP); containing virulent one or more antigen proteins, there is the particle with virus particle analog structure; not containing virulent genetic material; can not self-replicating, there is no infectivity; can be with native conformation and pattern submission antigen, effective stimulus body fluid and cellular immunization, induction body produces good immunoprotection.In view of the advantage of rhabdovirus expression vector and the characteristic of virus sample particle vaccines, utilize the baculovirus vector expression system honest and just horse pedigree of great expression O type FMDV current popular strain VP0, VP3 and VP1 gene in insect cell, and successful assembling assembly virus-like particle VLP, and prepare vaccine with this, the anti-system of immunity infecting for O type FMDV provides candidate vaccine strain.
Summary of the invention
An object of the present invention is to provide a kind of manually modified synthetic pig O type FMDV(Foot-and-mouth disease, FMDV, foot and mouth disease virus) immunogenic gene VP0, the VP1 of honest and just horse pedigree and the cDNA sequence of VP3, its sequence is shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
Another object of the present invention is to provide a kind of baculovirus transfer vector pFBDPHmVP013, and this carrier contains the honest and just horse pedigree of pig O type FMDV immunogenic gene VP0, VP1, VP3 and marker gene eGFP.
A further object of the invention is to provide a boar O type foot and mouth disease virus recombinant baculovirus, this strain is named as recombinant baculovirus QP-Ac-FVLP, belong to baculovirus (Baculovirus), this virus contains the honest and just horse pedigree of manually modified synthetic pig O type FMDV main immunogenic gene VP0, VP1 and VP3, can express the honest and just horse pedigree of manually modified synthetic pig O type FMDV VP0, VP1, VP3 albumen, August 16 in 2012, deliver Chinese Typical Representative culture collection center (CCTCC) preservation, preserving number is CCTCC NO:V201237, Classification And Nomenclature: recombinant baculovirus QP-Ac-FVLP Recominant Autographa californica multiple Nucleopolyhedrovirus QP-Ac-FVLP, address: Wuhan, China Wuhan University.
A further object of the invention has been to provide the application of a boar O type foot and mouth disease virus recombinant baculovirus in preparation prevention pig O type mouth disease virus infection subunit vaccine, will after this virus immunity mouse, can induce body to produce specific immune response.
In order to realize above-mentioned object, the present invention adopts following technical measures:
One boar O type foot and mouth disease virus recombinant baculovirus, its preparation process is as follows:
1, the structure of the manually modified synthetic and baculovirus transfer vector pFBDPHmVP013 of FMDV VP013 gene
With reference to FMDV O type epidemic strain O/HLJOC12/03 gene order (DQ119643.2), according to preferences synthetic VP0, the VP1 of codon and the sequence of VP3 gene, its nucleotide sequence is respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
With pFBDPHmHNM1P10eGFP plasmid (money equality, a kind of recombinant baculovirus of expressing manually modified synthetic influenza A H 1 N 1 virus HA-NA-M 1 gene, patent publication No. CN101624580A, patent No. ZL200910063217.6) be skeleton, by NcoI and KpnI enzyme, cut pFBDPHmHNM1P10eGFP, reclaim large fragment and obtain pFBDPHmHNM1.By XhoI and HindIII respectively enzyme cut VP0 gene and pFBDPHmHNM1, reclaim VP0 gene and skeleton carrier pFBDPHmHN, then by ligase enzyme Ligase, in 16 ℃, connect, transform bacillus coli DH 5 alpha competent cell, extract recombinant plasmid enzyme and cut evaluation, obtain baculovirus transfer vector pFBDPHmHNVP0.By SpeI and NotI enzyme, cut VP1 gene and pFBDPHmHNVP0, reclaim VP1 gene and pFBDPHmHVP0, then by ligase enzyme Ligase, connect, extract recombinant plasmid enzyme and cut evaluation, obtain transfer vector pFBDPHmHVP01.Again by BamHI and EcoRI respectively enzyme cut VP3 gene and pFBDPHmHVP01, reclaim VP3 gene and pFBDPHmVP01, then by ligase enzyme Ligase, connect, extract recombinant plasmid, enzyme is cut qualification result and is shown the final baculovirus transfer vector pFBDPHmVP013(Fig. 1 that obtains).
2, the Construction and identification of recombinant virus baculovirus QP-Ac-FVLP
(1) structure of recombinant virus baculovirus QP-Ac-FVLP
Get order-checking and identify correct baculovirus transfer vector pFBDPHmVP013 2.0 μ L and 100 μ L DH10Bac intestinal bacteria (Invitrogen tM) competent cell mixing, after ice bath 30min, in 42 ℃ of 45s water-baths, carry out heat shock, then ice bath 2min, adds 900 μ L LB liquid nutrient mediums (sodium chloride concentration is 10%), and 4h are cultivated in 37 ℃ of joltings, by 10 -1, 10 -2, 10 - 3after dilution, respectively get 100 μ L and coat three high salt tolerance LB dull and stereotyped (kantlex, gentamicin and tsiklomitsin, working concentration is pressed the Bac-to-Bac Baculovirus Expression System(of Invitrogen company test kit) process specifications), cultivate 24-48h for 37 ℃, by blue hickie, screen, after twice line purifying, obtain pure stable hickie, the positive bacterium colony of purifying, extracts recombinant shuttle vector (Bacmid) and further identifies by PCR.With VP0(F) (5 '-TTTTGGATCCACCATGGGCGCCGGGCAATCC-3 ') and M13(R) (5 '-AGCGGATAACAATTTCACACAGG-3 ') carry out PCR evaluation.Result shows to amplify the special object band of a 4200bp, shows that foreign gene VP013 has been recombined into (Fig. 2) in Baculovirus Gene group in intestinal bacteria.
Utilize the operation of liposome-mediated infection protocol (by the Bac-to-Bac Baculovirus Expression System(of Invitrogen company test kit) process specifications), by recombinant shuttle vector (Bacmid) warp after screening purifying in Rea gent transfection sf9 cell (purchased from Wuhan University's Chinese Typical Representative thing preservation center), 24h after transfection respectively, 48h, 72h utilizes fluorescence microscope recombinant baculovirus green fluorescence signal, after transfection 72h, collecting cell supernatant obtains recombinant baculovirus called after QP-Ac-FVLP, this virus is delivered Chinese Typical Representative culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province August 16 in 2012, its preserving number is CCTCC V201237, Classification And Nomenclature Recomina nt Autographa californica multiple Nucleopolyhedrovirus QP-Ac-FVLP.This virus can be preserved or-80 ℃ long-term preservations in 4 ℃.This recombinant baculovirus QP-Ac-PCV3ORF2 has and many embedding nuclear polyhedrosis virus of carrier baculovirus autographa california (Autographa califonica multiple nuclear polyhedrosis virus, AcMNPV) identical morphological characteristic, the functional performances such as cultural characters, viral genome is DNA, be shaft-like, have cyst membrane parcel; Its host range is mainly lepidopteran, Hymenoptera and dipteral insect.Vitro culture sf9 and the sf21 that derive from meadow high (Spod optera frugiperda) ovary at greedy night that use more, and the BTI-Tn-5B1-4 that derives from powder pattern night high (Trichoplusia ni) is Hi5 clone, cultivating optimum temperuture is 26-28 ℃, can reduce and mammalian cell between crossed contamination.
Extract F2 and use respectively VP1 primer (VP1 (F): 5 '-TTTTGTCGACCCACCATGACCACCTCTGCGGGTG-3 ' for the genome of recombinant baculovirus QP-Ac-FVLP, VP 1 (R): 5 '-CTGTAAGCTTTTACTGTTTTGCGGGTGCCAC-3 '), VP0(F) (5 '-TTTTGGATCCACCATGGGCGCCGGGCAATCC-3 ') and M13(R) (5 '-AGCGGATAACAATTTCACACAGG-3 ') two pairs of primers carry out PCR evaluation, expand respectively the fragment (Fig. 3) of 650bp and 4200bp, illustrate that the recombinant baculovirus QP-Ac-FVLP obtaining contains external source goal gene.
(2) evaluation of recombinant baculovirus QP-Ac-FVLP exogenous protein expression
(A) indirect immunofluorescence and Western-blot detect the expression of foreign protein
Just growing up on the sf9 cell of individual layer, the infection multiplicity inoculation recombinant baculovirus QP-Ac-FVLP with 3-5pfu/cell, collects sick cell ultrasonic treatment after fixed cell or 72h after 36 hours.On the one hand fixed cell carries out indirect immunofluorescence, mouse-anti FMDV O type VP1 monoclonal antibody prepared by this chamber professor Wu Bin of take as primary antibodie (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. tutor: Wu Bin), take Cy3 mark sheep anti-mouse igg antibody (Sigma) as two anti-, with DAPI, nucleus is dyeed, under Laser Scanning Confocal Microscope, observe, result shows with the cell of recombinate shape virus infection to be had compared with strong red fluorescence signal and normal sf9 cell redfree fluorescent signal (Fig. 4), shows that the foreign gene of recombinant baculovirus QP-Ac-FVLP is expressed in sf9 cell.On the one hand by the insect cell of collecting, after SDS-PAGE transferring film, one group with mouse-anti FMDV O type VP1 monoclonal antibody (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. lead teacher: Wu Bin) be primary antibodie, sheep anti-mouse igg-HRP(Sigma) be two anti-, another group take the anti-FMDV hyper-immune serum of rabbit IgG as primary antibodie (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. tutor: Wu Bin), goat anti-rabbit igg-HRP(Sigma) be two anti-, carry out Western blot analysis simultaneously, result shows first group of specific band (Fig. 5 (A)) that has a treaty 28KD, second group has two specific bands, one another treaty of treaty 28KD 36KD(5(B)), experimental result further shows that the foreign gene of recombinant baculovirus QP-Ac-FVLP all obtains effective expression (as Fig. 5) in sf9 cell.
(B) electron microscopic observation of virus particle
Just growing up on the sf9 cell of individual layer, the infection multiplicity inoculation recombinant baculovirus QP-Ac-FVLP with 3-5pfu/cell, collects sick cell ultrasonic treatment after 72 hours.Under 4 ℃ of conditions, the centrifugal 30min of 8000g, is then concentrated to 1/10 of original volume with sucrose, contains the VLP particle of expression in supernatant.By supernatant 27,000rpm/min, ultracentrifugation 3 hours, precipitation is resuspended in the PBS of 2ml, and repeatedly blows and beats with the syringe of 1ml, and then viral suspension is undertaken 27 by 20-60% sucrose density gradient, 00rpm/min ultracentrifugation 16 hours, the protein band of collecting the superiors carries out electron microscopic observation.At observed under electron microscope, arrive baculovirus (◇) and form typical FMDV sample virion (as shown in Figure 6).
The application of one boar O type foot and mouth disease virus recombinant baculovirus in preparation prevention pig O type mouth disease virus infection subunit vaccine, step is as follows:
1, the preparation of recombinant virus QP-Ac-FVLP subunit vaccine and immune mouse
In order to evaluate the immune effect of recombinant baculovirus QP-Ac-FVLP subunit vaccine in prevention O type mouth disease virus infection, the insect cell High Five by the recombinant baculovirus QP-Ac-FVLP obtaining with the infection multiplicity inoculation suspension culture of 3-5pfu/cell tM(purchased from Invitrogen company), collects virus liquid after 72h, the centrifugal 10min collecting cell of 2000rpm is also counted with cell counting count board, and with PBS, adjusting number of cells is 2 * 10 7individual/ml and 2 * 10 6individual/two of ml gradient, after multigelation 3 times with isopyknic Freund's complete adjuvant (Sigma) emulsification, put 4 ℃ temporary.
The subunit vaccine preparing is taked to the mode immune mouse of leg muscle injection, every immunity 100 μ L, twice of immunity, 3 weeks, interval, using not through virus infection and take the HighFive groups of cells of subunit vaccine same treatment mode as negative control group, usining commercialization O type FMD inactivated vaccine group as positive controls.
2, the detection of amynologic index
2.1 animal experiment serum ELISA antibody tests
Mice serum is added in antigen coated microplate after by 1:40 dilution proportion with PBST, and every hole 100 μ L, hatch 1h for 37 ℃, with the coated plate of PBST washing 3 times, each 5min, with PBST by sheep anti-mouse igg-HRP(Sigma) press 1:5000 and dilute, every hole adds 100 μ L, hatch 1h for 37 ℃, PBST washing plank 3 times, each 5min, every hole is taken up in order of priority and is added 50 μ L nitrite ion A and B, room temperature lucifuge effect 10min, last every hole adds 50 μ L stop buffers, by microplate reader, measures OD 630, result as shown in Figure 7.
2.2. real-time fluorescence quantitative PCR detects IFN-α, IFN-β, IFN-Y relative expression level in immune mouse spleen cell
2.2.1 the preparation of immune mouse spleen cell
Adopt cervical vertebra dislocation method to put to death mouse, under aseptic condition, take out spleen, add 5ml modified form 1640 use homogenizers to be ground to gently spleen and be dispersed into individual cells, and add the gentle and quiet 5min of putting of 5ml 1640 Culture Medium Laboratory, suct and transfer to clearly in another centrifuge tube, the centrifugal 10min of 1000rpm, abandon supernatant, add the aseptic 8.3g/L NH4Cl of 5ml resuspended, the centrifugal 10min of 1000rpm, precipitation is adjusted cell concn to 1 * 10 with the RPMI-1640 that contains 10%FBS 6individual/ml, adds the every hole 1ml of the resuspended liquid of cell in 12 porocyte culture plates, and it is former as stimulating that every hole adds the FMDV of 20 μ L uviolizing deactivations simultaneously, 37 ℃ of CO 2incubator is cultivated 20h, collects post-stimulatory mouse boosting cell to 1.5mlEP pipe ,-80 ℃ of preservations.
2.2.2 the extraction of the total RNA of immune mouse spleen cell
To the every pipe of mouse boosting cell of collecting, add 1ml SuPerfecTRI Total RNA Isolation Reagent, put upside down and mix the standing 5min of rear room temperature, every pipe adds 200 μ L chloroform concuss 15s, the standing 10min of room temperature, 4 ℃ of centrifugal 10min of 12000rpm, shift supernatant in another EP pipe; Add 500 μ L Virahols to mix, the standing 10min of room temperature, 4 ℃ of centrifugal 10min of 12000rpm; Abandon 75% ethanol (preparation of DEPC water) that supernatant adds 1ml now to join, the centrifugal 5min of 7000rpm, abandons supernatant, is deposited in Bechtop interior air-dry, and final 20 μ L DEPC water dissolution for RNA, measure RNA concentration and purity ,-80 ℃ of preservations.
2.2.3 real-time fluorescence quantitative PCR detects IFN mrna expression in immune mouse spleen cell
Adopt TRIzol method to extract total RNA of cell, utilize the method for SYBRGreen I real-time quantitative PCR to detect IFN-α, IFN-β, the IFN-Y mrna expression situation in immune mouse spleen cell.Result can find out that recombinant baculovirus immune group can stimulate the IFN-α of mouse induction generation higher level, and IFN-β, IFN-Y are without obviously increasing (Fig. 8).
Compared with prior art, the present invention has the following advantages:
1. the present invention is according to the preferences of codon, and manually modified immunogenic gene VP0, VP1 and the VP3 that synthesizes the honest and just horse pedigree of pig O type FMDV, has improved these genes expression level in baculovirus.
2. the present invention expresses after immunogenic gene VP0, the VP1 of the honest and just horse pedigree of synthetic pig O type FMDV and VP3 are placed in respectively to baculovirus PH promotor, has greatly improved the expression amount of foreign gene.
3. the expressed capsid protein of recombinant baculovirus of the present invention can successfully be assembled into virus-like particle (VLP) in insect cell, there is the particle with virus particle analog structure, containing virulent genetic material, can not self-replicating, there is no infectivity, thereby safer.
4. the virus-like particle (VLP) that the expressed packing of recombinant baculovirus of the present invention forms is prepared subunit vaccine, can induce body to produce specific immune response after immune mouse.
Accompanying drawing explanation
Fig. 1: for the enzyme of a kind of baculovirus vector pFBDPHmVP013 is cut evaluation collection of illustrative plates.
M1:15000bp DNA Marker; 1, pFBDPHmVP013/EcoRI enzyme is cut evaluation; 2, pFBDPHmVP013/KpnI enzyme is cut evaluation; 3, pFBDPHmVP013/SacII enzyme is cut evaluation; M2:5000Marker.
Fig. 2: for a kind of recombinant shuttle vector Bacmid PCR identifies.
Identify that correct baculovirus transfer vector pFBDPHmVP013 transforms DH10Bac, blue hickie screening obtains positive bacterium colony, extracts the electrophoresis picture that recombinant shuttle vector Bacmid carries out PCR evaluation.
1: be VP0(F)/M13(R) the PCR result of primer amplification recombinant shuttle vector Bacmid, clip size is about 4200bp;
M:15000Marker;
2: be VP0(F)/M13(R) the PCR result of primer amplification transfer vector pFBDPHmVP013;
3: be VP0(F)/M13(R) the PCR result of primer amplification water contrast.
Fig. 3: for the genomic PCR of a kind of recombinant baculovirus QP-Ac-FVLP identifies schematic diagram.
A figure: the PCR result of utilizing VP1 upstream and downstream primer amplification recombinant virus genomes;
B figure: the PCR result of utilizing VP0 (F) and M13 (R) upstream and downstream primer amplification recombinant virus genomes;
M1:2000bp DNA Marker; 1: recombinant virus QP-Ac-FVLP genome; 2: water contrast; M2:15000bp DNA Marker.
Fig. 4: be a kind of expression schematic diagram of indirect immunofluorescence evaluation recombinant baculovirus QP-Ac-FVLP foreign gene.
A figure: recombinant baculovirus QP-Ac-FVLP infects the indirect immunofluorescence result of sf9 cell;
B figure: the indirect immunofluorescence result of virus-free infection sf9 cell contrast;
A: red fluorescence CY3 signal; B: green fluorescence eGFP signal; C:DAPI staining cell core blue-fluorescence signal; D: three kinds of colour superimpositions.
Fig. 5: be a kind of expression schematic diagram of Western blot evaluation baculovirus QP-Ac-FVLP foreign gene.
The result that A:FMDV VP1 monoclonal antibody is primary antibodie; B: the result that the anti-FMDV hyper-immune serum of rabbit IgG is primary antibodie;
M: protein marker; 1: baculovirus QP-Ac-FVLP infects sf9 cell; 2: the contrast of virus-free infection sf9 cell.
Fig. 6: for a kind of baculovirus QP-Ac-FVLP infects the Electronic Speculum picture that sf9 cell conditioned medium forms virus-like particle.
Recombinant baculovirus QP-Ac-FVLP is with the infective dose inoculation sf9 cell of 3 ~ 5pfu/cell, after 72 hours, collect sick cell ultrasonic treatment and ultracentrifugation and carry out electron microscopic observation, except visible typical baculovirus particle (shown in ◇), also see a large amount of sizes be about 20nm without togavirus, form is identical with typical FMDV, shows assembling assembly virus-like particle VLP( shown in).
Fig. 7: be the ELISA antibody horizontal schematic diagram of a kind of different time mice serum for FMDV.
Cell control group, commercially available vaccine group, recombinant baculovirus low dosage (2 * 10 6individual cell/ml) and high dose group (2 * 10 7individual cell/ml) immune mouse immune mouse, the 21st day, 35 days and blood sampling in 42 days after exempting from head before head exempts from, separation of serum detects the ELISA antibody of the anti-FMDV in serum with the coated elisa plate of prokaryotic expression FMDV O type VP1 albumen.Result shows after head exempts from 21 days, and the anti-FMDV antibody horizontal of recombinant baculovirus immune group rises gradually, and high dose group antibody horizontal is higher than low dose group.
Fig. 8: analyze IFN in mouse boosting cell for a kind of real-time fluorescence quantitative PCR and express schematic diagram.
Cell control group, commercially available vaccine group, recombinant baculovirus low dosage (2 * 10 6individual cell/ml) and high dose group (2 * 10 7individual cell/ml) immune mouse, in head, exempt from dislocation execution afterwards in 42 days, spleen is taken out in aseptic technique, isolated cell adds the FMDV of deactivation to cultivate 20h as stimulating factor, adopt TRIzol method to extract total RNA of cell, utilize the method for SYBRGreenI real-time quantitative PCR to detect alpha-interferon, beta-interferon, the gamma-interferon mrna expression situation in immune mouse spleen cell, after result shows immunity, alpha-interferon mRNA level obviously rises.
Embodiment
1: one boar O type foot and mouth disease virus recombinant baculovirus of embodiment, its preparation process is as follows:
(1), the structure of recombinant baculovirus QP-Ac-FVLP
1, immunogenic gene VP0, the VP1 of pig O type FMDV, VP3's is manually modified synthetic
With reference to FMDV O type epidemic strain O/HLJOC12/03 gene order (DQ119643.2), according to the preferences synthetic VP0(SEQ ID NO.1 of codon), VP1(SEQ ID NO.2) and VP3(SEQ ID NO.3) sequence of gene.
2, the structure of baculovirus transfer vector pFBDPHmVP013
With pFBDPHmHNM1P10eGFP plasmid (money equality, a kind of recombinant baculovirus of expressing manually modified synthetic influenza A H 1 N 1 virus HA-NA-M 1 gene, patent publication No. CN200910063217.6) be skeleton, by NcoI and KpnI enzyme, cut pFBDPHmHNM1P10eGFP, reclaim large fragment and obtain pFBDPHmHNM1.By XhoI and HindIII respectively enzyme cut VP0 gene and pFBDPHmHNM1, reclaim VP0 gene fragment and pFBDPHmHN carrier, then connect, extract recombinant plasmid, enzyme is cut evaluation, obtains baculovirus transfer vector pFBDPHmHNVP0.And then cut VP1 gene and pFBDPHmHNVP0 by SpeI and NotI enzyme, and reclaim VP1 gene and pFBDPHmHVP0, then connect, extract recombinant plasmid, enzyme is cut evaluation, obtains baculovirus transfer vector pFBDPHmHVP01.Again by BamHI and EcoRI respectively enzyme cut VP3 gene and pFBDPHmHVP01, reclaim VP3 gene and pFBDPHmVP01, then connect, extract recombinant plasmid, enzyme cut identify show finally to obtain baculovirus transfer vector pFBDPHmVP013(as Fig. 1).
3, the structure of recombinant virus baculovirus QP-Ac-FVLP
(1) acquisition and the evaluation of recombinant shuttle vector (Bacmid)
Get order-checking and identify that correct baculovirus transfer vector pFBDPHmVP0132.0 μ L mixes with 100 μ L DH10Bac competent escherichia coli cells, after ice bath 30min, in 42 ℃ of 45s water-baths, carry out heat shock, then ice bath 2min, add 900 μ L LB liquid nutrient mediums (sodium chloride concentration is 10%), 4h is cultivated in 37 ℃ of joltings, press 10-1, 10-2, after 10-3 dilution, respectively get 100 μ L and coat the dull and stereotyped (kantlex of three high salt tolerance LB, gentamicin and tsiklomitsin, working concentration is pressed the Bac-to-Bac Baculovirus Expression System(of Invitrogen company test kit) process specifications), cultivate 24-48h for 37 ℃, by blue hickie, screen, the positive bacterium colony of purifying.Extract positive bacterium colony genome and by special primer VP0(F) (5 '-TTTTGGATCCACCATGGGCGCCGGGCAATCC-3 ') and M13(R) (5 '-AGCGGATAACAATTTCACACAGG-3 ') carry out PCR evaluation.
Pcr amplification condition is as follows:
Reaction conditions
94℃ 3min
72℃ 3.0min
72℃ 10min
4℃ 10min
The PCR product of amplification is through 0.8%(w/v) agarose gel electrophoresis analysis, result shows to increase, and to amplify size be the band of 4200bp, big or small with expect unanimously, result shows that external source goal gene is inserted in the genome of baculovirus (Fig. 2).
(2) extraction of recombinant shuttle vector (Bacmid)
Aseptic picking falls within high salt LB liquid nutrient medium containing the positive bacteria of restructuring Bacmid, while being cultured to bacterium logarithmic phase, collect 0.3mL solution I (50mmol/L glucose for thalline, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0)) resuspended, add 0.3mL solution II (0.2mol/L NaOH, 1%SDS, matching while using) slightly mix, the standing 5min of room temperature, slowly add the 0.3mL solution III (potassium acetate of 3mol/L, pH5.0) mix, ice bath 5-10min, the centrifugal 10min of 14000r/min, supernatant is added in 0.5mL Virahol, mix ice bath 5-10min, the centrifugal 15min of room temperature 14000r/min, 70% washing with alcohol precipitation, after dry, be dissolved in 40 μ L TE, can use immediately or-20 ℃ of preservations, avoid multigelation.
(4) recombinant baculovirus QP-Ac-FVLP obtains and PCR evaluation
Utilize the operation of liposome-mediated infection protocol (by the Bac-to-Bac Baculovirus Expression System(of Invitrogen company test kit) process specifications), by recombinant shuttle vector (Bacmid) warp after screening, purifying in Rea gent transfection sf9 cell (purchased from Wuhan University's Chinese Typical Representative thing preservation center), in 28 ℃ of cultivations, after transfection, 24h, 48h, 72h utilize fluorescence microscope recombinant baculovirus green fluorescence signal respectively, after transfection 72h, collecting cell supernatant obtains recombinant baculovirus QP-Ac-FVLP, in-80 ℃ of preservations.
Sf9 cell is inoculated to 6 porocyte culture plates, when cell grows up to 80-90% individual layer, with 1 MOI recombinant baculovirus QP-Ac-FVLP inoculation sf9 cell, establish control baculovirus simultaneously, observe day by day, infect more than 90% sf9 cell generation pathology after 72 hours.
Get 400 μ l culture supernatant and cell suspensions, with trypsinase, at 56 ℃, process 4 hours, with isopyknic phenol/chloroform extracting secondary, get that 3M acetic acid that supernatant adds 1/10 volume is received and the dehydrated alcohol precipitation of 2 times of volumes, the centrifugal supernatant of abandoning, with 500 μ l70% ethanol, wash precipitation once, after seasoning, add the deionized water of 50 μ l sterilizings resuspended as pcr template.Use respectively VP1 primer (VP1 (F): 5 '-TTTTGTCGACCCACCATGACCACCTCTGCGGGTG-3 ', VP 1 (R): 5 '-CTGTAAGCTTTTACTGTTTTGCGGGTGCCAC-3 '), VP0(F) (5 '-TTTTGGATCCACCATGGGCGCCGGGCAATCC-3) and M 13(R) (5 '-AGCGGATAACAATTTCACACAGG-3 ') two pairs of primers carry out PCR evaluation, the same recombinant shuttle vector of reaction conditions (Bacmid) PCR identifies.Pcr amplification product is through 0.8%(w/v) agarose gel electrophoresis detection, result shows to expand respectively the fragment (Fig. 3) of 650bp and 4200bp, consistent with test expected results, illustrates that the recombinant baculovirus QP-Ac-FVLP obtaining contains external source goal gene.
(2), the formation of the expression of recombinant baculovirus QP-Ac-FVLP foreign gene and hollow capsid particle VLP
1, indirect immunofluorescence is identified the expression of foreign gene
Just growing up on the sf9 cell of individual layer, with the infection multiplicity inoculation recombinant baculovirus QP-Ac-FVLP of 3-5pfu/cell, fixed cell after 36h, the mouse-anti FMDV O type VP1 monoclonal antibody of preparing with my chamber (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. tutor: Wu Bin) be primary antibodie, take Cy3 mark sheep anti-mouse igg antibody (Sigma) as two anti-, with DAPI, nucleus is dyeed, under Laser Scanning Confocal Microscope, observe, result shows with the cell that recombinant baculovirus QP-Ac-FVLP infects to be had compared with strong red fluorescence signal and normal sf9 cell redfree fluorescent signal (Fig. 4), shows that the foreign gene of recombinant baculovirus QP-Ac-FVLP is expressed in sf9 cell.
2, recombinant baculovirus QP-Ac-FVLP expresses the Western blot analysis of foreign protein
Collect the cell that QP-Ac-FVLP infects pathology after 72h, after cracking, carry out SDS-PAGE electrophoresis, go to after nitrocellulose filter, one group take mouse-anti FMDV O type VP1 monoclonal antibody as primary antibodie (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. tutor: Wu Bin), sheep anti-mouse igg-HRP(Sigma) be two anti-, another group take the anti-FMDV hyper-immune serum of rabbit IgG as primary antibodie (Master's thesis: li Renfeng; Detect the foundation of foot and mouth disease virus MAb mediated ELISA method and the amalgamation and expression of the many antigen genes of foot and mouth disease virus. tutor: Wu Bin), goat anti-rabbit igg-HRP(Sigma) be two anti-, carry out Western blot analysis simultaneously, result shows first group of specific band (Fig. 5 A) that has a treaty 28KD, second group has two specific bands, one another treaty of treaty 28KD 36KD(Fig. 5 B), result further shows that the foreign gene of recombinant baculovirus QP-Ac-FVLP obtains effective expression and has immunogenicity in sf9 cell.
3, the detection of the formation virus-like particle of recombinant baculovirus QP-Ac-FVLP in insect cell
Just growing up on the sf9 cell of individual layer, the infection multiplicity inoculation recombinant baculovirus QP-Ac-FVLP with 3-5pfu/cell, collects sick cell ultrasonic treatment after 72 hours.Under 4 ℃ of conditions, the centrifugal 30min of 8000g, is then concentrated to 1/10 of original volume with sucrose, contains the VLP particle of expression in supernatant.By supernatant 27,000rpm/min, ultracentrifugation 3 hours, precipitation is resuspended in the PBS of 2ml, and repeatedly blow and beat with the syringe of 1ml, then viral suspension is undertaken 27 by 20-60% sucrose density gradient, 00rpm/min ultracentrifugation 16 hours, the protein band of collecting the superiors carries out electron microscopic observation, result is presented at the typical baculovirus particle of observed under electron microscope and FMDV sample virion (Virus-like particles, VLPs), diameter is about 20nm, its form and size all to FMDV totivirus particle similar (Fig. 6).
Embodiment 2: preparation and the application of recombinant baculovirus QP-Ac-FVLP subunit vaccine
1, the preparation of recombinant baculovirus QP-Ac-FVLP subunit vaccine
In order to evaluate the immune effect of recombinant baculovirus QP-Ac-FVLP subunit vaccine in prevention O type mouth disease virus infection, the insect cell High Five by the recombinant baculovirus QP-Ac-FVLP obtaining with the infection multiplicity inoculation suspension culture of 3-5pfu/cell tM(purchased from Invitrogen company), collects virus liquid after 72h, the centrifugal 10min collecting cell of 2000rpm is also counted with cell counting count board, and with PBS, adjusting number of cells is 2 * 10 7individual/ml and 2 * 10 6individual/two of ml gradient, after multigelation 3 times with isopyknic Freund's complete adjuvant (Sigma) emulsification, put 4 ℃ temporary.
2, the immuning effect test of small white mouse
The subunit vaccine preparing is taked to the mode immune mouse of leg muscle injection, every immunity 100 μ L, twice of immunity, 3 weeks, interval, using not through virus infection and take the HighFive groups of cells of subunit vaccine same treatment mode as negative control group, usining commercialization O type FMD inactivated vaccine group as positive controls.
The ELISA antibody horizontal of 2.1 recombinant baculovirus QP-Ac-FVLP inductions
The 21st day, 35 days and blood sampling in 42 days after exempting from head before head exempts from, and prokaryotic expression FMDV O type VP1 albumen for separation of serum (Lu is suitable, Dong Xiaohui, and Zhang Chunjuan, but the Chinese is also, Zhang Keshan, Wu Bin, Chen Huanchun.A kind of development that detects the indirect ELISA reagent kit of swine foot-and-mouth disease virus antibody.2009, the ten nationwide scaleization hog mans want supervision of epidemic disease and purify symposium) coated plank detects the ELISA antibody in serum, and result is as Fig. 8.As can be seen from the figure: immune group Serum Antibody after booster immunization is in rising trend, wherein immunity 10 6the antibody horizontal of individual groups of cells is than immunity 10 5individual groups of cells ascendant trend is obvious and level is high, but the two difference not significantly (P>0.05) peaks in the time of latter 35 days substantially in immunity, rises subsequently more slow.In whole process of the test, cell negative control group does not detect specific ELISA antibody (Fig. 7).
2.2 real-time fluorescence quantitative PCRs detect IFN mrna expression in immune mouse spleen cell
Little mouse's head is exempted from dislocation execution afterwards in 42 days, spleen is taken out in aseptic technique, isolated cell adds the FMDV virus of deactivation to cultivate 20h as stimulating factor, adopt TRIzol method to extract total RNA of cell, utilize the method for SYBRGreen I real-time quantitative PCR to detect IFN-α, IFN-β, the IFN-Y mrna expression situation in immune mouse spleen cell.Result can find out that recombinant baculovirus immune group can stimulate the IFN-α of mouse induction generation higher level, and IFN-β, IFN-Y are without obviously increasing (Fig. 8).
Although content of the present invention is to describe in conjunction with the present embodiment, can not think limitation of the scope of the invention, scope of the present invention is limited by appended claims.In addition, in the scope that those skilled in the art limits at appended claims, the present invention is carried out to various changes or modification, these changes or modified forms fall within the scope of the present invention equally.
SEQUENCE LISTING
<110> Hua Zhong Agriculture University
<120> mono-boar O type foot and mouth disease virus recombinant baculovirus and preparation method and application
<130> mono-boar O type foot and mouth disease virus recombinant baculovirus and preparation method and application
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 915
<212> DNA
<213> synthetic
<400> 1
atgggcgccg ggcaatccag cccgacgact gggtcacaaa accagtctgg caacactggc 60
agcattatta acaattacta catgcagcag taccagaact caatggacac ccaacttggc 120
gacaacgcca tttgtggagg gtctaatgag ggctccacgg atactacctc cacccacacc 180
aacaacactc agaacaacga ctggttttca aaactggcta acaccgcttt tagcggcctc 240
ttcggtgccc ttcttgcaga caagaagacg gaagaaacca ccctcctcga agaccgcatc 300
ctcaccaccc gcaacgggca cacgacctcg acaacccagt ctagcgtcgg ggtgacgtac 360
gggtacgcaa cggctgaaga cttcgtgagt gggcctaaca cctccggtct tgagaccaga 420
gtcgttcagg ccgaacggtt cttcaaaacc cacctgttcg actgggtcac cagtgacccg 480
tttggacggt gtcacttgtt ggagctaccg accgaccaca aaggtgtcta cggtagcctg 540
accgactctt acgcgtacat gaggaatggt tgggacgttg aagtcaccgc agtggggaac 600
cagttcaacg gaggctgttt gctggtagcg atggtaccgg agctctgttc tatcaacaag 660
agagagttgt accaactcac gcttttcccc caccagttca tcaacccacg gacgaacatg 720
acggcacaca tcaccgtgcc ctaccccggt gttaacaggt acgaccagta caaggtacac 780
aaaccctgga ccctcgtggt catggttgtg gctcccctga cggttaacaa cgagggcgct 840
ccgcaaatca aggtgtatgc caacatcgcc cccaccaacg ttcacgtcgc aggcgagctc 900
ccttccaaag agtaa 915
<210> 2
<211> 639
<212> DNA
<213> synthetic
<400> 2
atgaccacct ctgcgggtga gtccgcggac cccgtgactg ccaccgttga gaactacggt 60
ggcgagacac aagcccagag acgccaacac acggacattt cgttcatact agacaggttc 120
gtgaaagtca agccaaagga acaagtcaac gtgttggacc tgatgcagat ccctgcccac 180
accttggtgg gggcgctcct gcgaacggcc acctactact tctctgacct ggaactggct 240
gtcaagcacg agggcgatct cacctgggtc ccaaacggtg cccctgagac agcactggac 300
aacactacca acccaacagc ttaccacaag gaaccgctca cacggctggc actgccttac 360
acggctccac accgtgtctt ggcgaccgtc tacaacggga gcagtaagta cggtgacacc 420
agcactaaca acgtgagagg tgaccttcaa gtgttggctc agaagacgga aagaactctg 480
cctacttcct tcaacttcgg tgccattaag gctactcgtg tgactgaact actctacaga 540
atgaagagag ccgagacgta ctgtcccagg ccccttctcg ctattcagcc gagtgacgcc 600
agacacaagc agaagattgt ggcacccgca aaacagtaa 639
<210> 3
<211> 666
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<213> synthetic
<400> 3
atggggatct tccccgtggc atgcagcgat ggttacggtg gcttggtgac cacggatccg 60
aagacggcag accccgtcta cgggaaagtg ttcaacccac cccgtaacct gttgccaggg 120
cggttcacga acctcctcga cgtggccgag gcgtgcccta cgttcctaca cttcgacggc 180
gacgttccgt acgtgaccac aaagacggac tcagacaggg tgttggccca gttcgacttg 240
tccctcgcgg cgaaacacat gtcgaacacc tttctcgcgg gtcttgccca gtactacaca 300
cagtacagtg gcaccattaa cctacacttc atgttcacgg ggcccaccga cgcgaaggca 360
cgctacatgg ttgcgtatgc ccctcctggc atggaaccgc cgaaaacgcc tgaggcggct 420
gcacactgta tccacgctga gtgggacaca gggttgaatt cgaagttcac gttttcaatc 480
ccataccttt cggcagccga ctacgcgtac accgcgtccg atgtcgccga gaccactaac 540
gttcagggat gggtctgtct gttccagata acacacggga aagcagacgg tgatgctctg 600
gtcgtgctgg ctagtgccgg caaagacttt gacttgcgcc tgccggttga cgcccggacc 660
caataa 666

Claims (3)

1. one kind contains pig O type foot and mouth disease virus recombinant baculovirus, it is characterized in that, recombinant baculovirus QP-Ac-FVLP Recominant Autographa californica multiple Nucleopolyhedrovirus QP-Ac-FVLP, CCTCC V201237.
2. Antigenicgene VP0, a VP1 who contains the honest and just horse pedigree coding of manually modified synthetic pig O type foot and mouth disease virus and the baculovirus transfer vector pFBDPHmVP013 of VP3 gene, the nucleotide sequence of described VP0, VP1 and VP3 gene is respectively SEQ ID NO:1, shown in SEQ ID NO:2 and SEQ ID NO:3.
3. the application of recombinant baculovirus claimed in claim 1 in preparation prevention pig O style of the shoe mouth epidemic disease viral genetic engineering subunit vaccine.
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