CN103589693A - Recombinant HVT (herpesvirus of turkey) expressing IBDV (infectious bursal disease virus) VP2 and bursin chimeric protein - Google Patents
Recombinant HVT (herpesvirus of turkey) expressing IBDV (infectious bursal disease virus) VP2 and bursin chimeric protein Download PDFInfo
- Publication number
- CN103589693A CN103589693A CN201310566984.5A CN201310566984A CN103589693A CN 103589693 A CN103589693 A CN 103589693A CN 201310566984 A CN201310566984 A CN 201310566984A CN 103589693 A CN103589693 A CN 103589693A
- Authority
- CN
- China
- Prior art keywords
- gene
- ibdv
- gpt
- rhvt
- bursin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241001502481 Meleagrid alphaherpesvirus 1 Species 0.000 title claims abstract description 27
- 108010026970 bursopoietin Proteins 0.000 title claims abstract description 21
- DIBAKBGYJUCGDL-UHFFFAOYSA-N 2,6-diamino-n-[1-[(2-amino-2-oxoethyl)amino]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]hexanamide Chemical compound NCCCCC(N)C(=O)NC(C(=O)NCC(N)=O)CC1=CN=CN1 DIBAKBGYJUCGDL-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 11
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 11
- 241000702626 Infectious bursal disease virus Species 0.000 title abstract description 16
- 241000700605 Viruses Species 0.000 claims abstract description 39
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 241000287828 Gallus gallus Species 0.000 claims abstract description 21
- 229960005486 vaccine Drugs 0.000 claims abstract description 18
- 101500001532 Avian infectious bursal disease virus Capsid protein VP2 Proteins 0.000 claims abstract description 16
- 208000015181 infectious disease Diseases 0.000 claims abstract description 10
- 230000036039 immunity Effects 0.000 claims abstract description 9
- 230000001681 protective effect Effects 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 210000001669 bursa of fabricius Anatomy 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 14
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 claims description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 9
- 238000012546 transfer Methods 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- 238000013461 design Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 101150106093 gpt gene Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 210000003837 chick embryo Anatomy 0.000 claims description 4
- 210000002950 fibroblast Anatomy 0.000 claims description 4
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- 101150098675 BS gene Proteins 0.000 claims description 3
- 241001260012 Bursa Species 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 3
- 239000004471 Glycine Substances 0.000 claims description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 claims description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 claims description 3
- 241000700584 Simplexvirus Species 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- 239000002502 liposome Substances 0.000 claims description 3
- 229930182817 methionine Natural products 0.000 claims description 3
- 238000004153 renaturation Methods 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 241000271566 Aves Species 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 238000013459 approach Methods 0.000 claims description 2
- 230000004060 metabolic process Effects 0.000 claims description 2
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 claims description 2
- 229960000951 mycophenolic acid Drugs 0.000 claims description 2
- 108020004707 nucleic acids Proteins 0.000 claims description 2
- 150000007523 nucleic acids Chemical class 0.000 claims description 2
- 102000039446 nucleic acids Human genes 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 claims description 2
- 101710192523 30S ribosomal protein S9 Proteins 0.000 claims 3
- 230000008774 maternal effect Effects 0.000 abstract description 9
- 230000002458 infectious effect Effects 0.000 abstract description 7
- 208000027312 Bursal disease Diseases 0.000 abstract description 5
- 208000006758 Marek Disease Diseases 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 5
- 230000002045 lasting effect Effects 0.000 abstract description 3
- 229940031416 bivalent vaccine Drugs 0.000 abstract 2
- 230000000694 effects Effects 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 19
- 235000013330 chicken meat Nutrition 0.000 description 18
- 241000282326 Felis catus Species 0.000 description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 101150023587 US10 gene Proteins 0.000 description 3
- 101150093578 VP2 gene Proteins 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 235000013594 poultry meat Nutrition 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000701047 Gallid alphaherpesvirus 2 Species 0.000 description 2
- 101100427969 Gallid herpesvirus 2 (strain GA) US639 gene Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 229940075420 xanthine Drugs 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010015856 Extrasystoles Diseases 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 208000030289 Lymphoproliferative disease Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 101150099105 alien gene Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000000554 iris Anatomy 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 210000000578 peripheral nerve Anatomy 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a recombinant HVT (herpesvirus of turkey) expressing IBDV (infectious bursal disease virus) VP2 and bursin chimeric protein, and belongs to the research field of genetic engineering and biological products. 10 copies of BS 10 (bursin 10) chimeric IBDV VP2 gene expression cassettes are combined with HVT, so that a brand-new recombinant HVT (rHVT-BS10-VP2) is obtained. The virus simultaneously is recombining bivalent vaccine of an MD (Marek's disease) and an infectious bursal disease. The recombining bivalent vaccine not only can have an immune protection effect on the MD of chicken, but also can induce anti-IBDV lasting protective immunity of a commercialized chicken crowd with a high IBDV maternal antibody level. Compared with conventional IBDV and HVT vector live vaccine (vHVT-013-69 strains), the protection rate (100%) of an rHVT-BS10-VP2 immunity class is higher than the protection rate (93.3%) of an rHVT-VP2 immunity class.
Description
one, technical field
The invention belongs to genetically engineered and biological products research field.Be specifically related to the recombinant herpesvirus of turkeys of a kind of IBDV of expression VP2 and bursa of Fabricius bursin chimeric protein.
two, background technology
Chicken Marek's disease (Marek ' s disease, MD) and infectious bursal disease (Infectious bursal
Disease, IBD) be two kinds and affect the modal immunosuppressive disease of poultry production.Wherein IBD is caused by infections chicken cloacal bursa virus (Infectious bursal disease virus, IBDV), acute, the height contagious disease of 3 weeks age～12 young chickens in week age of main harm.The appearance of variant and highly virulent strain (vvIBDV) strengthens this sick prevention and control difficulty, and IBD immunoprophylaxis failure has become the major issue in poultry diease prevention and control.
Current commercial infectious bursal disease malicious vaccine alive is divided into weak poison, poisoning and strong malicious three classes.Attenuated live vaccines
To SPF chicken safety, but to there being maternal antibody chicken group's immune protective efficiency very poor.The immunoprotection of mesogenic and strong malicious vaccine
Although it is high a lot of that power is wanted, and damages the fabricius bursa owing to having certain virulence.Meanwhile, due to interference and the IBDV of maternal antibody, traditional vaccine can form the inevitable Blank immunization phase in actual applications.Therefore,, in current I BD prevention and control real work, urgent need immune efficacy is strong, the high novel IBD vaccine of biological safety.
MD is the chicken lymphoproliferative disease being caused by marek's disease virus (Marek ' s disease virus, MDV), and with the lymphoid cell infiltration of peripheral nerve, iris, skin, muscle and internal organs, hyperplasia and tumour form feature.MDV vaccine strain can be used as live vector and carrys out expression alien gene, and its molecular biological research is shown, MDV genome is larger, and has determined virus replication nonessential region, many places, itself is good virus expression carrier.Compared to other virus vector, MDV has unique advantage as carrier, and MDV is strict Cell binding type, can break through the interference of maternal antibody, is applicable to early immune, by embryo immunization, and more convenient operation.And primary immune response can bring out lifelong cell and humoral immunoresponse(HI), cost-saving.Based on above feature, the living vaccine research that the MDV of take carries out as carrier gains great popularity, and herpes turkey virus (HVT) is a kind of α simplexvirus wherein, because of itself and MDV antigen dependency, simultaneously, again because HVT and MDV have obvious difference to chicken no pathogenicity, be widely used as preventing the living vaccine of Marek (MD).
The fabricius bursa (BF) is the distinctive immune organ of bird, for B cell development, maturation provide microenvironment, is that immunoglobulin gene changes place; Its immunologic active material is Bursin (Bursin, BS), and it can induce poultry and the differentiation of Mammals B cell precursor; Its target organ is BF, can the damage of antagonism IBD vaccine to BF, raising ND attenuated vaccine and oil seepage generation antibody horizontal.Therefore the herpes turkey virus (HVT) of how can take becomes a difficult problem of urgently capturing for efficient living vaccine research that carrier carries out infectious bursal disease (IBD).
three, summary of the invention
technical problem
The present inventor provides a kind of recombinant herpesvirus of turkeys (rHVT-BS10-VP2); not only can play good immanoprotection action to chicken MD, and the lasting protective immunity that can induce anti-IBDV in thering is the horizontal commercialization chicken of high IBDV maternal antibody group.
technical scheme
A recombinant herpesvirus of turkeys rHVT-BS10-VP2 for IBDV VP2 and bursa of Fabricius bursin chimeric protein, is characterized in that: its gene orders that are carried at the chimeric IBDV VP2 of 10 copy bursa of Fabricius bursin (BS10) expression casette under promoter sequence control are as shown in SEQ ID NO.1.
The recombinant herpesvirus of turkeys rHVT-BS10-VP2 of described expression IBDV VP2 and bursa of Fabricius bursin chimeric protein, its preparation method is as follows:
1) 10 copy bursa of Fabricius bursin BS10 oligonucleotide is synthetic: select the conventional codon of coding Methionin Lys, histidine and glycine Gly to be arranged in order BS gene: 5'-AAGCATGGC-3 ', continuously synthetic l0 BS, BS tandem gene strand and reverse complemental chain thereof, make its renaturation become double-stranded by annealing;
2) contain the structure of the herpes turkey virus HVT transfer vector of IBDV VP2 and BS10 mosaic gene expression cassette: according to the gpt gene order design primer of registered GenBank NO .X00221.1, with
e.colidH
5α DNA is that template adopts PCR method amplification gpt gene, with it as external source screening-gene, be connected structure gpt expression cassette CMV-gpt-poly A with CMV promotor and poly (A) in plasmid pEGFP, after enzyme is cut sequence verification, be inserted in the homology arm cloned plasmids pUAB that contains herpes turkey virus HVT nonessential region US10, SORF3 both sides gene, build the cloned plasmids pUAB-gpt with screening-gene expression cassette; BS10-VP2 mosaic gene is inserted in pUAB-gpt, obtains recombinant herpesvirus of turkeys transfer vector pUAB-gpt-BS10-VP2, size is 10100bp;
3) express the preparation of the recombinant herpesvirus of turkeys of chimeric protein: adopt liposome mediated-method, by recombinant transfer vector plasmid pUAB-gpt-BS10-VP2 and the primary chick embryo fibroblast CEF of HVT-FC126 pnca gene cotransfection, after there is viral plaque, utilize mycophenolic acid MPA blocking-up nucleic acid metabolism approach, through screening, obtain the recombinant virus rHVT-BS10-VP2 of purifying.
Described recombinant herpesvirus of turkeys rHVT-BS10-VP2, it induces the application of the protective immunity of anti-bird simplexvirus and infections chicken cloacal bursa virus in avian host, and it also can be used as vaccine strain application.
beneficial effect
1) position of the chimeric IBDV VP2 of 10 copy bursa of Fabricius bursin (BS10) expression casette being inserted in HVT genome is positioned between the nonessential region US10 gene and SORF3 gene of HVT FC126 genome (ACCESSION NO.NC_002641), position is between the 138687bp-138826bp position of HVT FC126 genome (ACCESSION NO.NC_002641) more specifically, obtained a kind of immunity enhancement type recombinant herpesvirus of turkeys rHVT-BS10-VP2, this virus is also the bigeminy vaccine of a kind of Marek and infectious bursal disease simultaneously, rHVT-BS10-VP2 not only can play good immanoprotection action to chicken MD, and the lasting protective immunity that can induce anti-IBDV in thering is the horizontal commercialization chicken of high IBDV maternal antibody group, compare with existing chicken infectivity bursa of Fabricius virus herpes turkey virus carrier living vaccine (vHVT-013-69 strains), ((100%) is higher than the protection ratio (93.3%) of rHVT-VP2 immune group for the protection ratio of rHVT-BS10-VP2 immune group.
2) take gpt can express gpt as the rHVT-BS10-VP2 recombinant virus that screening-gene builds, the external source that this enzyme utilizes external source xanthine to complete guanine is remedied synthetic, thereby copying of recombinant virus dna can normally be carried out, can be all dead through several screening wild viruses of taking turns, and recombinant virus can obtain enrichment and purifying gradually.
four, accompanying drawing explanation
Fig. 1 is the DNA sequential patterns graph of rHVT-BS10-VP2 recombinant virus;
Fig. 2 HVT genome inserts BS10-VP2 schematic diagram;
The expression cassette Egpt(CMV-gpt-poly A of Fig. 3 selection markers gene gpt) amplification electrophoresis schematic diagram;
Fig. 4 is BS10-VP2 pcr amplification result electrophoresis schematic diagram;
Fig. 5 is recombinant plasmid pUAB-gpt-BS10-VP2 building process schematic diagram.
five, embodiment
IBDV variant AH1 strain (document sees reference: Wang Yongshan etc. cause in the recent period the characterization of molecules of the infectivity bursa of Fabricius virus VP 2 gene of immuning failure. and Chinese veterinary science, 2008, 38 (12): 1013-1019) VP2 gene line causes clone the IBDV variant (AH1) of immuning failure in Anhui from December, 2007 by RT-PCR method, this variant has the whole significant amino-acid residue of IBDV virulent strain, VP2 gene seven peptide motifs are SWSASGS, 222, 253, 256, 279, 284, amino-acid residue on 294 and 299 is respectively A, Q, I, D, A, I and S, there is larger difference with the VP2 gene order of existing commercially available vaccine strain B87.Therefore, the prevention and control of current I BDV are had more to specific aim and practical significance.
experiment material
1.1 carriers, bacterial classification and main agentspMD18-T simple vector is purchased from precious biotechnology (Dalian) company limited; Expression plasmid Liu pEGFP-N1(of containing green fluorescence protein gene is drizzly, Yang Decheng, Zhou Guohui etc., the structure of coexpression O type FM DV capsid protein and green fluorescent protein recombinant adenovirus, 2012(24) 83-86); E.coli DH5 α and E.coli DH10B(are purchased from precious biotechnology (Dalian) company limited).AxyPrep plasmid in a small amount purification kit, DNA gel reclaims test kit purchased from Axygen, and X-gal, IPTG, restriction endonuclease, T4 DNA Ligase, ATP, dNTP and Taq archaeal dna polymerase are all purchased from precious biotechnology (Dalian) company limited; DMEM substratum is purchased from Invitrogen company; Pancreatin is purchased from Thermo company.
1.2SPF chicken embryo and virus9～10 age in days SPF chicken embryos, herpes turkey virus (Herpesvirus of turkey, HVT) Fc126 strain all purchased from
nanjing Tianbang Bio-industry Co., Ltd.; The standard strain of infectious bursa of Fabricius virus BC6/85 is purchased from China Veterinary Drugs Supervisory Inst.;
2 experimental techniques
2.1 design of primers are with synthetic
According to the HVT Fc126 strain full-length gene group sequence (AF291866) of delivering on GenBank, gpt sequence (X00221.1) and pEGFP-C1 plasmid sequence, designed following PCR primer (table 1), primer is synthesized by Invitrogen company.
The primer of table 1 recombinant virus transfer vector builds
the structure of 2.2 homology arm cloned plasmids pUAB
The HVT Fc126 strain infector of take is template for the total DNA of chick embryo fibroblast (CEF) sick cell, take A-F/A-R and B-F/B-R respectively as primer amplification recombinant virus transfer vector left/right homology arm A/B, reclaim the PCR product of left/right homology arm A/B, be cloned into pMD18-T simple vector, obtain positive colony plasmid pMD-A and pMD-B.With BamH I and EcoR I double digestion, analyze pMD-A, EcoR I, Sca I and Xba I three restriction analysis pMD-B, further order-checking is confirmed.Then, by EcoR I and Xba I double digestion for pMD-A, for pMD-B, EcoR I, Sca I and Xba I three enzymes are cut, reclaim pMD-A carrier segments and B fragment, T4 DNA Ligase connects, and transforms DH5 α competence bacterium, extracts recombinant plasmid, EcoR I and Xba I double digestion are identified, obtain the cloned plasmids pUAB that contains HVT left/right homology arm A/B.
the amplification of gene and Egpt gene
Take gpt-F/gpt-R as primer, the E.coli DH5 α DNA of take is template, adopt PCR method amplification gpt gene, with it as external source screening-gene, be connected structure gpt expression cassette (CMV-gpt-poly A) with CMV promotor and poly (A) in plasmid pEGFP-N1, after PCR sequence verification, be inserted in the homology arm cloned plasmids pUAB that contains herpes turkey virus (HVT) nonessential region US10, SORF3 both sides gene, build the cloned plasmids pUAB-gpt with screening-gene expression cassette.
the design of gene is with synthetic
Select the conventional codon of coding Methionin (Lys), Histidine (His) and glycine (Gly) to be arranged in order BS gene: 5'-AAGCATGGC-3 '.Continuously synthetic l0 BS (BS tandem gene strand (BS-F) and reverse complemental chain (BS-R) thereof, by annealing, make its renaturation become double-stranded, concrete sequence is as follows: BS-F: 5-gtc gac atg aag cat ggc aag cat ggc aag cat ggc aag cat ggc aag cat ggc aag cat ggc aag-3 (front end adds Sal I restriction enzyme site); BS-R:5-
ttg cag gtt cgc catgcc-atg-ctt gcc atg ctt gcc atg ctt gcc atg-ctt-gcc-atg-ctt-gcc-atg-3(
black italicpart is the complementary opposite sequence of primer I BDV VP2-F primer, overlapping extension PCR method (SOE method) amplification BS10-VP2 mosaic gene).
recombinant herpesvirus of turkeys shifts the structure that carries pUAB-gpt-BS10-VP2
According to IBDV VP2 sequences Design (GenBank NO.AF362747) the design primer of registration, the IBDV VP2 gene that increases complete from AH1 viral RNA by RT-PCR method.Primer sequence is as follows: VP2-F:5-
atg-gcg – aac-ctg-caa -gat – caa-ac-3; VP2-R:5-ggg-aag-ctt-cta-cta-cct-cct-tat-ggc-ccg-gat-tat-gtc-ttt-g-3 (front end adds HindIII restriction enzyme site).Adopt overlapping extension PCR method (SOE method), take BS10 and VP2 as template, BS-F and VP2 – R are primer amplification BS10-VP2 mosaic gene
.bS10-VP2 mosaic gene is inserted in pUAB-gpt, obtains recombinant herpesvirus of turkeys and shift a year pUAB-gpt-BS10-VP2, size is 10100bp.
the acquisition of recombinant virus
Adopt liposome Lipofectamine2000 mediated method, by recombinant transfer vector plasmid pUAB-gpt-BS10-VP2 and the primary chick embryo fibroblast of HVT-FC126 pnca gene cotransfection (CEF), when cytopathy reaches 70% left and right, pass on the individual layer CEF of fresh preparation, nutrient solution changed pressurization screening and culturing liquid into (containing 2% serum, 1% S/P before 1 hour, 300 ug/mL mycophenolic acids, 60 ug/mL xanthine and the hypoxanthic DMEM of 100 ug/mL), every 24 h change the screening and culturing liquid that once pressurizes.The individual layer CEF that passes to another fresh preparation when sick cell reaches 70% left and right is upper, through 6, takes turns the recombinant virus rHVT-BS10-VP2 that pressurization screening obtains purifying.
the evaluation of recombinant virus rHVT-BS10-VP2
2.7.1 PCR identifies
At the both sides of BS10-VP2 expression cassette design primer, primer sequence is as follows: rHVT-F: 5-atg-aag-cat-ggc-aag-cat – ggc-3; RHVT-R: 5-cta-cta-cct-cct-tat-ggc-3.The rHVT-BS10-VP2 of above-mentioned acquisition of take utilizes this primer as template and increases, can amplify the specific fragment of 1458bp, through sequence verification, the fragment of this 1458bp is IBDV-VP2 expression cassette sequence, in the recombinant virus rHVT-BS10-VP2 of known acquisition, has successfully inserted BS10-VP2 mosaic gene expression cassette;
2.7.2 indirect immunofluorescence assay (IFA) detects
The rHVT-BS10-VP2 of 100PFU virus quantity is inoculated on 6 porocyte culture plates.Cultivating 4d occurs after obvious plaque, growth media is outwelled, with cold acetone: ethanol (3: 2) stationary liquid is 10min fixedly, PBS washes 1 time, in each hole, add 0.5mL (dilution in 1: 100) IBDV-VP2 monoclonal antibody, be positioned in 37 ℃ of constant incubators and react 1h, with PBS, clean 3 times, every hole adds the anti-mouse IgG of 0.5mL FITC mark fluorescence antibody, be positioned in 37 ℃ of constant temperature biochemical cultivation cases and react 1h, with PBS, wash 3 times, under inverted fluorescence microscope, observe, rHVT-BS10-VP2 infects hole and has occurred specificity green fluorescence, and HVT infects control wells without fluorescence, show the correction that BS10-VP2 gene can be good.
immunization experiment in the commercial chicken that recombinant virus rHVT-BS10-VP2 is applied to contain maternal antibody
120 1 age in days commercial chickens, are divided into 4 groups at random, detect its maternal antibody positive by ELISA method.The
One group of inoculation rHVT-BS10-VP2, immunizing dose is 2500PFU/.Second group of inoculation rHVT-VP2 (chicken infectivity bursa of Fabricius virus herpes turkey virus carrier living vaccine vHVT-013-69 strains, purchased from Cimmeria animal health company limited), immunizing dose is for being 2500PFU/.The 3rd group is nonimmunely to attack malicious group, and the 4th group is nonimmunely non-ly to attack malicious group.35 ages in days carry out IBDV BC6/85 strong virus attack, and after attacking, 5d cuts open and kills, and carry out naked eyes pathological observation and get immune maincenter organ fabricius bursa tissue fixing to make pathological study with 10% formaldehyde.Immune effect is in Table 1, and known in containing the commercial chicken of maternal antibody by table 1 content, the protection ratio of rHVT-BS10-VP2 immune group reaches 100%, higher than the protection ratio (93.3%) of rHVT-VP2 immune group
| Immunity recombinant virus | Attack poison strain | Lethality rate | Fabricius bursa damage |
| rHVT-VP2 | BC6/85 | 0/30 | 2/30(6.7%) |
| rHVT-BS10-VP2 | BC6/85 | 0/30 | 0/30（0%） |
| - | BC6/85 | 7/30(23%) | 30/30(100%) |
| - | - | 0/30(0%) |
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> expresses IBDV VP2 and bursa of Fabricius bursin chimeric protein recombinant herpesvirus of turkeys
<130> 0
<160> 13
<170> PatentIn version 3.1
<210> 1
<211> 1458
<212> DNA
<213> is artificial
<220>
The DNA sequence of <221> rHVT-BS10-VP2 recombinant virus
<222> (1)..(1458)
<223>
<400> 1
atgaagcatg gcaagcatgg caagcatggc aagcatggca agcatggcaa gcatggcaag 60
catggcaagc atggcaagca tggcaagcat ggcatggcga acctgcaaga tcaaacccaa 120
cagattgttc cgttcatacg gagccttctg atgccaacaa ccggaccggc gtccattccg 180
gacgacaccc tagagaagca cactctcagg tcagagacct cgacctacaa tttgactgtg 240
ggggacacag ggtcagggct aattgtcttt ttccctggtt tccctggctc aattgtgggt 300
gctcactaca cactgcagag caatgggaac tacaagttcg atcagatgct cctgactgcc 360
cagaacctac cggccagcta caactactgc aggctagtga gtcggagtct cacagtgagg 420
tcaagcacac tccctggtgg cgtttatgca ctaaatggca ccataaacgc cgtgaccttc 480
cgaggaagcc tgagtgaact gacagatgtt agctacaatg ggttgatgtc tgcaacagcc 540
aacatcaacg acaaaatcag gaacgtccta gtgggggaag gggtgaccgt cctcagctta 600
cccacatcat atgatcttgg gtatgtgaga ctcggtgacc ccattcccgc tatagggctc 660
gacccaaaaa tggtagcaac atgtgacagc agtgacaggc ccagagtcta caccataact 720
gcagccgatg attaccaatt ctcatcacag taccaagcag gtggggtaac aatcacactg 780
ttctcagcta atatcgatgc catcacaagc ctcagcatcg ggggagaact cgtgtttcaa 840
acaagcgtcc aaggcctcat actgggtgct accatctacc ttataggctt tgatgggact 900
gcggtaatca cccgagctgt ggccgcagac aatgggctaa cggccggcac tgacaacctt 960
atgccattca acattgtgat tccaaccagc gagataaccc agccaatcac atccatcaaa 1020
ctggagatag tgacctccaa aagtggtggc caggcggggg atcagatgtc atggtcagca 1080
agtgggagcc tagcagtgac gatccacggt ggcaactatc caggggccct ccgtcccgtc 1140
acactagtag cctacgaaag agtggcaaca gggtctgtcg ttacggtcgc cggggtaagc 1200
aacttcgagc tgatcccaaa tcctgaacta gcaaagaacc tggtcacaga atacggccga 1260
tttgacccag gggccatgaa ctacacaaaa ttgatactga gtgagaggga ccgtcttggc 1320
atcaagaccg tatggccaac aagggagtac actgactttc gcgagtactt catggaggtg 1380
gccgacctca actctcccct gaagattgca ggagcatttg gcttcaaaga cataatccgg 1440
gccataagga ggtagtag 1458
<210> 2
<211> 28
<212> DNA
<213> is artificial
<220>
<221> A-F
<222> (1)..(28)
<223>
<400> 2
cgggatccac atcgggccac gttccgcc 28
<210> 3
<211> 42
<212> DNA
<213> is artificial
<220>
<221> A-R
<222> (1)..(42)
<223>
<400> 3
gctctagagc gtcgaccgga attcgatgag ctgacgtgtg ga 42
<210> 4
<211> 41
<212> DNA
<213> is artificial
<220>
<221> B-F
<222> (1)..(41)
<223>
<400> 4
ggaattcaag tcgacggaag cttccactaa tatgggcaca c 41
<210> 5
<211> 28
<212> DNA
<213> people
<220>
<221> B-R
<222> (1)..(28)
<223>
<400> 5
gctctagatg gcccatctag gtgattat 28
<210> 6
<211> 30
<212> DNA
<213> people
<220>
<221> gpt-F
<222> (1)..(30)
<223>
<400> 6
gcgctagcgt catgagcgaa aaatacatcg 30
<210> 7
<211> 29
<212> DNA
<213> people
<220>
<221> gpt-R
<222> (1)..(29)
<223>
<400> 7
actggatcct tagcgaccgg agattggcg 29
<210> 8
<211> 39
<212> DNA
<213> people
<220>
<221> Egpt-F
<222> (1)..(39)
<223>
<400> 8
gcgaattcta ttaatagtaa tcaattacgg ggtcattag 39
<210> 9
<211> 36
<212> DNA
<213> people
<220>
<221> Egpt-R
<222> (1)..(36)
<223>
<400> 9
gcgtcgaccg cgttaagata cattgatgag tttgga 36
<210> 10
<211> 66
<212> DNA
<213> people
<220>
<221> BS-F
<222> (1)..(66)
<223>
<400> 10
gtcgacatga agcatggcaa gcatggcaag catggcaagc atggcaagca tggcaagcat 60
ggcaag 66
<210> 11
<211> 66
<212> DNA
<213> people
<220>
<221> BS-R
<222> (1)..(66)
<223>
<400> 11
ttgcaggttc gccatgccat gcttgccatg cttgccatgc ttgccatgct tgccatgctt 60
gccatg 66
<210> 12
<211> 23
<212> DNA
<213> people
<220>
<221> VP2 -F
<222> (1)..(23)
<223>
<400> 12
atggcgaacc tgcaagatca aac 23
<210> 13
<211> 43
<212> DNA
<213> people
<220>
<221> VP2 -R
<222> (1)..(43)
<223>
<400> 13
gggaagcttc tactacctcc ttatggcccg gattatgtct ttg 43
Claims (5)
1. a recombinant herpesvirus of turkeys rHVT-BS10-VP2 who expresses IBDV VP2 and bursa of Fabricius bursin chimeric protein, is characterized in that: its gene orders that are carried at the chimeric IBDV VP2 of the 10 copy bursa of Fabricius bursin BS10 expression casette under promoter sequence control are as shown in SEQ ID NO.1.
2. the recombinant herpesvirus of turkeys rHVT-BS10-VP2 of expression according to claim 1 IBDV VP2 and bursa of Fabricius bursin chimeric protein, its preparation method is as follows:
1) 10 copy bursa of Fabricius bursin BS10 oligonucleotide is synthetic: select the conventional codon of coding Methionin Lys, histidine and glycine Gly to be arranged in order BS gene: 5'-AAGCATGGC-3 ', continuously synthetic l0 BS, BS tandem gene strand and reverse complemental chain thereof, make its renaturation become double-stranded by annealing;
2) contain the structure of the herpes turkey virus HVT transfer vector of IBDV VP2 and BS10 mosaic gene expression cassette: according to the gpt gene order design primer of registered GenBank NO .X00221.1, with
e.colidH
5α DNA is that template adopts PCR method amplification gpt gene, with it as external source screening-gene, be connected structure gpt expression cassette CMV-gpt-poly A with CMV promotor and poly (A) in plasmid pEGFP, after enzyme is cut sequence verification, be inserted in the homology arm cloned plasmids pUAB that contains herpes turkey virus HVT nonessential region US10, SORF3 both sides gene, build the cloned plasmids pUAB-gpt with screening-gene expression cassette; BS10-VP2 mosaic gene is inserted in pUAB-gpt, obtains recombinant herpesvirus of turkeys transfer vector pUAB-gpt-BS10-VP2, size is 10100bp;
3) express the preparation of the recombinant herpesvirus of turkeys of chimeric protein: adopt liposome mediated-method, by recombinant transfer vector plasmid pUAB-gpt-BS10-VP2 and the primary chick embryo fibroblast CEF of HVT-FC126 pnca gene cotransfection, after there is viral plaque, utilize mycophenolic acid MPA blocking-up nucleic acid metabolism approach, through screening, obtain the recombinant virus rHVT-BS10-VP2 of purifying.
3. the recombinant herpesvirus of turkeys rHVT-BS10-VP2 as described in claim 1 or 2, it induces the application of the protective immunity of anti-bird simplexvirus and infections chicken cloacal bursa virus in avian host.
4. the recombinant herpesvirus of turkeys rHVT-BS10-VP2 as described in claim 1 or 2, it is as the application of vaccine strain.
5. the recombinant herpesvirus of turkeys rHVT-BS10-VP2 as described in claim 3, it is as the application of vaccine strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310566984.5A CN103589693B (en) | 2013-11-13 | 2013-11-13 | A kind of expression IBDV VP2 and bursa of Fabricius bursin chimeric protein recombinant herpesvirus of turkeys |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310566984.5A CN103589693B (en) | 2013-11-13 | 2013-11-13 | A kind of expression IBDV VP2 and bursa of Fabricius bursin chimeric protein recombinant herpesvirus of turkeys |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103589693A true CN103589693A (en) | 2014-02-19 |
| CN103589693B CN103589693B (en) | 2016-02-03 |
Family
ID=50080009
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310566984.5A Expired - Fee Related CN103589693B (en) | 2013-11-13 | 2013-11-13 | A kind of expression IBDV VP2 and bursa of Fabricius bursin chimeric protein recombinant herpesvirus of turkeys |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103589693B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017027324A1 (en) | 2015-08-10 | 2017-02-16 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| CN109310750A (en) * | 2016-06-17 | 2019-02-05 | 英特维特国际股份有限公司 | Encode the recombination non-pathogenic marek's disease virus construct of infectious laryngotracheitis virus and infectious bursal disease virus antigen |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255189A (en) * | 2008-04-07 | 2008-09-03 | 广东大华农动物保健品有限公司 | Animal vaccine immunopotentiator and production method thereof |
| CN102000331A (en) * | 2010-11-09 | 2011-04-06 | 国家兽用生物制品工程技术研究中心 | Application of bursin as swine fever vaccine adjuvant |
| CN102634489A (en) * | 2012-03-22 | 2012-08-15 | 肇庆大华农生物药品有限公司 | Recombinant turkey herpesvirus and application thereof |
| CN102796200A (en) * | 2012-08-17 | 2012-11-28 | 南京大学 | Hybrid peptide bursin adjuvant and preparation method and application thereof |
-
2013
- 2013-11-13 CN CN201310566984.5A patent/CN103589693B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101255189A (en) * | 2008-04-07 | 2008-09-03 | 广东大华农动物保健品有限公司 | Animal vaccine immunopotentiator and production method thereof |
| CN102000331A (en) * | 2010-11-09 | 2011-04-06 | 国家兽用生物制品工程技术研究中心 | Application of bursin as swine fever vaccine adjuvant |
| CN102634489A (en) * | 2012-03-22 | 2012-08-15 | 肇庆大华农生物药品有限公司 | Recombinant turkey herpesvirus and application thereof |
| CN102796200A (en) * | 2012-08-17 | 2012-11-28 | 南京大学 | Hybrid peptide bursin adjuvant and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 邬向东等: "禽法氏囊三肤囊素免疫生物学及其同功能单链抗体研究进展", 《动物医学进展》, vol. 23, no. 2, 31 December 2002 (2002-12-31) * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017027324A1 (en) | 2015-08-10 | 2017-02-16 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| EP3334833A4 (en) * | 2015-08-10 | 2019-04-24 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| US10537628B2 (en) | 2015-08-10 | 2020-01-21 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| US10813991B2 (en) | 2015-08-10 | 2020-10-27 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| US11433130B2 (en) | 2015-08-10 | 2022-09-06 | The Texas A&M University System | Recombinant turkey herpesvirus vaccines and uses thereof |
| CN109310750A (en) * | 2016-06-17 | 2019-02-05 | 英特维特国际股份有限公司 | Encode the recombination non-pathogenic marek's disease virus construct of infectious laryngotracheitis virus and infectious bursal disease virus antigen |
| CN109310750B (en) * | 2016-06-17 | 2022-08-12 | 英特维特国际股份有限公司 | Recombinant nonpathogenic Marek's disease virus constructs encoding infectious laryngotracheitis virus and infectious bursal disease virus antigens |
| US11596687B2 (en) | 2016-06-17 | 2023-03-07 | Intervet Inc. | Recombinant non-pathogenic Marek's disease virus constructs encoding infectious laryngotracheitis virus and infectious bursal disease virus antigens |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103589693B (en) | 2016-02-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roohani et al. | Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens, and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV | |
| Gimeno | Marek's disease vaccines: a solution for today but a worry for tomorrow? | |
| CA3146727C (en) | Recombinant non-pathogenic marek's disease virus constructs encoding infectious laryngotracheitis virus and newcastle disease virus antigens | |
| JP5508252B2 (en) | Recombinant turkey herpesvirus containing avian influenza gene | |
| EP3185899B1 (en) | Improved hvt-vectored nd-ibd vaccine | |
| RU2593950C2 (en) | Recombinant nonpathogenic mdv vector providing poly-specific immunity | |
| Arnold et al. | Protective vaccination against infectious bursal disease virus with whole recombinant Kluyveromyces lactis yeast expressing the viral VP2 subunit | |
| Yi et al. | Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with largemouth bass virus (LMBV) | |
| CN107815441A (en) | A kind of II type Pseudorabies virus attenuated strain and its preparation method and application | |
| US20240123057A1 (en) | Recombinant Non-Pathogenic Marek's Disease Virus Constructs Encoding Infectious Laryngotracheitis Virus and Infectious Bursal Disease Virus Antigens | |
| CN114107228B (en) | Construction of attenuated African swine fever virus strain with twelve genes deleted and application of attenuated African swine fever virus strain as vaccine | |
| CN107432930A (en) | A kind of type aviadenovirus DNA vaccination of I group 4 and its preparation method and application | |
| JP2021500876A (en) | Recombinant non-pathogenic Marek's disease virus construct encoding multiple heterologous antigens | |
| CN109321535A (en) | A kind of heat-staple newcastle disease virus attenuated vaccine Candidate Strain | |
| CN113943714A (en) | Cat calicivirus strain and application thereof | |
| CN109266593B (en) | Ngpiwi protein-mediated avian pasteurella multocida gene knockout strain and construction method and application thereof | |
| JP6682441B2 (en) | Avian reovirus vaccine | |
| CN103589693B (en) | A kind of expression IBDV VP2 and bursa of Fabricius bursin chimeric protein recombinant herpesvirus of turkeys | |
| CN106047783B (en) | Kill sweetfish pseudomonad ExoU gene knockout mutant strain and its application | |
| CN102154224A (en) | Construction of recombinant chicken marek's disease vaccine virus SC9-1 strain and application thereof | |
| Sun et al. | DNA immunization against very virulent infectious bursal disease virus with VP2‐4‐3 gene and chicken IL‐6 gene | |
| CN106916832B (en) | O-type foot-and-mouth disease virus recombinant nucleic acid, recombinant vaccine strain, preparation method and application thereof | |
| CN102703369B (en) | Recombinant bifidobacterium for preparing EV (Enterovirus) 71 vaccine as well as preparation method and application of recombinant bifidobacterium | |
| CN105255841A (en) | Establishment method and application of H9 subtype avian influenza recombined duck enteritis virus rDEVdeltagE-H9 strain | |
| CN105385665B (en) | H9 subtype avian influenza recombinates UL2-H9 plants of duck enteritis virus rDEV Δ of building and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160203 |