CN106701693A - Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus - Google Patents

Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus Download PDF

Info

Publication number
CN106701693A
CN106701693A CN201710046543.0A CN201710046543A CN106701693A CN 106701693 A CN106701693 A CN 106701693A CN 201710046543 A CN201710046543 A CN 201710046543A CN 106701693 A CN106701693 A CN 106701693A
Authority
CN
China
Prior art keywords
virus
recombinant
rhvt
genes
serum
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.)
Pending
Application number
CN201710046543.0A
Other languages
Chinese (zh)
Inventor
毕建敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Bangzhuo Biological Science & Technology Co ltd
Original Assignee
Beijing Bangzhuo Biological Science & Technology Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beijing Bangzhuo Biological Science & Technology Co ltd filed Critical Beijing Bangzhuo Biological Science & Technology Co ltd
Priority to CN201710046543.0A priority Critical patent/CN106701693A/en
Publication of CN106701693A publication Critical patent/CN106701693A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The invention relates to construction and an application of a recombinant herpesvirus of turkey (HVT) for expressing a GB gene of a serum I type Marek's disease virus. The constructed recombinant chimeric herpesvirus of turkey rHVT-GB strain is a recombinant virus obtained by recombining a serum I type MDV GB (glycoprotein B, MDV1-GB) gene expression cassette to HVT; the recombinant virus can be used as a production virus seed for preparation of a Marek's disease vaccine; compared with the HVT, the recombinant virus plays better immune protection role and greatly enhances the immune protective ability to MD; the rHVT-GB recombinant virus strain can be used as a carrier for constructing viruses capable of expressing other avian pathogenic protective antigen genes (such as a newcastle disease virus F gene, an infectious laryngotracheitis GB gene and an infectious bursa of Fabricius VP2 gene) and used for preparation of live vector vaccines for preventing other corresponding poultry diseases.

Description

Express the recombinant herpesvirus of turkeys of the type marek's disease virus GB genes of serum I Build and its apply
Technical field
The present invention relates to a kind of structure of the recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I And its application.Animal biological product field,
Background technology
Chicken Marek's disease (Marek ' s disease, MD), is by chicken Marek's disease virus (Marek ' s disease Virus, MDV) a kind of chicken Marek's disease (Marek`s Disease, MD) for causing is cause chicken by chicken herpetoviruses one Most common lymphoproliferative diseases is planted, it is thin with the lymph sample of peripheral nerve, iris, skin, muscle and each internal organs Born of the same parents' infiltration, hyperplasia and tumour are formed and are characterized.
This disease was found first early in 1907 by Hungarian veterinary pathologist horse Garrick, and was named as horse in 1961 Vertical creutzfeldt jakob disease.There is prevalence each main poultry countries and regions in the present world, are also most important fowl on economic implications in China One of disease, serious threat and huge economic loss is caused to China's poultry production.
Marek's disease virus (Marek ' s disease virus, MDV) belongs to cell-associated herpesviral B groups.Disease Poison has two kinds of existence forms, i.e. nude particle (nucleocapsid) and has the complete virus particle of cyst membrane.The former virus nucleocapsid is in six Angular, a diameter of 85~100nm has strict cell-associated, leaves cytopathic and is remarkably decreased and loses, in the external world Survival activity is very low in environment, is mainly seen in renal tubule, the bursa of farbricius, nerve fiber and tumor tissues.Most of naked virals Particle is present in nucleus, occasionally in cytoplasm or extracellular fluid.The latter is primarily present in nuclear membrane nearby or core In vacuole, 130~170nm of diameter is mainly seen in feather capsule cuticular layer, and majority is the complete virus particle for having cyst membrane, non-thin Born of the same parents' associativity, can depart from cell and exist, and environmental resistance is strong to external world, is played an important role in terms of the propagation of this disease.
Bulow and Biggs in 1975 is neutralized using indirect immunofluorescence assay, agar gel immune precipitation and virus MDV and correlated virus (HVT), are divided into three kinds of serotypes by experiment, and subsequent this sorting technique is special by the MDV types that Lee is set up Specific monoclonal antibodies are confirmed.
Three kinds of serotypes according to MDV, current commercialization MDV vaccines are divided into I weak type CVI988/Rispens plants, nothing of cause II pathogenic type SB1 plants and FC126 plants of III type herpes turkey virus (Herpesvirus of turkey, HVT).Cause weak I Type CVI988/Rispens plants good to MD protecting effects, but it is used as strict cell mating type virus, it is necessary to which Liquid nitrogen storage is transported It is defeated, significantly increase vaccine cost.HVT can freeze preservation, save vaccine cost, but its protection to MD is weaker, Therefore the immune efficacy for improving HVT is very necessary.The GB genes of the type MDV of serum I are inserted into Ross the TK bases of HVT genomes Cause, but the recombinant virus multiplication capacity in vivo for building dies down.(Ross LJ,Binns MM,Tyers P,Pastorek J, Zelnik V,Scott S:Construction and properties of a turkey herpesvirus recombinant expressing the Marek's disease virus homologue of glycoprotein B of herpes simplex virus.The Journal of general virology 1993,74(Pt3):371- 377.), (Gimeno IM, Witter RL, Hunt HD, Reddy and the multiplication capacity of vaccine virus and protection are closely related SM,Reed WM:Biocharacteristics shared by highly protective vaccines against Marek's disease.Avian Pathol2004,33(1):59-68.).Therefore, normal proliferative is built and with guarantor higher The HVT for protecting power is highly desirable to.
Vaccine is one of important means of anti-livestock and poultry processed, and big more important fowl infection all can use corresponding at present Vaccine makees effective prevention.In order to further reduce production cost, improve product quality, the research of fowl new generation vaccine is compeled in eyebrow Eyelash.With becoming better and approaching perfection day by day for Protocols in Molecular Biology, to transform viral nucleic acid structure as the vaccine of means, such as gene-deleted strain epidemic disease Seedling, insertion mutation strain, virus live vector vaccine etc. have obtained great development, and wherein virus live vector vaccine is with production and application side Just, it is cheap, and security, stability and the aspect advantage, the focus as research such as immunity is stronger.Relative to other diseases Poisonous carrier (such as fowlpox virus, aviadenovirus, NDV), chicken Marek's disease virus (Marek ' s disease Virus, MDV) used as most potential vaccine carrier, its advantage for protruding is embodied in：MDV is strict cell mating type disease Poison, the carrier bacterin of structure is not disturbed by maternal antibody, overcomes the defect of numerous live vector vaccines；MDV molecules are big The small insertion for being 180kb or so, larger fragment gene being accommodated, the stability without influenceing recombinant virus；MDV vaccines are adapted to Early immune, the research and development to most virus particularly immunosuppressive virus vaccines bring dawn.Virus is built based on HVT living Carrier bacterin not only has above-mentioned advantage, can also realize that a seedling two is prevented, a seedling is prevented more, with wide market prospects.
The content of the invention
The purpose of the present invention is for there is provided a kind of chimeric herpes turkey virus (Herpesvirus of turkey, HVT) RHVT-GB, it is actually the base in Serum III type marek's disease virus (Marek ' s disease virus, MDV) HVT On plinth, by the expression cassette restructuring of serum I type MDV GB (glycoprotein B, MDV1-GB) gene to HVT, one is obtained Plant brand-new recombinant herpesvirus of turkeys rHVT-GB；It using rHVT-GB is carrier that the present invention is also simultaneously, and structure can express Other birds cause of disease protective antigen genes (such as F gene of NDV strain, the GB genes of infectious laryngotracheitis and infectiousness method The VP2 genes of family name's capsule) virus live vector vaccine, the vaccine can not only prevent MD, while other birds epidemic diseases can also be prevented Disease.
Technical scheme
1. it is a kind of express the type marek's disease virus GB genes of serum I recombinant herpesvirus of turkeys, it is characterised in that should The recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I is named as a kind of recombinant herpesvirus of turkeys RHVT-GB plants of (Herpesvirus of turkey), the strain sent Chaoyang District, Beijing City North Star west on 01 11st, 2017 The China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica of institute 3 of road 1 is protected Hide, its deposit number is：CGMCC No.13400.
2. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes of the present invention Build, it is characterised in that：By in the GB expression casettes restructuring of the type MDV of serum I to HVT genomes, recombination site is HVT (GenBank:AF291866.1) the US2 regions between HVT087-HVT088 genes of genome, the gene of the expression cassette Sequence such as Seq ID No:Shown in 1.
3. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes of the present invention Using, it is characterised in that the recombinant strain induces the application in the protective immunity of anti-Marek's disease in chicken body.
4. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes of the present invention Using, it is characterised in that the recombinant strain as production strain animal vaccine strain application.
5. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes of the present invention Using, it is characterised in that the recombinant virus is built the application in new recombinant vaccine virus as viral vectors.
6. feature of the present invention a kind of type marek's disease virus GB genes of expression serum I recombinant turkey bleb The application of virus,, in by the F expression casettes restructuring of NDV to rHVT-GB genomes, recombination site is HVT for it (GenBank:AF291866.1) between the HVT065 and HVT066 of genome, wherein the promoter sequence of F expression casettes is such as Seq ID No:Shown in 2；F gene orders such as Seq ID No:Shown in 3；PolyA sequences such as Seq ID No:Shown in 6, structure is obtained The recombinant virus for obtaining is named as rHVT-GB-NDVF plants of recombinant herpesvirus of turkeys (Herpesvirus of turkey), should Strain delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica on 01 11st, 2017 China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, its deposit number is：CGMCC No.13399.
7. feature of the present invention a kind of type marek's disease virus GB genes of expression serum I recombinant turkey bleb The application of virus, it induces anti-Marek's disease and newcastle disease in recombinant herpesvirus of turkeys rHVT-GB-NDVF plants in chicken Protective immunity in application and as production application of the strain in corresponding animal vaccine.
8. feature of the present invention a kind of type marek's disease virus GB genes of expression serum I recombinant turkey bleb The application of virus, it builds new recombinant vaccine virus as viral vectors in recombinant herpesvirus of turkeys rHVT-GB-NDVF plants Application.
9. feature of the present invention a kind of type marek's disease virus GB genes of expression serum I recombinant turkey bleb The application of virus, it is characterised in that by the GB expression casettes restructuring of avian infectious laryngotracheitis virus to rHVT-GB genomes In, recombination site is HVT (GenBank:AF291866.1) between the HVT065 and HVT066 of genome, ILTV-GB gene tables Up to the promoter sequence such as Seq ID No of box:Shown in 2, GB gene orders such as Seq ID No:Shown in 4, PolyA sequences such as Seq ID No:Shown in 6；Acquisition is named as recombinant herpesvirus of turkeys (Herpesvirus of turkey) rHVT-GB-ILTVGB Strain, the strain was delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism and is ground on 01 11st, 2017 The preservation of Jiu Suo China Committee for Culture Collection of Microorganisms's common micro-organisms centers, its deposit number is：CGMCC No.13398。
10. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes of the present invention Using, it is characterised in that recombinant herpesvirus of turkeys rHVT-GB-ILTVGB plants therein induced in chicken anti-Marek's disease and Application in the protective immunity of infectious laryngotracheitis of chicken and the application as production strain in corresponding animal vaccine.
A kind of 11. recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I of the present invention Using, it is characterised in that recombinant herpesvirus of turkeys rHVT-GB-ILTVGB plants therein builds new restructuring as viral vectors The application of vaccine virus.
A kind of 12. recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I of the present invention Using, it is characterised in that by the VP2 expression casettes restructuring of infections chicken cloacal bursa virus to rHVT-GB genomes, recombinate Site is HVT (GenBank:AF291866.1) between the HVT065 and HVT066 of genome, the promoter of VP2 expression casettes Sequence such as Seq ID No:Shown in 2, VP2 gene orders such as Seq ID No:Shown in 5, PolyA sequences such as Seq ID No:6 institutes Show；Acquisition is named as recombinant herpesvirus of turkeys (Herpesvirus of turkey) rHVT-GB-IBDVVP2 plants, the poison During strain delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica on 01 11st, 2017 State's Microbiological Culture Collection administration committee common micro-organisms center preservation, its deposit number is：CGMCC No.13397.
A kind of 13. recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I of the present invention Application, it is characterised in that wherein recombinant herpesvirus of turkeys rHVT-GB-IBDVVP2 plants induces anti-chicken Marek's in chicken Application in the protective immunity of disease and gumboro disease and the application as production strain in corresponding animal vaccine.
A kind of 14. recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I of the present invention Application, it is characterised in that wherein recombinant herpesvirus of turkeys rHVT-GB-IBDVVP2 plants builds new weight as viral vectors The application of group vaccine virus.
The specific embodiment of the invention
1. the structure of strain：
(1) the invention provides a kind of recombinant herpesvirus of turkeys (rHVT-GB), it is actually by the type MDV of serum I GB genes (MDV1-GB) are recombinated in herpes turkey virus genome, obtain a kind of chimeric herpes turkey disease of brand-new restructuring Malicious rHVT-GB, the virus is inserted into HVT by by the MDV1-GB genes (MDV1-GB expression casettes) under being controlled by promoter In genome, rHVT-GB can play immanoprotection action more more preferable than HVT to chicken MD.
The DNA sequence pattern figure of the recombinant virus is as shown in Figure 1, specific as follows：In the present invention, inventor will be contained In MDV1-GB expression casettes insertion HVT genomes, the region of insertion is the nonessential region of viral growth in HVT genomes US regions (between HVT genome HVT087-HVT088 genes).To achieve the above object, inventor constructs first For the recombinant plasmid of homologous recombination, the recombinant plasmid contains the homologous sequence of US2 genes both sides, with HVT genomes as template Expanded, the left and right arms sequence after amplification is cloned into PUC18 by restriction enzyme site, the restructuring containing left and right arms after clone Plasmid is named as PHVT-DS.Then by the weak malicious CVI988 pnca genes group (GenBank of the type MDV of serum I:DQ530348.1) it is mould Plate amplification obtains expression casette containing MDV1-GB, and the MDV1-GB expression cassettes after amplification are cloned into by PACI restriction enzyme sites In PHVT-DS, the successful plasmid of clone identification is for MDV1-GB to be recombinated the recombinant plasmid into HVT US2 regions, the matter Grain is named as PHVT-DS-MDV1-GB.Using recombinant plasmid (PHVT-DS-MDV1-GB) and HVT genome cotransfection CEF, treat After there is plaque, cell is passed into a generation, stay half liquid nitrogen cryopreservation, virus is diluted using limiting dilution assay after second half ultrasonication (dilution gradient is carried out, every hole is averagely only contained 1 plaque or less than 1 plaque) in reaching 96 porocyte culture plates, wait to lose After spot occurs, select the hole only containing 1 plaque and passed on, 1 hole passes 2 parallel holes.After after plaque appearance, wherein 1 Recombinant virus is detected in individual hole using indirect immunofluorescence (being screened using the monoclonal antibody of anti-MDV-GB), if the positive, correspondence The virus in another 1 hole then contain recombinant virus, carry out virus seed conservation identification through excessive wheel after purification, the virus is named as A kind of recombinant herpesvirus of turkeys rHVT-GB plants, and delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on 01 11st, 2017 The preservation of No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of Institute of Microorganism, Academia Sinica, its guarantor Hiding numbering is：CGMCC No.13400.
(2) while, the present invention using rHVT-GB be carrier platform, express respectively NDV (NDV) F genes, The GB genes of avian infectious laryngotracheitis virus (ILTV) and the VP2 genes of infections chicken cloacal bursa virus (IBDV).It is above-mentioned heavy The DNA sequence pattern figure of group virus is as shown in accompanying drawing 2, Fig. 3 and Fig. 4.Concrete scheme is as follows：In the present invention, inventor will contain In thering is NDV F genes, ILTV-GB genes and IBDV-VP2 expression casettes to insert rHVT-GB genomes respectively, the region of insertion It is HVT (GenBank:AF291866.1) between the HVT065 and HVT066 of genome.To achieve the above object, inventor Construct the recombinant plasmid for homologous recombination first, NDV F genes by artificial synthesized, in order to consider effect, the F bases of synthesis Because type belongs to the d hypotypes of NDV genes VII, while, it is contemplated that security, the amino acid sequence of its F gene cracking site is artificially changed It is GRQGRL to make, and meets the feature of low virulent strain；ILTV-GB gene references vaccine strain (GenBank:JN580317.1) GB sequences Row；IBDV-VP2 gene reference NCBI sequences (GenBank:HG974565.1), above-mentioned F genes, GB genes and VP2 genes by Commercial company synthesizes.Promoter with reference to the promoter sequence of herpesviral (Murid herpesvirus 1strain Smith) Row, terminator sequence is the PolyA terminator sequences commonly used in carrier for expression of eukaryon.Specific construction method is as follows：Carry according to a conventional method HVT virus genom DNAs are taken, according to the HVT-FC126 plants of (GenBank delivered in Genebank:AF291866.1 full base) Because of sequence, expand the left homology arm of left homology arm amplification and be connected to PMD19-Tsimple carriers, be built into plasmid PMD19-L.Design Primer expands promoter, and promoter is cloned into PMD19-L, is built into plasmid PMD19-L-PRO.Then design primer amplification VP2 genes, during VP2 gene clonings are entered into PMD19-L-PRO, are built into plasmid PMD19-L-PRO-VP2.Design primer amplification PolyA (with pCMVbeta plasmids as template, GenBank:U02451), PolyA is cloned into PMD19-L-PRO-VP2, structure Build up plasmid PMD19-L-PRO-VP2-PolyA；Finally design primer expands right homology arm, is cloned into plasmid PMD19-L-PRO- VP2-PolyA, is built into final recombinant plasmid PMD19-L-PRO-VP2-PolyA-R, and the plasmid is used for IBDV-VP2 weights Group is between the HVT065 and HVT066 in rHVT-GB genomes.Building the process of PMD19-L-PRO-VP2-PolyA-R In, VP2 genes two ends devise Not I restriction enzyme sites, can be by NDV-F and ILTV-GB gene clonings using this restriction enzyme site Enter PMD19-L-PRO-VP2-PolyA-R, respectively construction recombination plasmid PMD19-L-PRO-F-PolyA-R and PMD19-L-PRO- GB-PolyA-R.With reference to the construction method of rHVT-GB, respectively by PMD19-L-PRO-VP2-PolyA-R, PMD19-L-PRO-F- PolyA-R and PMD19-L-PRO-GB-PolyA-R recombinant plasmids and rHVT-GB genome cotransfection CEF, by indirectly immune Fluorescent technique clones recombinant virus using limiting dilution assay, and recombinant herpesvirus of turkeys is obtained respectively using the method RHVT-GB-NDVF plants of (Herpesvirus of turkey), recombinant herpesvirus of turkeys (Herpesvirus of turkey) RHVT-GB-ILTVGB plants and rHVT-GB-IBDVVP2 plants of recombinant herpesvirus of turkeys (Herpesvirus of turkey), should Three strain virus were delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism and are ground on 01 11st, 2017 Jiu Suo China Committee for Culture Collection of Microorganisms's common micro-organisms centers preservation, its deposit number is respectively and is：CGMCC No.13399, CGMCC No.13398 and CGMCC No.13397.
2. the checking of recombinant virus
RHVT-GB, rHVT-GB-NDVF, rHVT-GB-ILTVGB, rHVT-GB-IBDVVP2 and HVT are inoculated in 12 holes On Tissue Culture Plate.After obvious plaque occurs in culture, respectively with the monoclonal antibody of anti-MDV1-GB, NDV-F, ILTV-GB and IBDV-VP2 Or serum carries out indirect immunofluorescence identification, as a result shows, in MDV1-GB genes, the rHVT-GB-NDVF in rHVT-GB In MDV1-GB and ILTV-GB and rHVT-GB-IBDVVP2 in MDV1-GB and NDV-F genes, rHVT-GB-ILTVGB MDV1-GB and IBDV-VP2 obtain good correct expression.
3. prepared by vaccine
Above-mentioned recombinant virus is prepared into vaccine by inventor, and immune operation is carried out to facilitate, and further, inventor will weigh Group virus applications are in the immunoprotection experiment of chicken.
After rHVT-GB evaluates 1 age in days SPF chicken immunes rHVT-GB to the immune protection effectiveness of chicken prevention MD, 7 days after being immunized Chicken Marek's disease virus (Marek ' s disease virus, MDV) Md5 highly virulent strains of 1000PFU virus quantities are inoculated with respectively (ATCC, VR-987), rHVT-GB immune group vaccines protective index is 75, hence it is evident that better than HVT immune groups (protective index is 55).
RHVT-GB-NDVF evaluates 1 age in days SPF chicken immune rHVT-GB-NDVF to chicken prevention ND immune protection effectiveness, is immunized The strong poison Beijing Strain 0.5ml of each intramuscular injection NDV on the 21st (contains 10 afterwards5EID50, China Veterinery Drug Inspection Office, CVCC AV1611), 14d is observed, rHVT-GB-NDVF reaches 90% to the protective rate of ND.
RHVT-GB-ILTVGB evaluates 1 age in days SPF chicken immune rHVT-GB-ILTVGB to ILT immune protection effectiveness, is immunized 21 days afterwards, the strong poison Beijing Strain 0.2ml of each intratracheal injection infectious laryngotracheitis of chicken of chicken (contained 104EID50, CVCC AV China beast Pharmaceuticals supervise institute), 10d is observed after attacking poison, rHVT-GB-ILTVGB reaches 100% to ILT protective rates.
RHVT-GB-IBDVVP2 evaluates 1 age in days SPF chicken immunes rHVT-GB- to chicken prevention IBD immune protection effectiveness IBDVVP2,21 eye droppings of age in days every attack BC6/85 plants of IBDV (China Veterinery Drug Inspection Office, CVCC AV7) 10 MID, The cut open inspection bursa of farbricius after 72 hours, observes bursa of farbricius lesion.RHVT-GB-IBDVVP2 immune groups protect 17.HVT-GB immune groups There is lesion (bursa of farbricius serous coat occurs in gel-shaped or has different degrees of bleeding), rHVT- with the malicious control group bursa of farbricius is attacked GB-IBDVVP2 can form good protection to IBD.
In sum, the invention provides a kind of recombinant herpesvirus of turkeys (rHVT-GB), it is actually by serum I The expression cassette of type MDV GB genes is recombinated in HVT, obtains a kind of brand-new recombinant herpesvirus of turkeys rHVT-GB, the disease Poison is also simultaneously a kind of good Mareks disease vaccine, and used as vaccine, compared to HVT, rHVT-GB can be played preferably to MD Immanoprotection action.
The present invention is simultaneously carrier using rHVT-GB, and it is (new that structure can express other birds cause of disease protective antigen genes The VP2 genes of city epidemic disease virus F gene, the GB genes of infectious laryngotracheitis and infectious bursa of Fabricius) virus live vector vaccine, Obtain 3 recombinant herpesvirus of turkeys rHVT-GB-NDVF plants, rHVT-GB-ILTVGB plants and rHVT-GB-IBDVVP2 plants.Will Above-mentioned 3 recombinant viruses prepare vaccine, can not only prevent MD, while each can also effectively prevent other birds epidemic diseases.
Brief description of the drawings
Fig. 1 is rHVT-GB genome schematic diagrames；US2 regions in MDV1-GB expression cassettes insertion HVT genomes, insert position Point is between gene HVT087 and HVT088.
Fig. 2 is MDV1-GB expression cassettes integration site schematic diagram in HVT genomes；Strikethrough blackens Sequences upstream part It is gene HVT087, strikethrough blackens sequence downstream part for gene HVT088 (US2), and strikethrough part replaces for MDV1-GB HVT sequences.
Fig. 3 is NDV-F/ILTV-GB/IBDV-VP2 expression cassette integration site schematic diagrames；Blackening Sequences upstream part is HVT065, blackens sequence downstream part for HVT066, and strikethrough part replaces for NDV-F/ILTV-GB/IBDV-VP2 expression cassettes HVT sequences.
Fig. 4 is rHVT-GB-NDVF genome schematic diagrames；In NDV-F expression cassettes insertion HVT genomes, insertion point is base Because between HVT065 and HVT066.
Fig. 5 is rHVT-GB-ILTVGB genome schematic diagrames；In ILTV-GB expression cassettes insertion HVT genomes, insertion point For between gene HVT065 and HVT066.
Fig. 6 is rHVT-GB-IBDVVP2 genome schematic diagrames；In IBDV-VP2 expression cassettes insertion HVT genomes, position is inserted Point is between gene HVT065 and HVT066.
The present invention relates to biomaterial resource information
Recombinant herpesvirus of turkeys rHVT-GB plants, rHVT-GB-NDVF plants, rHVT-GB-ILTVGB plants of present invention structure Four strain virus are waited to deliver Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 on 01 11st, 2017 with rHVT-GB-IBDVVP2 plants Number preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center of Institute of Microorganism, Academia Sinica, its preservation Number and be：CGMCC No.13400, CGMCC No.13399, CGMCC No13398. and CGMCC No.13397；Chicken Marek's disease virus (Marek ' s disease virus) Md5 plants (ATCC, VR-987) is purchased from American Type Culture Collecti (American type culture collection,ATCC)；Newcastle disease virus (Newcastle v disease Irus) strong poison Beijing Strain (CVCC AV1611), avian infectious laryngotracheitis virus Infectious Beijing Strains (CVCC AV22) With chicken infectivity bursa of Fabricius virus (Infectious bursal disease virus) BC6/85 plants (CVCC AV7) see China Veterinery Drug Inspection Office, Chinese veterinary microorganism culture presevation administrative center write, Chinese animal doctor's strain catalogue (second Version), Scientia Agricultura Sinica technology publishing house, p142,137 and 135 in 2002.
Positive effect of the invention
The present invention relates to express the recombinant herpesvirus of turkeys of the type marek's disease virus GB genes of serum I structure and its Using the invention provides a kind of chimeric herpes turkey virus (Herpesvirus of turkey, HVT) rHVT-GB, in fact It is on the basis of Serum III type marek's disease virus (Marek ' s disease virus, MDV) HVT, by serum I on border The expression cassette of type MDV GB (glycoprotein B, MDV1-GB) gene is recombinated in HVT, obtains a kind of brand-new restructuring Herpes turkey virus rHVT-GB, the virus is also simultaneously a kind of good Mareks disease vaccine, by will be by promoter control Under MDV1-GB genes be inserted into HVT genomes, used as vaccine, compared to HVT, rHVT-GB can be to chicken Marek's disease (Marek ' s disease, MD) plays more preferable immanoprotection action.
Relative to the type MD vaccines of serum I, HVT can freeze preservation, greatly save transport storage cost.HVT is except energy Outside enough prevention MD, itself or excellent viral vectors, but HVT, in itself as MD vaccines, the protective capability to MD is weaker, and it is of the invention The embedded virus rHVT-GB of structure, greatly strengthen the immune protection to MD.The present invention is simultaneously load using rHVT-GB Body, structure can express other birds cause of disease protective antigen genes (such as F gene of NDV strain, infectious laryngotracheitis The VP2 genes of GB genes and infectious bursa of Fabricius) virus live vector vaccine, the vaccine can not only prevent MD, while can also Enough prevent other birds epidemic diseases.
Embodiment
Following examples are not construed as limiting to further illustrate the present invention to claimed technical scheme of the invention.
Embodiment 1
--- the structure of embedded virus rHVT-GB
1. the structure of recombinant plasmid PHVT-DS
The recombinant plasmid for homologous recombination is built first, and the recombinant plasmid contains the homologous sequence of US2 genes both sides, draws Thing (sequence 7, sequence 8, sequence 9 and sequence 10)；
Expanded by template of HVT genomes, the left and right arms sequence after amplification is cloned into PUC18 by restriction enzyme site In, the recombinant plasmid containing left and right arms after clone is named as PHVT-DS.
The structure of 2.PHVT-DS-MDV1-GB
By the weak malicious CVI988 pnca genes group (GenBank of the type MDV of serum I:DQ530348.1) for template amplification is contained MDV1-GB expression casettes, it is specific as follows：
The type GB expression casettes (MDV1-GB expression cassettes) of design primer (sequence 11 and sequence 12) amplification MDV serum I：
With CVI988 genomes as template, expanded, the MDV1-GB expression cassettes after amplification pass through PACI restriction enzyme sites gram It is grand enter PHVT-DS in, the successful plasmid of clone identification is for MDV1-GB to be recombinated the recombinant plasmid into HVT US2 regions, The plasmid is named as PHVT-DS-MDV1-GB.
HVT-US2left sense：5-GATgaattcA CATCGGGCCA CGTTCCGCC-3 29 (sequence 7)；
HVT-US2left antisense：5-ATAGTCGACt taattaaGAT GAGCTGACGT GTGGAAT-3 37 (sequence 8).
HVT-US2right sense：5-ATCgtcgacA CTAATATGGG CACACCCAC-3 29 (sequence 9)；
HVT-US2right antisense：(the sequences of 5-ATCaagcttT GGCCCATCTA GGTGATTAT-3 29 10)。
MDV1-GB-F:CggTTAATTA Agacctccct tggccatgat gaatgg 36 (sequence 11)；
MDV1-GB-R:(two ends add PACI enzymes to ccgTTAATTA Aagaaggaaa gcatcgagca atcac 36 Enzyme site, sequence 12).
3. the structure of recombinant virus
Using recombinant plasmid (PHVT-DS-MDV1-GB) and HVT genome cotransfection CEF, after plaque to appear, by cell A generation is passed, half liquid nitrogen cryopreservation is stayed, 96 hole cell culture are reached using limiting dilution assay dilution virus after second half ultrasonication (dilution gradient is carried out, every hole is averagely only contained 1 plaque or less than 1 plaque) in plate, after after plaque appearance, select and only contain The hole for having 1 plaque is passed on, and 1 hole passes 2 parallel holes.After after plaque appearance, wherein 1 hole is glimmering using being immunized indirectly Light (being screened using the monoclonal antibody of anti-MDV-GB) detects recombinant virus, and if the positive, the virus in corresponding another 1 hole then contains There is recombinant virus, carry out virus seed conservation identification through excessive wheel after purification, the Strain is named as recombinant herpesvirus of turkeys RHVT-GB plants (CGMCC No.13400).
Embodiment 2
--- the structure of recombinant herpesvirus of turkeys rHVT-GB-NDVF, rHVT-GB-ILTVGB and rHVT-GB-IBDVVP2 Build
It is carrier platform using rHVT-GB, F genes, the chicken infectivity throat tracheae of NDV (NDV) is expressed respectively The GB genes and the VP2 genes of infections chicken cloacal bursa virus (IBDV) of scorching virus (ILTV).Specific construction method is as follows：
1. recombinant herpesvirus of turkeys rHVT-GB-NDVF, rHVT-GB-ILTVGB and rHVT-GB-IBDVVP2 plants of structure Build
HVT virus genom DNAs are extracted according to a conventional method, according to HVT-FC126 plants delivered in Genebank (GenBank:AF291866.1 complete genome sequence), expands left homology arm, expands left homology arm and is connected to PMD19-Tsimple Carrier, is built into plasmid PMD19-L.Design primer (sequence 15 and sequence 16) amplification promoter, promoter is cloned into In PMD19-L, plasmid PMD19-L-PRO is built into.Then design primer (sequence 17 and sequence 18) expands VP2 genes, by VP2 Gene cloning enters in PMD19-L-PRO, is built into plasmid PMD19-L-PRO-VP2.Design primer (sequence 19 and sequence 20) expands Increase PolyA, PolyA is cloned into PMD19-L-PRO-VP2, be built into plasmid PMD19-L-PRO-VP2-PolyA；Finally set Meter primer HVT-R-F (sequence 21) and HVT-R-R (sequence 22) expand right homology arm, are cloned into plasmid PMD19-L-PRO-VP2- PolyA, is built into final recombinant plasmid PMD19-L-PRO-VP2-PolyA-R, and the plasmid is used for arriving IBDV-VP2 restructuring Between HVT065 and HVT066 in rHVT-GB genomes.During PMD19-L-PRO-VP2-PolyA-R is built, VP2 Gene two ends devise Not I restriction enzyme sites, NDV-F and ILTV-GB can be cloned into PMD19-L- using this restriction enzyme site PRO-VP2-PolyA-R, difference construction recombination plasmid PMD19-L-PRO-F-PolyA-R and PMD19-L-PRO-GB-PolyA- R.With reference to the construction method of rHVT-GB, respectively by PMD19-L-PRO-VP2-PolyA-R, PMD19-L-PRO-F-PolyA-R and PMD19-L-PRO-GB-PolyA-R recombinant plasmids and rHVT-GB genome cotransfection CEF, by IiT profit Recombinant virus is cloned with limiting dilution assay, recombinant herpesvirus of turkeys rHVT-GB-NDVF (CGMCC are obtained respectively using the method No.13399), rHVT-GB-ILTVGB (CGMCC No.13398) and rHVT-GB-IBDVVP2 (CGMCC No.13397).
HVT-L-F:5-GTTCCTTGAA ATGCCGACAA CTCTAAAAAC GGTATTCG-3 38 (sequence 13)；
HVT-L-R:5-GCTAGCGCGG CCGCGAATTC GTTTAATGTT AGTTTATT-3 38 (hold by anti-sense primer 5 ' Contain Nhe I/NotI/EcoRI restriction enzyme sites, sequence 14)；
PRO-F:-GGGGAATTCA CTAGTGGATC CCCCAACTCC GCCCGTTTTA TGACTAGA-3 48 (EcoRI, sequence 15)；
PRO-R:5-AAATTGCGGC CGCCTGCAGC GAGGAGCTCT GCGTTCTACG GTGGT-3 45 (Not I, Sequence 16)
Vp2-F:5-aatGCGGCCG CTCTAGAACT CGTCGATCGC AGCGATGACA AACCTGCAAG-3 50 (Not I, sequence 17)；
Vp2-R:5-GGCGCTAGCA AGCTTACCTC CTTATAGCCC GGATTATGTC TTTGAAGCCA-3 50 (HindIII/Nhe I, sequence 18),
polyA-F:5-AATAAGCTTG ATCTAGAGCG GCCGCGG-3 27 (HindIII, sequence 19)；
polyA-R:5-AATGCTAGCG TCGACTCTAG AGGATCCG-3 28 (Sal I/Nhe I, sequence 20),
HVT-R-F:5-GGCGGTCGAC AATTATTTTA TTTAAT-3 26 (sequence 21)；
HVT-R-R:5-GGCGCTAGCC TAGTGTTTCA ATTAT-3 25 (sequence 22)
Embodiment 3
--- the checking of recombinant virus
1.rHVT-GB recombinant viruses
The rHVT-GB and HVT of 50PFU virus quantities are inoculated in 12 porocyte culture plates.After there is obvious plaque in culture, Growth-promoting media is outwelled, with cold acetone:Ethanol (3:2) fixer fixes 10min, and PBS is washed 1 time, rHVT-GB and HVT inoculation holes In be separately added into 0.5mL (1:100 dilutions) MDV1-GB monoclonal antibodies, it is positioned in 37 DEG C of constant incubators and reacts 1h, with PBS 3 It is secondary, anti-mouse IgG fluorescence antibodies are marked plus 0.5mL FITC per hole, it is positioned in 37 DEG C of constant temperature biochemical cultivation cases and reacts 1h, use PBS is washed 3 times, is observed under inverted fluorescence microscope, and cell plaques occur in that specific green fluorescence, and control group HVT infects Unstressed configuration after CEF cells, as a result shows the correct expression that MDV1-GB genes can be good.
2.rHVT-GB-NDVF recombinant viruses
The rHVT-GB-NDVF and HVT of 50PFU virus quantities are inoculated in 12 porocyte culture plates.There is substantially erosion in culture After spot, growth-promoting media is outwelled, with cold acetone:Ethanol (3:2) fixer fixes 10min, and PBS washes 1 time, rHVT-GB-NDVF and 0.5mL (1 is added in HVT inoculation holes:100 dilutions) MDV1-GB monoclonal antibodies, added in rHVT-GB-NDVF and HVT inoculation holes in addition The anti-NDV-F mice serums of 0.5mL.It is positioned in 37 DEG C of constant incubators and reacts 1h, with PBS 3 times, 0.5mL is added per hole FITC marks anti-mouse IgG fluorescence antibodies, is positioned in 37 DEG C of constant temperature biochemical cultivation cases and reacts 1h, is washed 3 times with PBS, glimmering being inverted Viewed under light microscopy, cell plaques in rHVT-GB-NDVF inoculation holes add MDV1-GB monoclonal antibodies or anti-NDV-F mice serums Specific green fluorescence is occurred in that afterwards, and control group HVT infects unstressed configuration after CEF cells, shows MDV1-GB genes and NDV- F genes can be good correct expression.
3. recombinant virus rHVT-GB-ILTVGB
The rHVT-GB-ILTVGB and HVT of 50PFU virus quantities are inoculated in 12 porocyte culture plates.Culture occurs obvious After plaque, growth-promoting media is outwelled, with cold acetone:Ethanol (3:2) fixer fixes 10min, and PBS washes 1 time, rHVT-GB- 0.5mL (1 is added in ILTVGB and HVT inoculation holes:100 dilutions) MDV1-GB monoclonal antibodies, rHVT-GB-ILTVGB and HVT connect in addition Plant the addition anti-ILTV-GB mice serums of 0.5mL in hole.It is positioned in 37 DEG C of constant incubators and reacts 1h, with PBS 3 times, often Hole marks anti-mouse IgG fluorescence antibodies plus 0.5mL FITC, is positioned in 37 DEG C of constant temperature biochemical cultivation cases and reacts 1h, and 3 are washed with PBS It is secondary, observed under inverted fluorescence microscope, cell plaques in rHVT-GB-ILTVGB inoculation holes, add MDV1-GB monoclonal antibodies or anti- Specific green fluorescence is occurred in that after ILTV-GB mice serums, and control group HVT infects unstressed configuration after CEF cells, shows MDV1-GB genes and ILTV-GB genes can be good correct expression.
4.rHVT-GB-IBDVVP2 recombinant viruses
The rHVT-GB-IBDVVP2 and HVT of 50PFU virus quantities are inoculated in 12 porocyte culture plates.Culture occurs bright After aobvious plaque, growth-promoting media is outwelled, with cold acetone:Ethanol (3:2) fixer fixes 10min, and PBS washes 1 time, rHVT-GB- 0.5mL (1 is added in IBDVVP2 and HVT inoculation holes:100 dilutions) MDV1-GB monoclonal antibodies, in addition rHVT-GB-IBDVVP2 and HVT 0.5mL anti-ibd V-VP2 mice serums are added in inoculation hole.It is positioned in 37 DEG C of constant incubators and reacts 1h, with PBS 3 It is secondary, anti-mouse IgG fluorescence antibodies are marked plus 0.5mL FITC per hole, it is positioned in 37 DEG C of constant temperature biochemical cultivation cases and reacts 1h, use PBS is washed 3 times, is observed under inverted fluorescence microscope, cell plaques in rHVT-GB-IBDVVP2 inoculation holes, adds MDV1-GB mono- Specific green fluorescence is occurred in that after anti-or anti-ibd V-VP2 mice serums, and without glimmering after control group HVT infection CEF cells Light, shows the correct expression that MDV1-GB genes and IBDV-VP2 genes can be good.
Embodiment 4
--- recombinant virus prepares vaccine
By recombinant virus inoculated into chick embryo fibroblast, seed virus are prepared；Culture, inoculum density are enlarged with rolling bottle It is every 1cm2Area is inoculated with the recombinant virus of 1000PFU, about 50 hours after inoculation, has more than 70% cell monolayer to occur typical During cytopathy (CPE), nutrient solution is removed, add appropriate pancreatin digestive juice, 10min or so, cell monolayer are digested at room temperature Occur it is loose add the nutrient solution containing 10% cow's serum with digestive juice equivalent immediately when drawing in the net to approach disengaging bottle wall phenomenon, stop Only digest.Rolling bottle is shaken gently for, cell is all departed from bottle wall, added after the cell ultrasonic treatment being collected by centrifugation appropriate SPGA stabilizers, dispense lyophilized after shaking up.
Embodiment 5
--- rHVT-GB vaccines prevent chicken the immune protection effectiveness evaluation of MD
80 1 age in days SPF chickens, are randomly divided into 4 groups, and the 1st group of inoculation rHVT-GB, immunizing dose is 4000PFU/, the 2nd Group inoculation HVT, immunizing dose is 4000PFU/.3rd group is to attack malicious control group (nonimmune to attack poison), and the 4th group is blank Group (nonimmune non-attack malicious group).During 7 age in days, 1,2,3 groups of chickens are inoculated with the chicken Marek's of 1000PFU virus quantities through abdominal cavity respectively Sick virulent Md5 plants, four groups of chickens are all cutd open in 70 age in days and kill, statistics.Using immune chicken attack malicious protective index (PI) as Evaluate the index of vaccine immunity effect.Attack dead chicken before poison and be judged to nonspecific death, be not counted in test chicken number.Attack against each other The chicken counting of dead and off-test survival after poison and one by one cut open inspection, record each test group MD lesions positive chicken number in detail, Taking the suspicious chicken of MD lesions pathological material of disease carries out pathological examination.MD positive chickens refer to dead chicken is caused by MD, with observing MD Neoplastic lesion chicken and observe that MD neoplastic lesions are suspicious and Histopathological examination is the chicken of MD pathological changes.
Protective index (PI) expression of the immune efficacy of experimental vaccine,
Wherein：
Statistics is shown in Table 1, attacks the MD specific lesions that malicious control group occurs 100%.There is 45% MD in HVT immune groups Specific lesions, including the MD specificity of early stage is dead, and vaccine protective index is 55；RHVT-GB immune group vaccine protective indexes It is 75, hence it is evident that better than HVT immune groups.
Immune protective effects of the table 1rHVT-GB to SPF chickens
Embodiment 6
--- rHVT-GB-NDVF vaccines are evaluated chicken prevention ND immune protection effectiveness
80 1 age in days SPF chickens, are randomly divided into 4 groups, the 1st group of inoculation rHVT-GB-NDVF, and immunizing dose is 4000PFU/ Only, the 2nd group of inoculation rHVT-GB, immunizing dose is 4000PFU/, and the 3rd group is to attack malicious control group (nonimmune to attack malicious group), the 3rd Group is blank (nonimmune non-attack poison) group.21 days after immune, 1,2,3 groups of each intramuscular injection ewcastle disease Beijing Strains of chicken are malicious by force 0.5ml (contains 105EID50), 14d is observed, rHVT-GB-NDVF protective rates reach 90%.
Immune protective effects of the table 2rHVT-GB-NDVF to SPF chickens
Group Experimental animal number Attack poison strain ND falls ill or dead Protective rate (%)
RHVT-GB-NDVF vaccine immunity groups 20 NDV (Beijing Strain) 2 90
RHVT-GB vaccine immunity groups 20 NDV (Beijing Strain) 20 0
Attack malicious control group- 20 NDV (Beijing Strain) 20 0
Blank control group- 20 - 0 -
Embodiment 7
--- rHVT-GB-ILTVGB vaccines are evaluated ILT immune protection effectiveness
80 1 age in days SPF chickens, are randomly divided into 4 groups, the 1st group of inoculation rHVT-GB-ILTVGB vaccine, and immunizing dose is Only, the 2nd group of inoculation rHVT-GB vaccine, immunizing dose is 4000PFU/ to 4000PFU/, and the 3rd group (is attacked poison right to attack malicious control group According to group), the 3rd group is blank (nonimmune non-attack poison) group.21 days after immune, 1,2,3 groups of each intratracheal injection chickens of chicken are infected Property laryngotracheitis velogen strain 0.2ml (contain 104EID50), 10d is observed, rHVT-GB-ILTVGB protective rates reach 100%.
Immune protective effects of the table 3rHVT-GB-ILTVGB to SPF chickens
Group Experimental animal number Attack poison strain ILT morbidity numbers Protective rate (%)
RHVT-GB-ILTVGB vaccine immunity groups 20 ILTV (Beijing Strain) 0 100
RHVT-GB vaccine immunity groups 20 ILTV (Beijing Strain) 20 0
- attack malicious control group 20 ILTV (Beijing Strain) 20 0
Blank control group- 20 - 0 -
Embodiment 8
--- rHVT-GB-IBDVVP2 vaccines are evaluated chicken prevention IBD immune protection effectiveness
80 1 age in days SPF chickens, are randomly divided into 4 groups, the 1st group of inoculation rHVT-GB-IBDVVP2, and immunizing dose is Only, the 2nd group of inoculation rHVT-GB, immunizing dose is 4000PFU/ to 4000PFU/, and the 3rd group (nonimmune to attack poison to attack malicious control group Group), the 3rd group is blank (nonimmune non-attack poison) group.21 eye droppings of age in days every attack BC6/85 plants of 10 MID, 72 hours The cut open inspection bursa of farbricius, observes bursa of farbricius lesion afterwards.RHVT-GB-IBDVVP2 immune groups protect 17.2nd group (rHVT-GB is immunized Group) and the 3rd group of (attacking malicious control group) bursa of farbricius there is lesion (bursa of farbricius serous coat occur in gel-shaped or has different degrees of to go out Blood).
Immune protective effects of the table 4rHVT-GB-IBDVVP2 to SPF chickens
Group Experimental animal number Attack poison strain IBD lesion numbers Protective rate (%)
RHVT-GB-IBDVVP2 vaccine immunity groups 20 IBDV BC6/85 plants 3 85
RHVT-GB vaccine immunity groups 20 IBDV BC6/85 plants 20 0
- attack malicious control group 20 IBDV BC6/85 plants 20 0
- blank control group 20 - 0 -
Sequence table
　　<110>Beijing Bang Zhuo bio tech ltd
　　<120>The HVT for expressing the type MDV GB genes of serum I builds and its as virus live vector expression alien gene Using
　　<130>
　　<160> 22
　　<170> Patentin version 3.5
　　<210> 1
　　<211> 3870
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Build MDV1-GB expression cassette sequences used by rHVT-GB
　　<400> 1
gacctccctt ggccatgatg aatggttgga gatggtaaaa tcggcatcaa gtgatgtagc 060
tcgtagacgt aaaatgtatg cagagcgttt aacaaagaag tccctagcga gtctggataa 120
atgtattacc gaacaaagac atgaactaga aaaaatgttg cgtgtgaatg tatatggcga 180
agtattgata gactcttata cggcattatt caatgggttc cgttccagga aaagattatt 240
ggaagcggtt aaaaattgct gtgcaaacat cattgataat cgtaatagtg acgatgcatt 300
tgatgctcat cggttcatgc aaacatcatt actgaaacac agaatagatc ctgctatgct 360
tccaagtctc actcacaaat tttttcaact tgtgaatgga ccgatgttta gtcacgatag 420
acatcggttc gcccagccgt cgaatacagc attatatttt agtgttgaaa atgtagggct 480
gcttcctcac ttaaaggagg aaatggctcg attcatgttt catagcagta gaaaaacaga 540
ttggaccgtc agtaagttta gagggtttta tgactttagc actatagata atgtaactgc 600
ggcccatcgc atggcttgga aatatatcaa agaactgatt tttgcaacag ctttattttc 660
ttctgtattt aaatgtggcg aattgcacat ctgtcgtgcc gacagtttgc agatcaacag 720
caatggagac tatgtatgga aaaatggaat atatataaca tatgaaaccg aatatccact 780
tataatgatt ctggggtcag aatcaagcac ttcagaaacg caaaatatga ctgcaattat 840
tgatacagat gttttttcgt tgctttattc tattttgcag tatatggccc ccgttacggc 900
agatcaggtg cgagtagaac agattaccaa cagccacgcc cccatctgac ccgtccaata 960
ttcttgtgtc cctgcatttt atctcacaca atttatgaac agcatcatta agatcatctc 1020
actatgcact attttaggcg gaattgcatt tttttcctta tagttattct atatggtacg 1080
aactcatctc cgagtaccca aaatgtgaca tcaagagaag ttgtttcgag cgtccagttg 1140
tctgaggaag agtctacgtt ttatctttgt cccccaccag tgggttcaac cgtgatccgt 1200
ctagaaccgc cgcgaaaatg tcccgaacct agaaaagcca ccgagtgggg tgaaggaatc 1260
gcgatattat ttaaagagaa tatcagtcca tataaattta aagtgacgct ttattataaa 1320
aatatcattc agacgacgac atggacgggg acgacatata gacagatcac taatcgatat 1380
acagatagga cgcccgtttc cattgaagag atcacggatc taatcgacgg caaaggaaga 1440
tgctcatcta aagcaagata ccttagaaac aatgtatatg ttgaagcgtt tgacagggat 1500
gcgggagaaa aacaagtact tctaaaacca tcaaaattca acacgcccga atctagggca 1560
tggcacacga ctaatgagac gtataccgtg tggggatcac catggatata tcgaacggga 1620
acctccgtca attgtatagt agaggaaatg gatgcccgct ctgtgtttcc gtattcatat 1680
tttgcaatgg ccaatggcga catcgcgaac atatctccat tttatggtct atccccacca 1740
gaggctgccg cagaacccat gggatatccc caggataatt tcaaacaact agatagctat 1800
ttttcaatgg atttggacaa gcgtcgaaaa gcaagccttc cagtcaagcg taactttctc 1860
atcacatcac acttcacagt tgggtgggac tgggctccaa aaactactcg tgtatgttca 1920
atgactaagt ggaaagaggt gactgaaatg ttgcgtgcaa cagttaatgg gagatacaga 1980
tttatggccc gtgaactttc ggcaacgttt atcagtaata cgactgagtt tgatccaaat 2040
cgcatcatat taggacaatg tattaaacgc gaggcagaag cagcaatcga gcagatattt 2100
aggacaaaat ataatgacag tcacgtcaag gttggacatg tacaatattt cttggctctc 2160
gggggattta ttgtagcata tcagcctgtt ctatccaaat ccctggctca tatgtacctc 2220
agagaattga tgagagacaa caggaccgat gagatgctcg acctggtaaa caataagcat 2280
gcaatttata agaaaaatgc tacctcattg tcacgattgc ggcgagatat tcgaaatgca 2340
ccaaatagaa aaataacatt agacgacacc acagctatta aatcgacatc gtctgttcaa 2400
ttcgccatgc tccaatttct ttatgatcat atacaaaccc atattaatga tatgtttagt 2460
aggattgcca cagcttggtg cgaattgcag aatagagaac ttgttttatg gcacgaaggg 2520
ataaagatta atcctagcgc tacagcgagt gcaacattag gaaggagagt ggctgcaaag 2580
atgttggggg atgtcgctgc tgtatcgagc tgcactgcta tagatgcgga atccgtcact 2640
ttgcaaaatt ctatgcgagt tatcacatcc actaatacat gttatagccg accattggtt 2700
ctattttcat atggagaaaa ccaaggaaac atacagggac aactcggtga aaacaacgag 2760
ttgcttccaa cgctagaggc tgtagagcca tgctcggcta atcatcgtag atattttctg 2820
tttggatccg gttatgcttt atttgaaaac tataattttg ttaagatggt agacgctgcc 2880
gatatacaga ttgctagcac atttgtcgag cttaatctaa ccctgctaga agatcgggaa 2940
attttgcctt tatccgttta cacaaaagaa gagttgcgtg atgttggtgt attggattat 3000
gcagaagtag ctcgccgcaa tcaactacat gaacttaaat tttatgacat aaacaaagta 3060
atagaagtgg atacaaatta cgcgtttatg aacggtttgg ccgaattgtt taacggtatg 3120
ggtcaggtag ggcaagctat aggcaaagtt gtagtagggg ctgccggtgc aatcgtatct 3180
accatatctg gtgtctctgc tttcatgtca aatccctttg gggctttggc aatcggttta 3240
atcattatag caggactcgt ggctgcattt ttagcatatc gttatgtaaa caagcttaaa 3300
agcaatccaa tgaaagccct ttatcctatg acaacagaag tgcttaaggc acaggcaacg 3360
cgtgagttgc atggcgagga atcagatgat ttggaacgaa catctattga tgaaagaaaa 3420
ttagaagaag ctagagaaat gataaaatat atggcgttag tctccgcgga agaacgccac 3480
gagaaaaaac tgcggagaaa gaggcgaggc actaccgccg ttctatcgga ccacctggca 3540
aaaatgagga ttaaaaatag taaccctaaa tatgataagt tacctactac atattcagac 3600
tcagaagatg atgctgtgta agtgggcact attatatttg aactgaataa aacgcataga 3660
gcatgatatg gtttactcat ttattgcgag atataaagca tattcaatac gatatattgc 3720
gaacgtgatg ctaaaaacat agctccctgt attattgatg cgccatcatt tgattaataa 3780
atacatcgac gccggcatca ctggtgcggt gtataccagc tacggcgcta gcattcatgg 3840
tatcccgtga ttgctcgatg ctttccttct 3870
　　<210> 2
　　<211> 1391
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Build recombinant herpesvirus of turkeys rHVT-GB-NDVF, rHVT-GB- Promoter sequence used by ILTVGB and rHVT-GB-IBDVVP2
　　<400> 2
aactccgccc gttttatgac tagaaccaat agtttttaat gccaaatgca ctgaaatccc 060
ctaatttgca aagccaaacg ccccctatgt gagtaatacg gggacttttt acccaatttc 120
ccaagcggaa agccccctaa tacactcata tggcatatga atcagcacgg tcatgcactc 180
taatggcggc ccatagggac tttccacata gggggcgttc accatttccc agcatagggg 240
tggtgactca atggccttta cccaagtaca ttgggtcaat gggaggtaag ccaatgggtt 300
tttcccatta ctggcaagca cactgagtca aatgggactt tccactgggt tttgcccaag 360
tacattgggt caatgggagg tgagccaatg ggaaaaaccc attgctgcca agtacactga 420
ctcaataggg actttccaat gggtttttcc attgttggca agcatataag gtcaatgtgg 480
gtgagtcaat agggactttc cattgtattc tgcccagtac ataaggtcaa tagggggtga 540
atcaacagga aagtcccatt ggagccaagt acactgcgtc aatagggact ttccattggg 600
ttttgcccag tacataaggt caatagggga tgagtcaatg ggaaaaaccc attggagcca 660
agtacactga ctcaataggg actttccatt gggttttgcc cagtacataa ggtcaatagg 720
gggtgagtca acaggaaagt cccattggag ccaagtacat tgagtcaata gggactttcc 780
aatgggtttt gcccagtaca taaggtcaat gggaggtaag ccaatgggtt tttcccatta 840
ctggcacgta tactgagtca ttagggactt tccaatgggt tttgcccagt acataaggtc 900
aataggggtg aatcaacagg aaagtcccat tggagccaag tacactgagt caatagggac 960
tttccattgg gttttgccca gtacaaaagg tcaatagggg gtgagtcaat gggtttttcc 1020
cattattggc acgtacataa ggtcaatagg ggtgagtcat tgggtttttc cagccaattt 1080
aattaaaacg ccatgtactt tcccaccatt gacgtcaatg ggctattgaa actaatgcaa 1140
cgtgaccttt aaacggtact ttcccatagc tgattaatgg gaaagtaccg ttctcgagcc 1200
aatacacgtc aatgggaagt gaaagggcag ccaaaacgta acaccgcccc ggttttcccc 1260
tggaaattcc atattggcac gcattctatt ggctgagctg cgttctacgt gggtataaga 1320
ggcgcgacca gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga acgcagagct 1380
cctcgctgca g 1391
　　<210> 3
　　<211> 1662
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：NDV-F gene orders
　　<400> 3
atgggctcca aaccttctac caggatccca gcacctctga tgctggtcac ccggattatg 060
ctgatattag gctgtattcg ttcgacaagc tctcttgacg gcaggcctct tgcagctgca 120
ggaattgtag taacaggaga taaggcagtc aatgtataca cctcgtctca gacagggtca 180
atcatagtca agttgctccc gaatatgccc agggataagg aggcgtgtgc gaaagcccca 240
ttagaggcat ataacagaac actgactact ttgctcactc ctcttggcga ctccatccgc 300
aagattcaag ggtctgtgtc cacgtctgga ggagggagac aggggcgcct tataggtgct 360
gttattggca gtgtagctct tggggttgca acagcggcac agataacagc agctgcggcc 420
ctaatacaag ccaacaagaa tgctgccaac atccttcggc ttaaggagag cattgctgca 480
accaatgaag ctgtgcatga agtcaccgac ggattatcac aactatcagt ggcagttggg 540
gagatgcagc agtttgtcaa tgaccagttt aataatacgg cgcgagaatt ggactgtata 600
aaaatcacac aacaggttgg tgtagaactc aacctatacc taactgaatt gactacagta 660
ttcgggccac agatcacctc tcctgcatta actcagctga ccatccaggc actttataat 720
ttagctggtg gcaatatgga ttacttatta actaagttag gtatagggaa caatcaactc 780
agctcattaa ttggtagcgg cctgatcact ggttacccta tactgtatga ctcacagact 840
caactcttgg gcatacaagt gaatttgccc tcagtcggga acttaaataa tatgcgtgcc 900
acctatttgg agaccttatc tgtgagtaca accaaaggat atgcctcagc acttgtcccg 960
aaagtagtga cacaagtcgg ttctgtgata gaagagcttg acacctcaca ctgtatagag 1020
tccgatctgg atttatattg tactagaata gtgacattcc ccatgtcccc aggtatttat 1080
tcctgtttga gcggcaacac atcagcttgc atgtattcaa agactgaagg cgcactcact 1140
acgccgtata tggcccttaa aggctcagtt attgccaatt gtaagataac aacatgtaga 1200
tgtacagacc ctcctggtat catatcgcaa aattatggag aagctgtatc cctgatagat 1260
agacattcgt gcaatgtctt atcattagac gggataactc tgaggctcag cggagaattt 1320
gatgcaactt atcaaaagaa catctcaata ctagattctc aagtcatcgt gacaggcaat 1380
cttgatatat caaccgaact tggaaacgtc aacaattcaa tcagcaatgc cttggataag 1440
ttggcagaaa gcaacagtaa gatagaaaaa gtcaatgtca gattaaccag cacatctgct 1500
ctcattacct atattgttct aactgtcatt tctctatttt tcggtgcact taatctgggt 1560
ttagcgtgtt acctgatgta caaacagaag gcacaacaaa agaccttgct atggcttggg 1620
aataatgccc ttgatcagat gagagccact acaagagcat ga 1662
　　<210> 2
　　<211> 2652
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：ILTV-GB gene orders
　　<400> 4
atgcaatcct acatcgccgt gaacattgac atggctagct tgaaaatgct gatctgcgtg 060
tgcgtggcaa tcctgatccc atctacccta tctcaagatt cacacggaat tgctggaata 120
atagaccctc gtgatacagc cagcatggat gttggaaaaa tctctttctc cgaagccatt 180
gggtcggggg caccgaaaga accccagatt agaaacagaa tttttgcgtg ctcatctcca 240
actggcgcca gtgttgcgag gcttgcccag ccacgacatt gtcaccgaca tgccgattcg 300
actaacatga ctgaaggaat tgccgtagtc ttcaagcaaa acattgcccc gtacgtcttt 360
aatgtgactc tatactataa acatataacc acagttacta cgtgggcatt attctcaaga 420
ccccaaataa caaatgagta cgtgaccagg gttccaatag actatcatga aattgtcagg 480
attgatcgat cgggagaatg ctcatccaaa gcaacgtatc ataaaaattt catgtttttt 540
gaagcttacg acaatgatga agcagaaaaa aaattgcccc tggttccatc actgttaaga 600
tcaactgtct ccaaggcgtt tcatacaact aactttacta agcgacatca aaccctggga 660
taccgaacgt ctacatcggt cgactgtgtt gtggaatatc tacaggctag atctgtatac 720
ccgtatgatt actttggaat ggcgacaggt gatacagtag aaatttctcc tttttatacc 780
aaaaacacga ccggaccaag gcgtcacagt gtctacagag actatagatt tctcgaaatc 840
gcaaattatc aagtcaggga tttggaaacc ggacaaataa gaccccctaa aaaaagaaac 900
tttctaacag atgaacaatt cactataggc tgggatgcaa tggaagaaaa ggaatctgta 960
tgtactctca gtaaatggat tgaagtcccg gaagcagttc gtgtttcgta caaaaacagt 1020
taccactttt cacttaaaga tatgactatg acgttctcgt ccggaaaaca accttttaac 1080
atcagcaggc ttcatttggc tgaatgcgtt cctaccatag cctcggaggc catagatggc 1140
atctttgcca gaaagtatag ttcgactcat gtccgttctg gggacatcga atactatctc 1200
ggtagtggcg gatttctgat cgcatttcag aaactcatga gccatggctt ggctgaaatg 1260
tacctagaag aggcacaaag acaaaatcat ctcccgagag ggagagagcg tcgccaagcc 1320
gcaggtcgcc gcacggcgtc gctgcagtct ggacctcagg gtgatagaat tactacccac 1380
agttctgcaa catttgccat gttacaattt gcatacgaca aaatccaagc ccatgttaac 1420
gagcttatcg gaaatttgtt ggaagcgtgg tgtgagcttc agaaccgcca actgattgta 1500
tggcatgaga tgaagaaact aaacccgaac tcactgatga catctttgtt cggacaacct 1560
gtaagcgcca ggctattggg agacatcgta gcggtatcaa aatgtataga aattccaatc 1620
gaaaatatta ggatgcagga ttccatgcgc atgccagggg acccaaccat gtgctatacc 1680
agaccagtac ttattttcag gtattcgtcc tcccctgagt cacagttttc tgcgaactca 1740
acagaaaacc acaatcttga catattaggc caactcggag aacataatga aattttacaa 1800
gggcggaatt tgatagaacc atgcatgatc aatcacagac ggtactttct gttgggagaa 1860
aactaccttc tttacgaaga ctatacattt gttagacaag taaatgcttc cgagatcgaa 1920
gaagtgagca tattcatcaa cttgaacgcc actatactag aagatttgga ctttgtgccc 1980
gtcgaagtat acactcgcga ggaactcaga gatactggga ctttaaacta tgatgatgtg 2040
gtcagatatc aaaatattta taacaaaagg ttcagagaca ttgacactgt aatacgtgga 2100
gataggggag atgcaatctt tagagcaata gcagattttt ttggcaacac tcttggagaa 2160
gtaggaaagg cattgggaac tgtagtgatg acagccgcgg cagcagtaat ttctacagta 2220
tctggcatcg cctcatttct ttctaacccg ttcgccgcac tcggaattgg gatagcggtg 2280
gtggtgagca ttattttagg actgctggcg ttcaaatatg taatgaacct gaaatcaaac 2340
ccagttcagg ttctgttccc aggcgcagtt cccccggccg gaactcctcc acgaccctct 2400
agacgttact acaaggatga ggaggaggtt gaggaggata gtgatgagga cgacaggata 2460
cttgccacca gagttctgaa aggccttgag cttctacaca aggatgaaca gaaagctcga 2520
agacagaaag cgcggttttc tgcttttgct aaaaatatga gaaacctatt tcgcagaaaa 2580
ccccgaacca aggaagatga ctaccccctg ctcgaatacc cttcgtgggc agaagaaagc 2640
gaagacgaat aa 2652
　　<210> 5
　　<211> 1359
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：IBDV-VP2 gene orders
　　<400> 5
Atgacaaacc tgcaagatca aacccaacag attgttccgt tcatacggag ccttctgatg 060
Ccaacaaccg gaccggcgtc cattccggac gacaccctgg agaagcacac tctcaggtca 120
Gagacctcga cctacaattt gactgtgggg gacacagggt cagggctaat tgtctttttc 180
Cctggattcc ctggctcaat tgtgggtgct cactacacac tgcagagcaa tgggaactac 240
Aagttcgatc agatgctcct gactgcccag aacctaccgg ccagctacaa ctactgcaga 300
Ctagtgagtc ggagtctcac agtgaggtca agcacactcc ctggtggcgt ttatgcacta 360
Aacggcacca taaacgccgt gaccttccaa ggaagcctga gtgaactgac agatgttagc 420
tacaatgggt tgatgtctgc aacagccaac atcaacgaca aaattgggaa tgtcctggta 480
ggggaagggg tcactgtcct cagcctaccc acatcatatg atcttgggta tgtgaggctt 540
ggtgacccca ttcccgctat agggcttgac ccaaaaatgg tagctacatg cgacagcagt 600
gacaggccca gagtctacac cataactgca gccgatgatt accaattctc atcacagtac 660
caaccaggtg gggtaacaat cacactgttc tcagccaaca ttgatgctat cacaagcctc 720
agcattgggg gagagctcgt gtttcaaaca agcgtccaag gccttgtact gggcgccacc 780
atctacctta taggctttga tgggactgcg gtaatcacca gagctgtagc cgcagataat 840
gggctgacgg ccggcaccga caatcttatg ccattcaatc ttgtcattcc aaccaatgag 900
ataacccagc caatcacatc catcaaactg gagatagtga cctccaaaag tggtggtcag 960
gcaggggatc agatgtcatg gtcggcaagt gggagcctag cagtgacgat ccatggtggc 1020
aactatccag gggccctccg tcccgtcaca ctagtagcct acgaaagagt ggcaacagga 1080
tccgtcgtta cggtcgctgg ggtgagtaac ttcgagctga ttccaaatcc tgaactagca 1140
aagaacctgg ttacagaata cggccgattt gacccaggag ccatgaacta cacaaaattg 1200
atactgagtg agagggaccg tcttggcatc aagaccgtct ggccaacaag ggagtacact 1260
gattttcgtg agtacttcat ggaggtggcc gacctcaact ctcccctgaa gattgcagga 1320
gcatttggct tcaaagacat aatccgggct ataaggagg 1359
　　<210> 6
　　<211> 232
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：PolyA sequences
　　<400> 6
tctagagcgg ccgcggggat ccagacatga taagatacat tgatgagttt ggacaaacca 060
Caactagaat gcagtgaaaa aaatgctcta tttgtgaaat ttgtgatgct attgctttat 120
ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt catcttatgt 180
ttcaggttca gggggaggtg tgggaggttt tttcggatcc tctagagtcg ac 232
　　<210> 7
　　<211> 29
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：The primer HVT-US2 left of US2 genes left and right arms sequence after amplification sense
<400> 7
GATgaattcA CATCGGGCCA CGTTCCGCC-3 29
　　<210> 8
　　<211> 37
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：The primer HVT-US2 left of US2 genes left and right arms sequence after amplification antisense
<400> 8
ATAGTCGACt taattaaGAT GAGCTGACGT GTGGAAT-3 37
　　<210> 9
　　<211> 29
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：The primer HVT-US2 right of US2 genes left and right arms sequence after amplification sense
　　<400> 9
　　ATCgtcgacA CTAATATGGG CACACCCAC-3 29；
　　<210> 10
　　<211> 29
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：The primer HVT-US2 right of US2 genes left and right arms sequence after amplification antisense
<400> 10
ATCaagcttT GGCCCATCTA GGTGATTAT-3 29
　　<210> 11
　　<211> 36
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：MDV1-GB upstream region of gene primers MDV1-GB-F
<400> 11
cggTTAATTA Agacctccct tggccatgat gaatgg 36
　　<210> 12
　　<211> 36
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：The anti-sense primer MDV1-GB-R of MDV1-GB genes
<400> 12
ccgTTAATTA Aagaaggaaa gcatcgagca atcac（Two ends add PACI restriction enzyme sites） 36
　　<210> 13
　　<211> 38
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand left homology arm sense primer HVT-L-F
　　<400> 13
　　gttccttgaa atgccgacaa ctctaaaaac ggtattcg 38
　　<210> 14
　　<211> 38
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand left homology arm anti-sense primer HVT-L-R
　　<400> 14
GCTAGCGCGG CCGCGAATTC gtttaatgtt agtttatt (contain Nhe I/ in the end of anti-sense primer 5 ' NotI/EcoRI restriction enzyme sites) 38
　　<210> 15
　　<211> 48
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer PRO-F of promoter
　　<400> 15
　　GGGGAATTCA CTAGTGGATC CCCCAACTCC GCCCGTTTTA TGACTAGA (EcoRI) 48
　　<210> 16
　　<211> 45
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer PRO-R of promoter
　　<400> 16
　　AAATTGCGGC CGCCTGCAGC GAGGAGCTCT GCGTTCTACG GTGGT(Not I) 45
　　<210> 17
　　<211> 50
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer Vp2-F of Vp2 genes
　　<400> 17
　　aatGCGGCCG CTCTAGAACT CGTCGATCGC AGCGATGACA AACCTGCAAG (Not I) 50
　　<210> 18
　　<211> 50
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer Vp2-R of VP2 genes
　　<400> 18
　　GGCGCTAGCA AGCTTACCTC CTTATAGCCC GGATTATGTC TTTGAAGCCA (HindIII/Nhe I) 50
　　<210> 19
　　<211> 27
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer polyA-F of polyA
　　<400> 19
　　AATAAGCTTG ATCTAGAGCG GCCGCGG (HindIII) 27
　　<210> 20
　　<211> 28
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer polyA-R of polyA
　　<400> 20
　　AATGCTAGCG TCGACTCTAG AGGATCCG (Sal I/Nhe I) 28
　　<210> 21
　　<211> 26
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the sense primer HVT-R-F of right homology arm
　　<400> 21
　　GGCGGTCGAC aattatttta tttaat 26
　　<210> 22
　　<211> 32
　　<212> DNA
　　<213>Artificial sequence
　　<223>Description to artificial sequence：Expand the anti-sense primer HVT-R-R of right homology arm
　　<400> 22
　　GGCGCTAGCc tagtgtttca attat 25
4

Claims (14)

1. it is a kind of express the type marek's disease virus GB genes of serum I recombinant herpesvirus of turkeys, it is characterised in that the expression The recombinant herpesvirus of turkeys of the type marek's disease virus GB genes of serum I is named as herpes turkey virus (Herpesvirus Of turkey) rHVT-GB plants of recombinant virus, the strain delivered BeiChen West Road, Chaoyang District, BeiJing City 1 on 01 11st, 2017 Number No. 3 China Committee for Culture Collection of Microorganisms's common micro-organisms centers of Institute of Microorganism, Academia Sinica of institute preservation, Its deposit number is：CGMCC No.13400.
2. a kind of recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I as claimed in claim 1 Structure, it is characterised in that：By in the GB expression casettes restructuring of the type MDV of serum I to HVT genomes, recombination site is HVT (GenBank:AF291866.1) the US2 regions between HVT087-HVT088 genes of genome, the gene of the expression cassette Sequence such as Seq ID No:Shown in 1.
3. a kind of recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I as claimed in claim 1 Application, it is characterised in that the recombinant strain induces the application in the protective immunity of anti-Marek's disease in chicken body.
4. the recombinant turkey blister sore of the type marek's disease virus GB genes of a kind of expression serum I as described in claim 1 and 3 Poison application, it is characterised in that the recombinant strain as production strain animal vaccine strain application.
5. the recombinant turkey blister sore of the type marek's disease virus GB genes of a kind of expression serum I as described in claim 1 and 3 The application of poison, it is characterised in that the recombinant virus is built the application in new recombinant vaccine virus as viral vectors.
6. as described in claim 1 and 5 feature a kind of type marek's disease virus GB genes of expression serum I recombinant turkey The application of herpesviral,, in by the F expression casettes restructuring of NDV to rHVT-GB genomes, recombination site is for it HVT(GenBank:AF291866.1) between the HVT065 and HVT066 of genome, the wherein promoter sequence of F expression casettes Such as Seq ID No:Shown in 2；F gene orders such as Seq ID No:Shown in 3；PolyA sequences such as Seq ID No:Shown in 6, build The recombinant virus of acquisition is named as herpes turkey virus (Herpesvirus of turkey) rHVT-GB-NDVF plant weights group disease Poison, the strain was delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism and is ground on 01 11st, 2017 The preservation of Jiu Suo China Committee for Culture Collection of Microorganisms's common micro-organisms centers, its deposit number is：CGMCC No.13399。
7. feature as claimed in claim 6 a kind of type marek's disease virus GB genes of expression serum I recombinant turkey blister The application of exanthema virus, it induces anti-Marek's disease and chicken new city in recombinant herpesvirus of turkeys rHVT-GB-NDVF plants in chicken Application in the protective immunity of epidemic disease and the application as production strain in corresponding animal vaccine.
8. feature as claimed in claim 7 a kind of type marek's disease virus GB genes of expression serum I recombinant turkey blister The application of exanthema virus, it builds new recombinant vaccine disease as viral vectors in recombinant herpesvirus of turkeys rHVT-GB-NDVF plants The application of poison.
9. as described in claim 1 and 5 feature a kind of type marek's disease virus GB genes of expression serum I recombinant turkey The application of herpesviral, it is characterised in that by the GB expression casettes restructuring of avian infectious laryngotracheitis virus to rHVT-GB bases Because in group, recombination site is HVT (GenBank:AF291866.1) between the HVT065 and HVT066 of genome, ILTV-GB bases Because of the promoter sequence such as Seq ID No of expression cassette:Shown in 2, GB gene orders such as Seq ID No:Shown in 4, PolyA sequences are such as Seq ID No:Shown in 6；Acquisition is named as herpes turkey virus (Herpesvirus of turkey) rHVT-GB-ILTVGB Strain recombinant virus, the strain delivered the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences on 01 11st, 2017 The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology, its deposit number is：CGMCC No.13398。
10. a kind of recombinant herpesvirus of turkeys for expressing serum I type marek's disease virus GB genes as claimed in claim 9 Using, it is characterised in that recombinant herpesvirus of turkeys rHVT-GB-ILTVGB plants therein induced in chicken anti-Marek's disease and Application in the protective immunity of infectious laryngotracheitis of chicken and the application as production strain in corresponding animal vaccine.
A kind of 11. recombinant herpesvirus of turkeys for expressing the type marek's disease virus GB genes of serum I as claimed in claim 9 Using, it is characterised in that recombinant herpesvirus of turkeys rHVT-GB-ILTVGB plants therein builds new restructuring as viral vectors The application of vaccine virus.
12. as described in claim 1 and 5 it is a kind of express the type marek's disease virus GB genes of serum I recombinant turkey blister sore The application of poison, it is characterised in that recombinate in rHVT-GB genomes the VP2 expression casettes of infections chicken cloacal bursa virus, Recombination site is HVT (GenBank:AF291866.1) between the HVT065 and HVT066 of genome, VP2 expression casettes are opened Promoter sequences such as Seq ID No:Shown in 2, VP2 gene orders such as Seq ID No:Shown in 5, PolyA sequences such as Seq ID No:6 It is shown；Acquisition is named as herpes turkey virus (Herpesvirus of turkey) rHVT-GB-IBDVVP2 plant weight groups, should Strain delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica on 01 11st, 2017 China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, its deposit number is：CGMCC No.13397.
A kind of 13. recombinant turkey blister sores for expressing the type marek's disease virus GB genes of serum I as claimed in claim 12 The application of poison, it is characterised in that wherein recombinant herpesvirus of turkeys rHVT-GB-IBDVVP2 plants induces anti-chicken horse Garrick in chicken Application in the protective immunity of family name's disease and gumboro disease and the answering in corresponding animal vaccine as production strain With.
A kind of 14. recombinant turkey blister sores for expressing the type marek's disease virus GB genes of serum I as claimed in claim 12 The application of poison, it is characterised in that wherein recombinant herpesvirus of turkeys rHVT-GB-IBDVVP2 plants new as viral vectors structure The application of recombinant vaccine virus.
CN201710046543.0A 2017-01-19 2017-01-19 Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus Pending CN106701693A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710046543.0A CN106701693A (en) 2017-01-19 2017-01-19 Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710046543.0A CN106701693A (en) 2017-01-19 2017-01-19 Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus

Publications (1)

Publication Number Publication Date
CN106701693A true CN106701693A (en) 2017-05-24

Family

ID=58909361

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710046543.0A Pending CN106701693A (en) 2017-01-19 2017-01-19 Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek's disease virus

Country Status (1)

Country Link
CN (1) CN106701693A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099513A (en) * 2017-06-22 2017-08-29 北京邦卓生物科技有限公司 The structure of HVT of coexpression NDV HN and IBDV VP2 genes a kind of and its application
WO2018112051A1 (en) * 2016-12-14 2018-06-21 Merial, Inc. Recombinant hvt vectors expressing multiple antigens of avian pathogens, and vaccines comprising them
WO2020127964A1 (en) * 2018-12-21 2020-06-25 Ceva Sante Animale Recombinant avian herpes viruses containing multiple foreign genes
RU2812979C2 (en) * 2018-12-21 2024-02-06 Сева Санте Анималь Recombinant avian herpes viruses containing several alien genes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1192783A (en) * 1995-07-07 1998-09-09 日本是恩株式会社 Marek's disease virus genes and their use in vaccines for protection against marek's disease
CN105920598A (en) * 2011-11-30 2016-09-07 梅里亚有限公司 Recombinant Hvt Vectors Expressing Antigens Of Avian Pathogens And Uses Thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1192783A (en) * 1995-07-07 1998-09-09 日本是恩株式会社 Marek's disease virus genes and their use in vaccines for protection against marek's disease
CN105920598A (en) * 2011-11-30 2016-09-07 梅里亚有限公司 Recombinant Hvt Vectors Expressing Antigens Of Avian Pathogens And Uses Thereof

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
AFONSO CL等: "登录号：AF291866.1", 《GENBANK》 *
GAO H等: "Expression of HA of HPAI H5N1 Virus at US2 Gene Insertion Site of Turkey Herpesvirus Induced Better Protection than That at US10 Gene Insertion Site", 《PLOS ONE》 *
LJN ROSS等: "Construction and properties of a turkey herpesvirus recombinant expressing the Marek"s disease virus homologue of glycoprotein B of herpes simplex virus", 《JOURNAL OF GENERAL VIROLOGY》 *
REDDY SK等: "Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek"s diseases in specific pathogen-free chickens", 《VACCINE》 *
ROSS LJ: "Nucleotide sequence and characterization of the Marek"s disease virus homologue of glycoprotein B of herpes simplex virus", 《J GEN VIROL》 *
ROSS LJ等: "登录号：D13713.1", 《GENBANK》 *
ROSS N等: "Influence of chicken genotype on protection against Marek"s disease by a herpesvirus of turkeys recombinant expressing the glycoprotein B (gB) of Marek"s disease virus", 《VACCINE》 *
SPATZ SJ等: "登录号：EF523390.1", 《GENBANK》 *
孙燕: "表达传染性喉气管炎病毒gB、gD基因的重组火鸡疱疹病毒", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *
魏雪涛: "含有MDVgB和IL-2重组火鸡疱疹病毒的构建及其免疫保护作用", 《中国优秀博硕士学位论文全文数据库 (硕士) 农业科技辑》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018112051A1 (en) * 2016-12-14 2018-06-21 Merial, Inc. Recombinant hvt vectors expressing multiple antigens of avian pathogens, and vaccines comprising them
US10822620B2 (en) 2016-12-14 2020-11-03 Boehringer Ingelheim Animal Health USA Inc. Recombinant HVT vectors expressing multiple antigens of avian pathogens and uses thereof
EP4085928A1 (en) * 2016-12-14 2022-11-09 Boehringer Ingelheim Animal Health USA Inc. Recombinant hvt vectors expressing multiple antigens of avian pathogens, and vaccines comprising them
EP4112072A1 (en) * 2016-12-14 2023-01-04 Boehringer Ingelheim Animal Health USA Inc. Recombinant hvt vectors expressing multiple antigens of avian pathogens, and vaccines comprising them
CN107099513A (en) * 2017-06-22 2017-08-29 北京邦卓生物科技有限公司 The structure of HVT of coexpression NDV HN and IBDV VP2 genes a kind of and its application
CN107099513B (en) * 2017-06-22 2020-07-14 北京邦卓生物科技有限公司 Construction and application of HVT co-expressing NDV HN and IBDV VP2 genes
WO2020127964A1 (en) * 2018-12-21 2020-06-25 Ceva Sante Animale Recombinant avian herpes viruses containing multiple foreign genes
CN113226363A (en) * 2018-12-21 2021-08-06 法国诗华大药厂 Recombinant avian herpesvirus containing multiple foreign genes
US20220040287A1 (en) * 2018-12-21 2022-02-10 Ceva Sante Animale Recombinant avian herpes viruses containing multiple foreign genes
RU2812979C2 (en) * 2018-12-21 2024-02-06 Сева Санте Анималь Recombinant avian herpes viruses containing several alien genes

Similar Documents

Publication Publication Date Title
CN101514334B (en) Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof
CN101935637B (en) Recombinant low-virulent vaccine strain of chicken infectious bursal disease viruses (IBDV) and application thereof
CN102220287B (en) Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof
WO2021103421A1 (en) Gene vii type newcastle disease virus attenuated strain and use thereof
CN102533674B (en) Recombinant duck virus enteritis virus vaccine strain (rDEVul41HA) for expressing avian influenza virus hemagglutinin (HA) genes and construction method as well as application thereof
CN102363769B (en) Chicken Marek&#39;s disease Meq gene deleted vaccine strain, construction method thereof, and application thereof
CN102373180A (en) Recombinant duck enteritis virus (DEV) vaccine strain for expressing avian influenza virus haemagglutinin (HA) gene and constructing method and application thereof
CN107384874A (en) Pseudorabies virus epidemic strain gI/gE gene deletion mutants and structure and application
CN106701693A (en) Construction and application of recombinant herpesvirus of turkey for expressing GB gene of serum I type Marek&#39;s disease virus
CN102559610B (en) Recombined duck virus enteritis viral vaccine strain CCTCC for expressing bird flu virus hemagglutinin (HA) gene (rDEVus78Ha) as well as establishing method and application thereof
CN103224913B (en) Duck plague live vaccine and preparation method thereof
CN103382460B (en) Strain of Marek&#39;s disease virus vaccine strain, and separation and identification and application thereof
CN106754761A (en) The structure and its application of a kind of recombinant marek&#39;s disease virus meq and the dual-gene gene-deleted strains of vIL 8
CN102634489B (en) Recombinant turkey herpesvirus and application thereof
CN107099513B (en) Construction and application of HVT co-expressing NDV HN and IBDV VP2 genes
CN102154224A (en) Construction of recombinant chicken marek&#39;s disease vaccine virus SC9-1 strain and application thereof
CN102399756A (en) Chicken Marek&#39;s disease virus (MDV) miRNA (micro Ribonucleic Acid) deletion vaccine strain and application thereof
CN110499296A (en) A kind of heat-resisting serum 8b type aviadenovirus recombinant vaccine Candidate Strain and its construction method
CN101979503B (en) Recombinant brucella for expressing Asia type-I foot and mouth disease virus VP1 genes and method for producing vaccines thereof
CN107296956A (en) A kind of genetic recombination live vector vaccine
CN114395536B (en) Avian adenovirus type 4, 8 and 11 trivalent vaccine and preparation method and application thereof
CN108342367B (en) Recombinant Marek&#39;s virus strain SCA13 strain and application thereof
CN104312982A (en) Duck plague virus recombinant vaccine strain rDEVTK-EGFP for expressing enhanced green fluorescent protein genes and constructing method and application therefore
CN102363770A (en) Recombinant baculovirus capable of expressing porcine circovirus type 2 Cap protein and somatostatin in fusion manner, and subunit vaccine thereof
CN101974472A (en) Marker-free gene deletion attenuated mutant strain of Edwardsiella tarda wild strain as well as relevant preparations and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170524

RJ01 Rejection of invention patent application after publication