CN104561049A - Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application - Google Patents

Recombinant baculovirus expressing porcine parvovirus VP2 protein as well as preparation method and application Download PDF

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CN104561049A
CN104561049A CN201510031795.7A CN201510031795A CN104561049A CN 104561049 A CN104561049 A CN 104561049A CN 201510031795 A CN201510031795 A CN 201510031795A CN 104561049 A CN104561049 A CN 104561049A
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ppvp2
recombinant baculovirus
ppv
gene
cell
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钱平
李祥敏
钱苏红
石林
陈焕春
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Huazhong Agricultural University
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Huazhong Agricultural University
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Abstract

The invention discloses recombinant baculovirus expressing porcine parvovirus VP2 protein as well as a preparation method and an application. The method comprises the following steps: artificially synthesizing VP2 gene by referring to the VP2 gene sequence of a porcine parvovirus (PPV) isolate; with pFBDPHmHNM1P10eGFP plasmid as a skeleton, connecting the synthesized VP2 gene to the plasmid to obtain a baculovirus transfer vector pFBDPHm3VP2 and then obtain recombinant bacmid rBacmid-PPVP2; and transfecting the bacmid with sf9 cell to obtain recombinant baculovirus Ac-PPVP2. The recombinant baculovirus Ac-PPVP2 efficiently expresses PPV VP2 protein and successfully forms virus-like particles. The protein expressed by the recombinant baculovirus disclosed by the invention is used for preparing a subunit vaccine; and after the subunit vaccine immunizes an animal, the body can be induced to generate a specific immunoreaction, and the porcine body can be fully protected from the attack of strong poison of parvovirus.

Description

A kind of express PPV VP 2 protein recombinant baculovirus and preparation method and application
Technical field
The present invention relates to biotechnology and field of virology.Be specifically related to a kind of recombinant baculovirus Ac-PPVP2 of pig parvoviral, also relate to the preparation method of a kind of recombinant baculovirus Ac-PPVP2 of pig parvoviral, also relate to the application of the recombinant baculovirus Ac-PPVP2 subunit vaccine of pig parvoviral.The pig parvoviral VP2 gene utilizing recombinant baculovirus expression of the present invention manually modified is also assembled into pig parvoviral sample virion VLP, may be used for preparing pig parvoviral subunit vaccine safely and effectively.
Background technology
Within 1996, Mary and Mahnel finds pig parvoviral (Porcine parvovirus when carrying out Pestivirus suis tissue culture, PPV) (Mary et al., 1968), within 1967 subsequently, Cartwright goes out pig parvoviral from infertile with isolation identification miscarriage sow pathological material of disease, and demonstrate its pathogenic (Cartwright et al., 1969).Porcine parvovirus is a kind of important breeding difficulty sexually transmitted disease of the global pig industry of harm, and especially progestational first farrowing sow, causes huge financial loss to pig industry.Since the nineties in last century, along with developing rapidly of pig industry and emerging in multitude of Compact Develop, PPV infects the trend presenting expansion in China, rise.In recent years, there is new epidemic characteristic in PPV, PPV and JEV, PCV2, PRRSV and PRV polyinfection can occur, and PPV, JEV and PRV tri-kinds of viruses also can polyinfection, make the state of an illness complicated, bring extreme difficulties (Huang Gang etc., 2010) to clinical diagnosis and prevention and control.Synthetical prevention must be carried out, to put prevention first: (1) adheres to importing into from the external world to stop PPV from numerous autotrophy pattern for PPV; (2) the immunization work of pig parvoviral is carried out in strict accordance with immune programme for children; (3) regularly immune antibody monitoring is carried out; (4) porcine parvovirus pig must effectively be isolated with healthy swinery and be eliminated, must harmless treatment be carried out to the ight soil of sick pig, dirt, feed, and strengthen sterilization and periodic monitoring; (5) strengthen feeding and management, carry out health and epidemic prevention disinfection etc. in real earnest.Present stage, the prevention and corntrol to porcine parvovirus mainly used vaccine, along with the development of Protocols in Molecular Biology, made porcine parvovirus vaccine research just gradually from traditional deactivation and attenuated vaccine to recombinant vaccine transition.
Pig parvoviral (Procine parvovirus, PPV) member that Parvoviridae (Parvoviridae) parvovirus belongs to is belonged to, its genome is strand linear DNA molecule, genome encoding three kinds of structural protein (VP1, VP2 and VP3) and three kinds of Nonstructural Proteins (NS1, NS2 and NS3) (Xu and Li, 2007).Wherein Viral structural protein VP2 is the main component forming capsid protein, the polypeptide energy oneself of VP2 genes encoding is assembled into virus-like particle, is good antigen delivery carrier, also has good immunogenicity simultaneously, very strong cellular immunization (Adriaan et al., 2006) can be induced.The diseases related China's pig industry of giving of PPV brings tremendous economic to lose, the current control infected PPV is mainly through strengthening feeding and management and bio-security, but PPV infection rate in swinery is higher and the resistibility of PPV environment is to external world comparatively strong, is thus infected by the PPV of vaccine immunity prevention safely and effectively and seems very important.Virus sample particle vaccines (the Virus likeparticles that development in recent years is got up; VLP); containing virulent one or more antigen protein, there is the particle with virus particle analog structure; not containing virulent genetic material; can not self-replicating, there is no infectivity, can with native conformation and pattern present antigen, effective stimulus humoral and cellular immune response; induction body produces good immunoprotection (Li YJ et al., 2007).
Baculovirus expression system have simple to operate, security is high, the goal gene of large fragment can be held, express foreign protein effect high, have the effect of posttranslational modification, the immunogenicity of expressing protein, advantage (Anderson et al., 1995 such as biological activity and natural protein similar; Wang et al., 2001; Ribeiro et al., 2001).In recent years, rhabdovirus expression vector, with the advantage of self, account for leading status on expression vector.In view of the advantage of rhabdovirus expression vector and the characteristic of virus sample particle vaccines, utilize baculovirus vector expression system great expression PPV VP2 gene in insect cell, and successful assembling assembly virus-like particle VLP, prepare subunit vaccine with this, the immune me chanism infecting relative disease for PPV provides important candidate vaccine strain.
Summary of the invention
An object of the present invention is to provide a kind of cDNA sequence of pig parvoviral (Porcine parvovirus, PPV) VP2 gene of manually modified synthesis, its sequence is as shown in SEQ ID NO.1, and the protein of coding is for shown in SEQ ID NO.2.
Another object of the present invention is to provide a kind of baculovirus transfer vector pFBDPHm3VP2, and this carrier contains cDNA sequence (shown in SEQ ID NO.1 sequence) and the marker gene eGFP of 3 copy PPV immunogenic gene VP2.
A further object of the invention is just to provide a kind of recombinant baculovirus of expressing PPV VP 2 protein, this strain is named as recombinant baculovirus Ac-PPVP2, belong to baculovirus (Baculovirus), this virus is containing PPV immunogenic gene VP2, this virus delivers China typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on September 9th, 2013, its preserving number is CCTCC NO:V201339, Classification And Nomenclature: recombinant baculovirus (Recominant Autographa californica multiple Nucleopolyhedrovirus) Ac-PPVP2, address: Wuhan, China Wuhan University.
A further object of the invention there is provided the preparation method of a kind of pig parvoviral recombinant baculovirus Ac-PPVP2, and method is simple, easy, is applicable to scale operation.
A further object of the invention there is provided a kind of pig parvoviral recombinant baculovirus Ac-PPVP2 and is preparing the application in subunit vaccine, by body can be induced after this virus immunity animal to produce specific immune response, possesses the strong malicious characteristic of anti-PPV.
In order to achieve the above object, the present invention takes following technical measures:
Express a recombinant baculovirus for PPV VP 2 protein, prepared by following steps:
1, the manually modified synthesis of PPV main immunogenic gene VP2
With reference to the VP2 gene order (GenBank:AY583318) of pig parvoviral Chinese strain, according to the VP2 gene of the preferences synthetic PPV of codon, its nucleotides sequence is classified as shown in SEQ ID NO.1.
2, the structure of baculovirus transfer vector pFBDPHm3VP2
With pFBDPHmHNM1P10eGFP plasmid for skeleton, cut pFBDPHmHNM1P10eGFP by BamHI and EcoRI enzyme, reclaim large fragment and obtain pFBDPHmHNM1.By BamHI and EcoRI respectively enzyme cut PPV VP2 gene and pFBDPHmHNM1, reclaim VP2 gene and pFBDPHmHN, then connected in 16 DEG C by ligase enzyme Ligase, extract recombinant plasmid, enzyme cuts qualification, obtains baculovirus transfer vector pFBDPHmHNVP2.And then cut PPV VP2 gene and pFBDPHmHNVP2 by SpeI and NotI enzyme, reclaim VP2 gene and pFBDPHmHVP2, then connected in 16 DEG C by ligase enzyme Ligase, extract recombinant plasmid, enzyme cuts qualification, obtains baculovirus transfer vector pFBDPHmH2VP2.And then by SaLI and HindIII respectively enzyme cut PPV VP2 gene and pFBDPHmH2VP2, reclaim VP2 gene and pFBDPHm2VP2, then connect, after extracting recombinant plasmid, enzyme cuts the baculovirus transfer vector pFBDPHm3VP2 that qualification shows to obtain 3 VP2 genes.
3, the structure of recombinant virus baculovirus Ac-PPVP2
(1) to recombinate the acquisition of rod granule rBac-PPVP2 and qualification
Get the correct baculovirus transfer vector pFBDPHm3VP22.0 μ L of order-checking qualification to mix with 100 μ L DH10Bac competent escherichia coli cells (GiBCO BRL Products), after ice bath 30min, heat shock is carried out in 42 DEG C of 45s water-baths, then ice bath 2min, add 900 μ L LB liquid nutrient mediums (sodium chloride concentration is 10%), 37 DEG C of joltings cultivate 4h, by 10 -1, 10 -2, 10 -3after dilution, respectively get 100 μ L and coat three high salt tolerance LB flat board (kantlex, gentamicin and tsiklomitsins, working concentration presses Invitrogen company Bac-to-Bac Baculovirus Expression System (test kit) process specifications), cultivate 24-48h for 37 DEG C, by blue hickie screening, the positive bacterium colony of purifying, extract restructuring rod granule rBacmid-VP2.
(2) recombinant baculovirus Ac-PPVP2 obtains
Utilize liposome mediated transfection method, by in the restructuring rod granule rBacmid-VP2 transfection sf9 cell after screening purifying, in 28 DEG C of cultivations, 24h, 48h, 72h utilize fluorescence microscope recombinant baculovirus green florescent signal after transfection respectively, after transfection 72h, namely collecting cell supernatant obtains recombinant baculovirus Ac-PPVP2, and then on insect cell, inoculate this recombinant virus, during the 4th generation, poison valency reaches 1.0 × 10 8.125, be placed in-80 DEG C as seed culture of viruses and save backup.
So far the recombinant baculovirus that PPV VP 2 protein is expressed in a strain is obtained, this virus has delivered China typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on September 9th, 2013, its preserving number is CCTCC NO:V201339, Classification And Nomenclature: recombinant baculovirus (Recominant Autographa californica multipleNucleopolyhedrovirus) Ac-PPVP2, address: Wuhan, China Wuhan University.
This restructuring bar virus Ac-PPVP2 has and embeds the identical morphological characteristic of nuclear polyhedrosis virus (AcMNPV) and cultural characters with parent's strain autogra-phacalifornica many.
Pig parvoviral recombinant baculovirus Ac-PPVP2 is preparing the application in subunit vaccine, by body can be induced after this vaccine immunity animal to produce specific immune response, has the ability characteristics of anti-PPV strong virus attack.
Concrete scheme refers to embodiment.
The invention has the beneficial effects as follows:
1. the present invention is according to the preferences of baculovirus codon, manually modified gene order of having synthesized pig parvoviral VP2 gene.
2. the present invention expresses under the pig parvoviral VP2 gene of synthetic is placed in baculovirus pH promotor, improves the expression amount of foreign gene.Being inserted under baculovirus transfer vector being placed in baculovirus pH promotor and expressing, is the expression amount that 3 copy forms can improve foreign gene because inserting.
3. expressed by recombinant baculovirus of the present invention, pig parvoviral VP2 gene successfully can form pig parvoviral sample virion (VLPs).
4. the expressed chimeric VLPs of recombinant baculovirus of the present invention prepares subunit vaccine, and body generation can be induced after immune animal for the specific immune response of pig parvoviral.
Accompanying drawing explanation
Fig. 1: for the enzyme of a kind of baculovirus vector pFBDPHm3VP2 cuts qualification collection of illustrative plates;
M:15000bp DNA Marker; 1 & 2:pFBDPHm3VP2BamHI and HindIII double digestion.
Fig. 2: for one restructuring rod granule rBacmid-VP2PCR identifies schematic diagram;
Identify that correct baculovirus transfer vector pFBDPHm3VP2 transforms DH10Bac, blue hickie screens positive bacterium colony, extracts the electrophoresis picture that rod granule rBacmid-VP2 carries out PCR qualification.
M1 and M2:DNA Marker
1-5:rBacmid-VP2 (the PCR result of M13 (F)/VP2 (R1) primer), clip size is about 5100bp
7-10:rBacmid-VP2 (the PCR result of VP2 (F3)/M13 (R) primer), clip size is about 2500bp
6 & 11: water contrast water control
Fig. 3: for a kind of Western blot analyzes the schematic diagram that recombinant baculovirus Ac-PPVP2 expresses VP2 albumen;
M: protein marker; The Western blotting of collecting cell after 1: baculovirus Ac-PPVP2 infection sf9 cell; The Western blotting of collecting cell after 2:sf9 cell
Fig. 4: be a kind of expression schematic diagram of indirect immunofluorescence analysis recombinant baculovirus Ac-PPVP2VP2 albumen;
A: the sf9 cell do not infected; The sf9 cell that B:Ac-eGFP infects; The sf9 cell that C:Ac-PPVP2 infects;
EGFP: be green fluorescence basis of microscopic observation; Red: be red fluorescence basis of microscopic observation (CY3); Merge: red and green fluorescence basis of microscopic observation overlay chart
Fig. 5: for a kind of recombinant baculovirus Ac-PPVP2 expresses the mensuration of VP2 protein content.
On the sf9 cell just growing up to individual layer, with 1.0MOI dose inoculation recombinant baculovirus Ac-PPVP2, after infecting, 96h receives poison, the expression amount of quantitative assay PPV VP2 albumen after SDS-PAGE electrophoresis, simultaneously and adherent HF cell carried out contrast experiment.
1-3:BSA standard protein sample: 36.25 μ g, 62.5 μ g, 125 μ g
5-6: suspension culture HF cell infects the sample after Ac-PPVP2: 92 μ g/mL
7-8: the sample after the HF cellAc-PPVP2 of adherent culture: 56 μ g/mL;
4: normal sf9 cell controls
Fig. 6: for forming the Electronic Speculum schematic diagram of virus-like particle after a kind of recombinant baculovirus Ac-PPVP2 infection sf9 cell.
Recombinant baculovirus Ac-PPVP2 is with the infective dose of 3 ~ 5pfu/cell inoculation sf9 cell, 72 h before harvest sick cells ultrasonic treatment and ultracentrifugation carry out electron microscopic observation, in typical icosahedral symmetry structure, without cyst membrane, diameter is about 23 ~ 28nm, its form size is all similar to pig parvoviral particle, shows assembling assembly virus-like particle VLPs.
Fig. 7: be a kind of Detection of Stability schematic diagram of recombinant baculovirus Ac-PPVP2 expression alien gene.
Ac-PPVP2 is inoculated sf9 cell and carry out continuous passage to 20 generation.Get F5, F7, F9, F11, F13, F15, F17 and F20 and carry out the expression of Western blot analysis VP2 protein stabilizes for virus infected cell as detection sample.
M: albumen marker; 1 ~ 8:F5, F7, F9, F11, F13, F15, F17 and F20 infect the Western blot detected result of sf9 cell sample for Ac-PPVP2.
Fig. 8: be the ELISA antibody schematic diagram of the anti-PPV2 of different time after mouse immune
Subunit vaccine, porcine parvovirus inactivated vaccines (commercial vaccine prepared by the different adjuvant of recombinant baculovirus Ac-PPVP2, positive control) and cell controls immune mouse after within 0,14,28,42 and 56 day, gather serum, detect the ELISA antibody horizontal of anti-PPV2 in different time serum.
Fig. 9: be the ELISA antibody schematic diagram of the anti-PPV2 of different time after rabbit immunity
The subunit vaccine that recombinant baculovirus Ac-PPVP2 is prepared through ISA206VG adjuvant emulsion, porcine parvovirus inactivated vaccines (commercial vaccine, positive control) and cell controls immunize rabbit body after within 0,14,28,42 and 56 day, gather serum, detect the ELISA antibody horizontal of anti-PPV2 in different time serum.
Table 1: the hemagglutination inhibition antibody being the anti-PPV of different times after a boar immunity
After recombinant baculovirus Ac-PPVP2 subunit vaccine, pig parvoviral commercialized vaccine and cell controls immunity piglet, different time points gathers serum, detects the hemagglutination inhibition antibody of anti-PPV.
Embodiment
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.Technical scheme of the present invention, if not otherwise specified, is the conventional scheme of this area, described reality, if not otherwise specified, is commercialization or published reagent material.
Embodiment 1:
A kind of structure of expressing the immunogenic gene VP2 recombinant baculovirus Ac-PPVP2 of the PPV2 of manually modified synthesis
(1) structure of recombinant baculovirus Ac-PPVP2
1, the manually modified synthesis of PPV main immunogenic gene VP2
With reference to the VP2 gene order (GenBank:AY583318) of pig parvoviral Chinese strain, according to the VP2 gene of the preferences synthetic PPV of codon, its nucleotides sequence is classified as shown in SEQ ID NO.1, and the protein of coding is for shown in SEQ IDNO.2.
2, the structure of baculovirus transfer vector pFBDPHm3VP2
With pFBDPHmHNM1P10eGFP plasmid (money equality, a kind of recombinant baculovirus of expressing the influenza A H 1 N 1 virus HA-NA-M 1 gene of manually modified synthesis, patent publication No. CN101624580A, patent No. ZL200910063217.6) be skeleton, cut pFBDPHmHNM1P10eGFP by BamHI and EcoRI enzyme, reclaim large fragment and obtain pFBDPHmHNM1.By BamHI and EcoRI respectively enzyme cut PPV VP2 gene and pFBDPHmHNM1, reclaim VP2 gene and pFBDPHmHN, then connected in 16 DEG C by ligase enzyme Ligase, extract recombinant plasmid, enzyme cuts qualification, obtains baculovirus transfer vector pFBDPHmHNVP2.And then cut PPV VP2 gene and pFBDPHmHNVP2 by SpeI and NotI enzyme, reclaim VP2 gene and pFBDPHmHVP2, then connected in 16 DEG C by ligase enzyme Ligase, extract recombinant plasmid, enzyme cuts qualification, obtains baculovirus transfer vector pFBDPHmH2VP2.And then by SaLI and HindIII respectively enzyme cut PPV VP2 gene and pFBDPHmH2VP2, reclaim VP2 gene and pFBDPHm2VP2, then connect, after extracting recombinant plasmid, enzyme cuts the baculovirus transfer vector pFBDPHm3VP2 that qualification shows to obtain 3 VP2 genes.
3, the structure of recombinant virus baculovirus Ac-PPVP2
(1) to recombinate the acquisition of rod granule rBac-PPVP2 and qualification
Get the correct baculovirus transfer vector pFBDPHm3VP22.0 μ L of order-checking qualification to mix with 100 μ L DH10Bac competent escherichia coli cells (GiBCO BRL Products), after ice bath 30min, heat shock is carried out in 42 DEG C of 45s water-baths, then ice bath 2min, add 900 μ L LB liquid nutrient mediums (sodium chloride concentration is 10%), 37 DEG C of joltings cultivate 4h, by 10 -1, 10 -2, 10 -3after dilution, respectively get 100 μ L and coat three high salt tolerance LB flat board (kantlex, gentamicin and tsiklomitsin, working concentration presses Invitrogen company Bac-to-Bac Baculovirus Expression System (test kit) process specifications), cultivate 24-48h for 37 DEG C, screened by blue hickie, the positive bacterium colony of purifying, extract restructuring rod granule rBacmid-VP2, by two pairs of PCR primer amplification qualifications: M13 upstream primer (M13F) and PPV F3 (PPV F3), PPV F4 upstream primer (PPV F4) and M13 downstream primer (M13R).
M13F:5’-CCCAGTCACGACGACGTTGTAAAACG-3’
M13R:5’-AGCGGATAACAATTTCACACAGG-3’
VP2(R1)5’-CCCGAATTCTTAGTACAGCTTTCTTGGAATC-3’
VP2(F3)5’-TTGTCGACCACCATGAAGCACAAGGTCAG-3’
Its PCR amplification system is as follows: 10 × LA buffer 5.0 μ l, LA Taq enzyme 0.5 μ l, rod granule 5.0 μ l, specific upstream primer 1.0 μ l, specific Down Stream primer 1.0 μ l, dNTPs 2.0 μ l, ddH2O 35.5 μ l.Reaction conditions is as follows: 94 DEG C of 3min; 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 3.0min, 30 circulations; 72 DEG C of 10min, 4 DEG C of 10min.The PCR primer increased is through the agarose gel electrophoresis analysis of 0.8% (w/v), result shows to amplify two special object bands of 5100bp and 2500bp, size is consistent with expection, show that foreign gene VP2 has been recombined in Baculovirus Gene group in intestinal bacteria, result as shown in Figure 2.
Aseptic picking falls within high salt LB liquid nutrient medium containing the positive bacteria of restructuring rod granule rBac-PPVVP2, when being cultured to bacteria log vegetative period, collect thalline 0.3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol/LTris-Cl (pH8.0)) resuspended, add 0.3mL solution II (0.2mol/L NaOH, 1%SDS, matching while using) slightly mix, room temperature leaves standstill 5min, 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, mixing ice bath 5-10min, the centrifugal 15min of room temperature 14000r/min, 70% washing with alcohol precipitation, be dissolved in 40 μ L TE after drying, can use immediately or-20 DEG C of preservations, avoid multigelation.
(2) recombinant baculovirus Ac-PPVP2 obtains
Utilize liposome mediated transfection method (by the operation of Invitrogen company Bac-to-Bac Baculovirus Expression System (test kit) process specifications), by the restructuring rod granule rBacmid-VP2 warp after screening purifying in Reagent transfection sf9 cell (purchased from Wuhan University's Chinese Typical Representative thing preservation center), in 28 DEG C of cultivations, 24h, 48h, 72h utilize fluorescence microscope recombinant baculovirus green florescent signal after transfection respectively, after transfection 72h, namely collecting cell supernatant obtains recombinant baculovirus Ac-PPVP2, and then on insect cell, inoculate this recombinant virus, during the 4th generation, the malicious valency of recombinant virus reaches 1.0 × 10 8.125, be placed in-80 DEG C as seed culture of viruses and save backup.
So far the recombinant baculovirus that pig parvoviral VP2 gene is expressed in a strain is obtained, this virus delivers China typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on September 9th, 2013, its deposit number is CCTCC NO:V201339, Classification And Nomenclature: recombinant baculovirus (Recominant Autographa californica multipleNucleopolyhedrovirus) Ac-PPVP2, address: Wuhan, China Wuhan University.
This restructuring bar virus Ac-PPVP2 has and embeds the identical morphological characteristic of nuclear polyhedrosis virus (AcMNPV) and cultural characters with parent's strain autogra-phacalifornica many.
(2), the expression of recombinant baculovirus Ac-PPVP2 foreign gene and the formation of virus-like particle (VLPs)
1, the Westerning blot of recombinant baculovirus Ac-PPVP2 expression VP2 albumen analyzes
Sf9 cell is inoculated 6 porocyte culture plates, when cell grows up to 80-90% individual layer, inoculate sf9 cell with 1 MOI recombinant baculovirus Ac-PPVP2, infect the sf9 cell generation pathology of after 72 hours more than 90%, collect sick cell and ultrasonic treatment.After SDS-PAGE transferring film, VP2 albumen resists for primary antibodie (this laboratory preparation more, PPV VP2 albumen is carried out immunize New Zealand rabbit preparation after prokaryotic expression, purifying), the goat anti-rabbit igg (Sigma be) two of HRP mark is anti-carry out Western-blotting detection VP2 because of expression.There is the specific band of a treaty 67kD in result display recombinant virus Ac-PPVP2 cells infected sample, and sf9 cell controls group is without this specific band, confirms that VP2 albumen obtains to express, and result as shown in Figure 3.
2, the expression of indirect immunofluorescence qualification foreign gene
On the sf9 cell just growing up to individual layer, with the infection multiplicity of 3-5pfu/cell inoculation recombinant baculovirus Ac-PPVP2, fixed cell after 36h, with the polyclonal antibody of PPV VP2 albumen (after PPV VP2 albumen being carried out prokaryotic expression, purifying prepared by immunize New Zealand rabbit) for primary antibodie, two resist for CY3-goat anti-rabbit igg (Sigma) carries out indirect immunofluorescence, observe under the most rearmounted Laser Scanning Confocal Microscope.Infect in the sf9 cell of Ac-PPVP2 and all have very strong red fluorescent, and sf9 cell does not have red fluorescent, shows that recombinant baculovirus Ac-PPVP2 can express VP2 albumen and have good immunocompetence, result as shown in Figure 4.
3, the mensuration of recombinant baculovirus VP2 expressing quantity
On the sf9 cell just growing up to individual layer, with the 1.0MOI dose inoculation recombinant baculovirus Ac-PPVP2 (cell about 2.4 × 10 of virus infection 6individual every milliliter), after infecting, 96h receives poison, and after SDS-PAGE electrophoresis, the expression amount of quantitative assay display PPV VP2 albumen is about 92 micrograms per millilitre (μ g/mL).Simultaneously and adherent HF cell carried out contrast experiment, result shows the optimum cell (as shown in Figure 5) that suspension HF cell is amplifying target genes.
4, the Detection of Stability of recombinant baculovirus Ac-PPVP2 expression alien gene
Recombinant baculovirus Ac-PPVP2 is inoculated sf9 cell, carries out being passaged to for 20 generations.Get F1, F2, F5, F10, F15, F20 for virus infected cell as detection sample, with PPV VP2 albumen monoclonal antibody for primary antibodie, it is two anti-carry out Western blot analysis that HRP marks sheep anti-mouse igg, all can there is the specific band of a treaty 67kD in result display F1, F2, F5, F10, F15, F20, show that VP2 protein stabilizes expresses (result such as Fig. 7 shows) for cells infected sample on recombinant virus.
5, recombinant baculovirus Ac-PPVP2 forms the detection of virus-like particle in insect cell
On the sf9 cell just growing up to individual layer, with the infection multiplicity of 3-5pfu/cell inoculation recombinant baculovirus Ac-PPVP2,72 h before harvest sick cells ultrasonic treatment.Under 4 DEG C of conditions, the centrifugal 30min of 8000g, is then concentrated to 1/10 of original volume with sucrose, namely containing the VLP particle of expressing 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 20-60% sucrose density gradient carries out 27,00rpm/min ultracentrifugation 16 hours, the protein band collecting the superiors carries out electron microscopic observation, and result is presented at observed under electron microscope to a large amount of virus-like particle (Virus-like particles, VLPs), diameter is about 25nm, its form and size are all similar to PPV totivirus particle, and show successfully to form VLPs, result as shown in Figure 6.
Embodiment 2:
The security of recombinant baculovirus Ac-PPVP2 subunit vaccine, immune efficacy experiment
(1), the safety testing of the recombinant baculovirus Ac-PPVP2 subunit vaccine of invention
1. the preparation of recombinant baculovirus Ac-PPVP2 subunit vaccine
(the insect cell High Five of suspension culture is inoculated with the infection multiplicity of 3-5pfu/cell with the recombinant baculovirus that embodiment 1 obtains tMpurchased from Invitrogen company) sf9 cell, collecting cell and supernatant after 72h, by removing cell debris centrifugal after cell crushing instrument process, through binary ethylenimine (BEI) deactivation 36 ~ 48 hours of 7mM, to add in the Sulfothiorine of equivalent and rear and oily adjuvant emulsion is prepared into subunit vaccine, be stored in 4 DEG C for subsequent use.In the vaccine of final acquisition, foreign protein PPV VP2 albumen is 20 micrograms per millilitre (μ g/ml), for safety testing.
2. newborn piglet safety testing
Subunit vaccine prepared by laboratory 4 batches, often criticize and get 3 bottles immediately, after mixing, select healthy 1 age in days newborn piglet, often criticize vaccine inoculation 10 pigs, each intramuscular injection 2 multiple dose (multiple dose is the vaccine of 20 micrograms per millilitre, and every pig injects 2 milliliters) parvovirus oil-emulsion subunit vaccine, i.e. the vaccine of step 1 acquisition of every pig injection 4mL; Separately set 4 pigs as blank.All piglets before vaccinate and after vaccinate 1 week, measure 2 body temperature every day and observe the upgrowth situation of pig, spirit and appetite, Continuous Observation 14 days.Vaccine inoculation body temperature rise on same day 0.2-0.5 DEG C, recovers normal for second day, and spirit and appetite normally, have no adverse reaction, show that vaccine is safe to newborn piglet.
3. pregnant sow safety testing
Subunit vaccine prepared by laboratory 4 batches, often criticize and get 3 bottles immediately, after mixing, select healthy pregnant sow, inoculation when pregnant 40 days, often criticize vaccine inoculation 10 pigs, (multiple dose is each intramuscular injection 2 multiple dose: the vaccine of 20 micrograms per millilitre, every pig injects 2 milliliters) parvovirus oil-emulsion subunit vaccine, the vaccine that namely step 1 of every pig injection 4mL is obtained; Separately set 2 pigs as blank.Body temperature, spirit, appetite and the impact on sow reproductive performance is observed after vaccine inoculation.Result shows that body temperature, spirit and appetite are all good; Pregnant sow is after this vaccine of inoculation, and pregnancy status is normal, does not occur the clinical symptom of the breeding difficultys such as miscarriage, fetus dissolving, shows that this subunit vaccine is safe to pregnant sow.
(2), the immunogenicity experiments of recombinant baculovirus Ac-PPVP2 subunit vaccine
1. the Study On Immunogenicity of recombinant baculovirus Ac-PPVP2 subunit vaccine immunity to mouse
In order to evaluate the immunogenicity of Ac-PPVP2 subunit vaccine for pig parvoviral of above-mentioned preparation, buying female Ba/bc mouse 18 in 6 week age, being divided into 5 groups at random, often organize 6.The negative group of PBS, positive group (the veterinary drug new word (2006) 170041059 of pig parvoviral deactivation commercial vaccine group, as follows), and the Ac-PPVP2 subunit vaccine group of ISA201VG, ISA206VG (purchased from Seppic company, as follows) and the emulsification of AL (OH) 3 adjuvant difference.Immunizing dose is Ac-PPVP2 group every mouse immune 3.0 microgram VP2 albumen; Pig parvoviral deactivation commercial vaccine group is every mouse 100 microlitre.Immunization route is hind leg muscle injection, after 3 weeks with same dosage booster immunization once.Within 0,14,28,40 days, carry out tail vein blood after head exempts from, evaluate its immune effect.
Pig parvoviral ELISA antibody kit detects the display of (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) result: recombinant baculovirus Ac-PPVP2 can induce the ELISA antibody produced for PPV, after head exempts from, antibody horizontal is always in rising trend, after two immunity, two weeks antibody horizontals reach peak value, present downtrending afterwards.There is some difference to its immune effect for different adjuvants: antibody horizontal presents ISA206VG adjuvant higher than ISA201VG adjuvant, and ISA201VG is higher than AL (OH) 3, AL (OH) 3 antibody horizontal of immunity 28 days test group Ac-PPVP2 does not have difference (P>0.05) compared with feminine gender, pole significant difference (P<0.01) is all there is in all the other test group three kinds of adjuvant antibody horizontals compared with negative group, but do not have difference (P>0.05) between three kinds of adjuvants, result such as Fig. 8 shows.The effect of subunit vaccine prepared by ISA201VG emulsification of the present invention is suitable with the immune effect of commercialization inactivated vaccine, but the subunit vaccine prepared due to the present invention is by bionic method great expression VP2 albumen, also can avoid mass propgation parvovirus on cell.
2. recombinant baculovirus Ac-PPVP2 subunit vaccine is to the Study On Immunogenicity of rabbit
Showing ISA206 according to the immune Study On Immunogenicity result to mouse of recombinant baculovirus Ac-PPVP2 subunit vaccine in above-mentioned experimental program is best adjuvant bag water-in-oil two-phase adjuvant, and then utilizes ISA206 emulsification to prepare vaccine further immune effect evaluating this subunit vaccine in rabbit body.Choose 2 monthly age safety Japanese screech owl White Rabbits 18, be divided into 3 groups at random, often organize 6.Vaccine immunity group is subunit vaccine prepared by ISA206 emulsification that the present invention develops, amount containing VP2 albumen is 15.0 micrograms per millilitre, and positive control is the tiny other living vaccine of pig (veterinary drug new word (2006) 170041059) that Ke Qian biological products limited-liability company produces; The HF cell of negative control.Every rabbit leg muscle injection 2mL vaccine (making VP2 albumen be 30.0 micrograms/rabbit), the business inactivated vaccine of the same immunization of positive controls 2 milliliters, the HF cell sample of negative group injection 2 milliliters.Animal immune injection head exempts from rear 28d booster immunization once.Respectively after immunity 0,14,28,42, the blood sampling of 56d ear vein.(purchased from Wuhan Ke Qian biological products limited liability company) is detected to being separated the serum sample pig parvoviral ELISA antibody kit gathered.
Result shows: recombinant baculovirus Ac-PPVP2 and commercial vaccine immune group can both induce the ELISA antibody produced for PPV, after head exempts from, antibody horizontal is always in rising trend, after booster immunization, two weeks antibody horizontals reach peak value, keep very higher antibody horizontal afterwards.All there is pole significant difference (P<0.01) after immunity 14d compared with negative group, result such as Fig. 9 shows.The effect of this subunit vaccine and commercialization inactivated vaccine, but due to the subunit of this development be by bionic method great expression VP2 albumen, mass propgation parvovirus on cell can be avoided, thus there is good application prospect.
3, recombinant baculovirus Ac-PPVP2 subunit vaccine is to the Study On Immunogenicity of pig
The healthy growing and fattening pigs at the subunit vaccine immunity 3-4 monthly age of being prepared by recombinant baculovirus Ac-PPVP2, often organize 5, subunit vaccine prepared by vaccine musculi colli injection ISA206 emulsification, make PPV VP2 albumen be 40 micrograms/pig, immunity 2 times, 4 weeks, interval.Establish inactivated vaccine positive control and nonimmune group of negative control simultaneously, after exempting from head before immunity, within the 2nd, 4,6 and 8 week, gather serum, detect PPV hemagglutination inhibition antibody.After result display vaccine immunity 14 days, HI antibody reached 60, within 4 weeks, reaches 128, after booster immunization 2 weeks and 4 weeks, and antibody horizontal is promoted significantly, reaches 289 and 428 respectively.Result is as shown in table 1.Although the antibody horizontal that the induction of this subunit vaccine produces all produces a little less than inactivated vaccine induction, differ not obvious, and all can meet clinical application requirement.
Table 1: recombinant baculovirus Ac-PPVP2 inactivated vaccine immune swine antibody horizontal
Note: the hemagglutination inhibition antibody detecting PPV, is judged to the positive during HI > 16.
(3), the immune efficacy experiment of recombinant baculovirus Ac-PPVP2 subunit vaccine
The negative 6 monthly age replacement gilt (Landrace×Large White Sows) 9 of PPV serum antibody, is divided into 3 groups at random.The subunit vaccine prepared of immune ISA206 adjuvant emulsion, makes VP2 albumen be 30 micrograms/pig respectively; Porcine parvovirus inactivated vaccines 2 milliliters/pig positive group and physiological saline blank group.First immunisation is after 28 days, with identical dosage booster immunization once, booster immunization is bred for 21 days later.Inoculation 1 × 10 during sow gestation 40 days 6.0tCID 50the strong poison of PPV (Ph D dissertation: the clone of pig parvoviral genome sequence determination and structural protein gene, express and study with genetic immunization, Zhao Junlong, 2001), after this observation sow gestation situation, treats a point puerperium observation farrowing situation.
Sow gestation after attacking poison of subunit vaccine and porcine parvovirus inactivated vaccines immune group is normal.Lower 31 of subunit vaccine immune group common property is lived young, and be good for young 29, weak young 1,1, stillborn foetus, PPV PCR detection is carried out in stillborn foetus sampling, and result is negative, and confirms what its dead not PPV infection caused; Lower 30 of porcine parvovirus inactivated vaccines immune group common property is lived young, weak young 1, without stillborn foetus; And the sow of blank group diminishes a bit at the abdominal circumference of later pregnancy, a point puerperium only has 5 to live young, weak young 4,24, stillborn foetus.This test shows, subunit vaccine prepared by recombinant baculovirus Ac-PPVP2 can provide the opposing PPV immunoprotection of strong virus attack for pregnant sow.
Although content of the present invention is described in conjunction with the present embodiment, can not think limitation of the scope of the invention, scope of the present invention is defined by the appended claims.In addition, those skilled in the art carries out various change or modification to the present invention in the scope that appended claims limits, and these are changed or modified forms falls within the scope of the present invention equally.
SEQUENCE LISTING
 
<110> Hua Zhong Agriculture University
 
<120> mono-kind expresses the recombinant baculovirus of PPV VP 2 protein and preparation method and application
 
<130> mono-kind expresses the recombinant baculovirus of PPV VP 2 protein and preparation method and application
 
<160> 2
 
<170> PatentIn version 3.1
 
<210> 1
<211> 1740
<212> DNA
<213> artificial sequence
 
<400> 1
atgagtgaaa atgtggaaca acacaaccct attaatgcag gcactgaatt gtctgcaaca 60
 
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gtgtctacag gtagtttcaa taatcaaaca gaatttcaat acttggggga gggcttggtt 180
 
agaatcactg cacacgcatc aagactcata catctaaata tgccagaaca cgaaacatac 240
 
aaaagaatac atgtactaaa ttcagaatca ggggtggcgg gacaaatggt acaagacgat 300
 
gcacacacac aaatggtaac accttggtca ctaatagatg ctaacgcatg gggagtgtgg 360
 
ttcaatccag cggactggca gttaatatcc aacaacatga cagaaataaa cttagttagt 420
 
tttgaacaag caatattcaa tgtagtactt aaaacaatta cagaatcagc aacctcacca 480
 
ccaaccaaaa tatataataa tgatctaact gcaagcttaa tggtcgcact agacaccaat 540
 
aacacacttc catacacacc agcagcacct agaagtgaaa cacttggttt ttatccatgg 600
 
ttacctacaa aaccaactca atacagatat tacctatcat gcatcagaaa cctaaatcca 660
 
ccaacataca ctggacaatc acaacaaata acagactcaa tacaaacagg actacacagt 720
 
gacattatgt tctacacaat agaaaatgca gtaccaattc atcttctaag aacaggagat 780
 
gaattctcca caggaatata tcactttgac acaaaaccac taaaattaac tcactcatgg 840
 
caaacaaaca gatctctagg actgcctcca aaactactaa ctgaacctac cacagaagga 900
 
gaccaacacc caggaacact accagcagct aacacaagaa aaggttatca ccaaacaatt 960
 
aataatagct acacagaagc aacagcaatt aggccagctc aggtacgata taatacacca 1020
 
tacatgaatt ttgaatactc caatggtgga ccatttctaa ctcctatagt accaacagca 1080
 
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aaatgtggaa gagctccaaa gcaacaattt aatcaacagg caccactaaa cctagaaaat 1260
 
acaaataatg gaacactttt accttcagat ccaataggag ggaaatctaa catgcatttc 1320
 
atgaatacac tcaatacata tggaccatta acagcactaa acaatactgc acctgtattt 1380
 
ccaaatggtc aaatatggga taaagaactt gatacagatc taaaacctag actacatgtt 1440
 
acagctccat ttgtttgtaa aaacaatcca ccaggacaac tatttgtaaa aatagcacca 1500
 
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aaccctattc aacaacacac aacaacagca gaaaacattg gtaaatatat tcctacaaat 1680
 
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Met Ser Glu Asn Val Glu Gln His Asn Pro Ile Asn Ala Gly Thr Glu
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Leu Ser Ala Thr Gly Asn Glu Ser Gly Gly Gly Gly Gly Gly Gly Gly
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Gly Arg Gly Ala Gly Gly Val Gly Val Ser Thr Gly Ser Phe Asn Asn
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Gln Thr Glu Phe Gln Tyr Leu Gly Glu Gly Leu Val Arg Ile Thr Ala
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His Ala Ser Arg Leu Ile His Leu Asn Met Pro Glu His Glu Thr Tyr
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Lys Arg Ile His Val Leu Asn Ser Glu Ser Gly Val Ala Gly Gln Met
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Val Gln Asp Asp Ala His Thr Gln Met Val Thr Pro Trp Ser Leu Ile
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Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Ala Asp Trp Gln Leu
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Ile Ser Asn Asn Met Thr Glu Ile Asn Leu Val Ser Phe Glu Gln Ala
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Ile Phe Asn Val Val Leu Lys Thr Ile Thr Glu Ser Ala Thr Ser Pro
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Pro Thr Lys Ile Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
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Leu Asp Thr Asn Asn Thr Leu Pro Tyr Thr Pro Ala Ala Pro Arg Ser
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Glu Thr Leu Gly Phe Tyr Pro Trp Leu Pro Thr Lys Pro Thr Gln Tyr
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Arg Tyr Tyr Leu Ser Cys Ile Arg Asn Leu Asn Pro Pro Thr Tyr Thr
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Gly Gln Ser Gln Gln Ile Thr Asp Ser Ile Gln Thr Gly Leu His Ser
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Asp Ile Met Phe Tyr Thr Ile Glu Asn Ala Val Pro Ile His Leu Leu
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Arg Thr Gly Asp Glu Phe Ser Thr Gly Ile Tyr His Phe Asp Thr Lys
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Pro Leu Lys Leu Thr His Ser Trp Gln Thr Asn Arg Ser Leu Gly Leu
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Pro Pro Lys Leu Leu Thr Glu Pro Thr Thr Glu Gly Asp Gln His Pro
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Gly Thr Leu Pro Ala Ala Asn Thr Arg Lys Gly Tyr His Gln Thr Ile
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Asn Asn Ser Tyr Thr Glu Ala Thr Ala Ile Arg Pro Ala Gln Val Arg
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Tyr Asn Thr Pro Tyr Met Asn Phe Glu Tyr Ser Asn Gly Gly Pro Phe
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Leu Thr Pro Ile Val Pro Thr Ala Asp Thr Gln Tyr Tyr Asp Asp Glu
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Pro Asn Gly Ala Ile Arg Phe Thr Met Gly Tyr Gln His Gly Gln Leu
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Thr Thr Ser Ser Gln Glu Leu Glu Arg Tyr Thr Phe Asn Pro Gln Ser
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Lys Cys Gly Arg Ala Pro Lys Gln Gln Phe Asn Gln Gln Ala Pro Leu
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Asn Leu Glu Asn Thr Asn Asn Gly Thr Leu Leu Pro Ser Asp Pro Ile
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Gly Gly Lys Ser Asn Met His Phe Met Asn Thr Leu Asn Thr Tyr Gly
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Pro Leu Thr Ala Leu Asn Asn Thr Ala Pro Val Phe Pro Asn Gly Gln
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Ile Trp Asp Lys Glu Leu Asp Thr Asp Leu Lys Pro Arg Leu His Val
465 470 475 480
 
 
Thr Ala Pro Phe Val Cys Lys Asn Asn Pro Pro Gly Gln Leu Phe Val
485 490 495
 
 
Lys Ile Ala Pro Asn Leu Thr Asp Asp Phe Asn Ala Asp Ser Pro Gln
500 505 510
 
 
Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp Trp Lys Gly Thr Leu
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Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met Trp Asn Pro Ile Gln
530 535 540
 
 
Gln His Thr Thr Thr Ala Glu Asn Ile Gly Lys Tyr Ile Pro Thr Asn
545 550 555 560
 
 
Ile Gly Gly Ile Lys Met Phe Pro Glu Tyr Ser Gln Leu Ile Pro Arg
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Lys Leu Tyr Glx
580
 
 

Claims (4)

1. a gene for synthetic, its sequence is for shown in SEQ ID NO.1.
2. express a recombinant baculovirus for PPV VP 2 protein, it is characterized in that, recombinant baculovirus (Recominant autographa californicamultiple Nucleopolyhedrovirus) Ac-PPVP2, its deposit number is CCTCC NO:V201339.
3. include the pFBDPHm3VP2 plasmid of sequence shown in 3 copy claims 1.
4. recombinant baculovirus according to claim 2 is preparing the application in pig parvoviral genetic engineering subunit vaccine.
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CN105820216A (en) * 2016-05-28 2016-08-03 青岛易邦生物工程有限公司 Porcine parvovirus VP2 protein
CN105879024A (en) * 2016-05-28 2016-08-24 青岛易邦生物工程有限公司 Porcine parvovirus subunit vaccine
CN106148358A (en) * 2016-07-15 2016-11-23 河南省农业科学院 A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application
CN107119330A (en) * 2017-04-27 2017-09-01 河南牧业经济学院 A kind of preparation method and application method of the genetic chip for detecting pig parvoviral
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105820216A (en) * 2016-05-28 2016-08-03 青岛易邦生物工程有限公司 Porcine parvovirus VP2 protein
CN105879024A (en) * 2016-05-28 2016-08-24 青岛易邦生物工程有限公司 Porcine parvovirus subunit vaccine
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CN106148358A (en) * 2016-07-15 2016-11-23 河南省农业科学院 A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application
CN107119330A (en) * 2017-04-27 2017-09-01 河南牧业经济学院 A kind of preparation method and application method of the genetic chip for detecting pig parvoviral
CN110845580A (en) * 2019-11-05 2020-02-28 中国农业科学院兰州兽医研究所 Method for assembling porcine parvovirus-like particles and identifying immunogenicity thereof
CN113150126A (en) * 2021-04-20 2021-07-23 开江县动物疫病预防控制中心 Rabbit-derived porcine parvovirus 6-type VP2 protein antibody and preparation method thereof

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