CN109022373A - The end of duck plague virus UL56 gene 3 ' missing and LORF5 gene deletion mutants and its construction method and application - Google Patents

The end of duck plague virus UL56 gene 3 ' missing and LORF5 gene deletion mutants and its construction method and application Download PDF

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CN109022373A
CN109022373A CN201810864918.9A CN201810864918A CN109022373A CN 109022373 A CN109022373 A CN 109022373A CN 201810864918 A CN201810864918 A CN 201810864918A CN 109022373 A CN109022373 A CN 109022373A
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gene
duck plague
lorf5
plague virus
duck
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CN109022373B (en
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杨承槐
毛娅卿
刘丹
侯力丹
黄小洁
李俊平
***
王乐元
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China Institute of Veterinary Drug Control
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Abstract

The present invention relates to the end of duck plague virus UL56 gene 3 ' missing and LORF5 gene deletion mutants and its construction method and applications; the present invention provides the recombination Duck plague virus strains of a kind of end of UL56 gene 3 ' missing and LORF5 gene delection; the strain has the virulence weakened; to duck no pathogenicity; and there is good immunogenicity, it can be realized the immunoprotection of anti-duck plague virus.Attenuation Duck plague virus strains provided by the invention can be used in preparing Duck plague live vaccine, or prepare genetically engineered live vector vaccine through being inserted into the antigen gene of other cause of diseases as live vector, have very big application potential and market.

Description

The end of duck plague virus UL56 gene 3 ' missing and LORF5 gene deletion mutants and its structure Construction method and application
Technical field
The present invention relates to field of biotechnology, and in particular to the end of duck plague virus UL56 gene 3 ' missing and LORF5 gene lack Lose mutant strain and its construction method and application.
Background technique
Duck plague virus also known as duck enteritis virus (Duck enteritis virus, DEV), can infected duck, goose and other wild geese Shape mesh birds cause injury of blood vessel, tissue bleeding, gastrointestinal mucosal to damage, lymphoid organ is impaired and parenchymatous organ's degeneration The acute of lesion, contact, Septic blood infectious disease-duck viral enteritis (Duck viral enteritis, DVE) or duck Pest (Duck plague, DP) (Sandhu and Metwally, 2008).From nineteen twenty-three by since finding for the first time (Baudet, 1923), duck plague betide in succession France, India, Belgium, Britain, Thailand and Canada etc. it is national (Yin Zhen and Liu Jinghua, 1997), the type of Epidemic Scope and infected poultry has gradually widened trend.Have now been found that the birds at least 48 kinds of Anseriformes (Kaleta et al., 2007) susceptible to DEV.Duck plague is a kind of infectious disease of extensive thermophilic systemic infection, disease incidence and dead It is all very high to die rate, water supply poultry breeding industry brings massive losses.Immunity inoculation is to prevent and control the important measures that the disease is broken out, mesh Preceding China is mainly to study successful chicken embryo reduction live vaccine the sixties in last century for the vaccine of duck plague prevention.DEV has good Good immunogenicity, immunity generate quickly, can resist within 3 days the virulent attack of duck plague after immune, and duration of immunity was up to 1 year As long as.
DEV belong to herpetoviridae, A type herpesviral subfamilies, Marek's disease poison category, 1 type of duck herpesviral (http: // www.ictvonline.org).Li Yufeng etc. completes DEV-China's duck plague commercialized vaccine using Shot-gun method for the first time The genome sequencing of (DEV VAC).DEV VAC full-length genome is that 158,089bp, G+C content is 44.91%, by long only Special zone (Unique Long, UL) and short distinct zones (Unique Short, US) composition, the area US both ends are a pair of of inverted repeat sequence Column, be referred to as internal repeat (Internal Repeat, IR) and terminal repeat (Terminal Repeat, TR).Genome structure is UL-IR-US-TR, in typical D type hsv gene group structure (Li et al., 2009).Hair Bright people completes the whole genome sequence measurement of the virulent Reference Strains of China duck plague virus, the strain genome in the research of early stage Overall length is 162,131bp, totally 78 ORF, encodes 76 albumen (Yang et al., 2014).
With the development of technique for gene engineering, recombinant vaccines become the hot spot of current new generation vaccine research. DEV has the advantage that (1) gene pool-size is big as live vaccine vectors, can be inserted into and express various exogenous genes；(2) energy It is enough to generate immunity rapidly, it can produce certain protection within 3 days after immune；(3) duration of immunity was long, up to 1 year；(4) host range Extensively, the birds (Kaleta et al., 2007) in 48 kinds of Anseriformes can at least be infected.Therefore, carry out duck plague recombinant virus and The research of duck plague recombinant vaccines is of great significance.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide the ends of duck plague virus UL56 gene 3 ' to lack It becomes estranged LORF5 gene deletion mutants and its construction method and application.
Firstly, the present invention provides a kind of Duck plague virus strains of attenuation, the UL56 gene inactivation of the Duck plague virus strains Or UL56 gene and LORF5 gene inactivate simultaneously.
Wherein, the Duck plague virus strains of attenuation refer to that the virulence of host drops compared with the virulence of parent's strain in Duck plague virus strains Low or Duck plague virus strains do not have host pathogenic.
The inactivation can be such that the deactivated genetic modification of UL56 gene coded protein realizes by any, including But it is not limited to the insertion or replacement of all or part of one or more nucleotide for lacking UL56 gene or UL56 gene.
Preferably, the inactivation holds 60bp and LORF5 gene 5 ' end 2638bp for missing UL56 gene 3 '.
Further, LORF5 gene inactivates while the UL56 gene 3 ' of attenuation provided by the invention holds missing, and inserts Enter foreign gene.
Preferably, the foreign gene is the gene of riddled basins or non-duck plague virus antigen, it is preferable that the sieve Selecting marker gene is fluorescence protein gene.
The gene of the non-duck plague virus antigen refers to the antigen encoding gene other than the antigen in duck plague virus source, Preferably Avian Influenza Virus HA Gene, virus hepatitis VP1 gene, parvovirus VP3 gene or duck tembusu virus E protein base Cause.
The fluorescin includes but is not limited to the common fluorescin known in this field such as RFP, GFP or YFP.
On the other hand, the present invention also provides the construction method of the Duck plague virus strains of the attenuation, include the following steps:
(1) building UL56 gene 3 ' end missing and LORF5 gene inactivation or the end of UL56 gene 3 ' missing and LORF5 gene Inactivate and be inserted into the transfer vector of foreign gene；
(2) transfer vector and duck plague virus parent's strain cotransfection obtained step (1) is into host cell, through homologous Recombination, screening obtains the end of UL56 gene 3 ' missing and LORF5 gene inactivation or the end of UL56 gene 3 ' missing and LORF5 gene loses Duck plague virus strains that are living and being inserted into foreign gene in the position.
Preferably, the transfer vector is that UL56 gene 3 ' is linked in sequence using pUC-18 cloning vector as skeleton carrier The left homology arm and right homology arm of end missing and LORF5 gene delection；Or the end of UL56 gene 3 ' missing and LORF5 is linked in sequence Left homology arm sequence, exogenous gene expression box and the right homology arm sequence of gene delection；
Specifically, the construction method of the Duck plague virus strains of the attenuation, includes the following steps:
(1) using duck plague virus CVCC AV1221 genome as template, using primer SEQ ID NO.10 and SEQ ID NO.11 expands the left homology arm of homologous recombination, expands right homology arm using primer SEQ ID NO.12 and SEQ ID NO.13, will Left homology arm and right homology arm are sequentially connected to pUC-18 carrier, or by left homology arm, exogenous gene expression box and right homology arm It is sequentially connected to pUC-18 carrier, constructs transfer vector.Preferably, its table when foreign gene is red fluorescent protein encoding gene Up to box sequence as shown in SEQ ID NO.5.
(2) by the transfer vector of step (1) building and parent's strain cotransfection to host cell, the recombination disease tentatively obtained Recombinant virus is carried out plaque purification by poison, obtains the recombination that missing UL56 gene 3 ' holds 60bp and LORF5 gene 5 ' end 2638bp Duck plague virus.
Wherein, parent's strain is duck plague virus CVCC AV1221 velogen strain or duck plague virus CVCC AV1221 is strong The low virulent strain for the clone purification that strain is obtained through chicken embryo fibroblasts and chicken embryo continuous passage；The host cell be chicken embryo at Fibrocyte (CEF) or duck embryo fibroblasts (DEF)；
Whether UL56 gene and LORF5 gene have function relevant to virus virulence unknown at present, and inventor is carrying out In the research of duck plague virus virulence, the duck plague that missing UL56 gene 3 ' holds 60bp and LORF5 gene 5 ' end 2638bp is chanced on The virulence of virus stain is substantially reduced, or even is not had to duck pathogenic.However it much can be with the gene of attenuated virus virulence Missing frequently results in the forfeiture of virus antigenicity, it is surprising that missing UL56 gene 3 ' holds 60bp and LORF5 gene 5 ' end 2638bp will not impact the antigenicity of duck plague virus, i.e., duck plague virus UL56 gene 3 ' holds 60bp and LORF5 gene 5 ' The mutant strain of 2638bp missing is held with significantly reduced virulence and there is good immunogenicity, is required for preparing vaccine Strain ideal chose.
The present invention confirms that the end of UL56 gene 3 ' missing and LORF5 gene delection can weaken the poison of duck plague virus for the first time Power, the end of UL56 gene 3 ' missing and LORF5 gene delection strain remain good immunogenicity to duck safety, therefore, The recombinant virus can be used for preparing Duck plague live vaccine, prevent duck plague.In addition, in the preparation of recombinant live-vector vaccine, very much There is incompatible phenomenon after importing live vector in exogenous antigen, i.e., foreign antigen genes can not great expression, lead to external source The immunogenicity of antigen is very low, cannot achieve its immune effect.The present invention has also demonstrated in the end of UL56 gene 3 ' and LORF5 gene Position insertion foreign antigen genes do not influence the immunogenicity of duck plague virus not only, and can be realized a large amount of of exogenous antigen Expression, therefore, the Duck plague virus strains of attenuation provided by the invention can also be used to prepare by the antigen gene of insertion external source Genetically engineered live vector vaccine.
Therefore, on the other hand, the present invention provides preparation duck plague vaccine or recombinate live vector vaccine in application, The duck plague vaccine is duck plague list seedling or duck plague combined vaccine.
Preferably, the application in recombination live vector vaccine is in the UL56 gene of the Duck plague virus strains Other foreign antigen genes are inserted into 3 ' ends and the position of LORF5 gene delection.
Further, the present invention provides a kind of recombination live vector, for duck plague virus strain attenuation provided by the invention Strain.
And a kind of duck plague virus or live recombinant vectors viral vaccine comprising Duck plague virus strains or in institute The recombinant virus strain of exogenous antigen encoding gene preparation is inserted into the Duck plague virus strains stated.
Preferably, the exogenous antigen encoding gene be Avian Influenza Virus HA Gene, it is virus hepatitis VP1 gene, thin Small virus VP3 gene or duck tembusu virus E protein gene.
Present invention firstly discovers that the end of duck plague virus UL56 gene 3 ' missing and LORF5 gene delection can weaken duck plague virus Virulence, it was demonstrated that UL56 gene 3 ' end and/or LORF5 gene it is related to the virulence of duck plague virus.
Therefore, the present invention provides duck plague virus UL56 gene or UL56 gene association LORF5 gene in regulation duck plague virus Virulence prepares application in vaccine, the UL56 gene inactivation for preparing duck plague virus in vaccine or UL56 gene and LORF5 Gene inactivates simultaneously.
Preferably, the UL56 gene with the nucleotide sequence as described in SEQ ID NO.1 or has such as SEQ ID The coding identical function albumen that replacement, missing or insertion of the nucleotide sequence described in NO.1 through one or more nucleotide obtain Nucleotide sequence；The LORF5 gene is with the nucleotide sequence as described in SEQ ID NO.2 or has such as SEQ ID The coding identical function albumen that replacement, missing or insertion of the nucleotide sequence described in NO.2 through one or more nucleotide obtain Nucleotide sequence.
Present invention demonstrates that UL56 gene or LORF5 gene are related to the virulence of duck plague virus, its antigenicity system can use Standby monoclonal antibody.
Therefore, the present invention also provides a kind of monoclonal antibody, corresponding epitope contains UL56 gene or LORF5 base Because of the amino acid sequence of coding.
And application of the monoclonal antibody in preparation duck plague virus detection kit.
The beneficial effects of the present invention are:
(1) present invention firstly discovers that UL56 gene 3 ' end missing and LORF5 gene delection can attenuated virus virulence, and protect Good immunogenicity is held, and then can be used in preparing live vaccine or as live vector, prepare genetic engineering live vector epidemic disease Seedling.
(2) Duck plague virus strains of attenuation provided by the invention do not have duck pathogenic, and have good immunogene Property, it can be realized 100% immunoprotection.
Detailed description of the invention
Fig. 1 is to recombinate duck plague virus rDEV Δ UL56-RFP in the expression knot of fluorescence microscopy microscopic observation red fluorescent protein Fruit, the gray area in figure are the red fluorescence that red fluorescent protein expression generates.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Wherein, make AV1221 plants of duck plague virus CVCC of parent's strain from Chinese veterinary microorganism culture presevation administrative center (Beijing sea The veterinary microorganism culture presevation administrative center of China of China Veterinery Drug Inspection Office of shallow lake area Zhong Guan-cun South Street 8；Referring to: China Veterinary microorganism culture presevation administrative center writes, Chinese animal doctor's strain catalogue, the second edition, and Scientia Agricultura Sinica technology is published Society, 2002, p138).PMD-18T carrier has for the building of cloning and sequencing and transfer vector purchased from precious bioengineering (Dalian) Limit company.
The building of 1 duck plague virus rDEV Δ UL56 of embodiment
(1) extraction of DEV genomic DNA
The 437.5 μ L of virus liquid of duck plague virus CVCC AV1221 is taken, Proteinase K (20mg/mL) 12.5 μ L and 10% is added SDS 50μL；56 DEG C of water-bath 1h；It is respectively extracted 1 time with phenol, phenol/chloroform/isoamyl alcohol (25:24:1), chloroform respectively；Supernatant is taken, is added Enter the 3M sodium acetate of 1/10 volume and the dehydrated alcohol of 2 times of volumes, -20 DEG C of placement 30min；12000 × g is centrifuged 10min, precipitating With 70% ethanol washing 1 time, precipitating is dissolved in 30 μ L deionized waters, and -20 DEG C save backup.
(2) building of transfer vector pT-UL56
Using duck plague virus CVCC AV1221 genomic DNA as PCR amplification template, with primer UA3F1 (SEQ ID NO.10) With the upstream 1387bp of UA3R1 (SEQ ID NO.11) amplification UL56 gene (its nucleotide sequence is as shown in SEQ ID NO.1) Segment, as left homology arm (shown in SEQ ID NO.3).
With the downstream of primer DA3F1 (SEQ ID NO.12) and DA3R1 (SEQ ID NO.13) amplification UL56 gene 1421bp segment (shown in SEQ ID NO.4), as right homology arm.Left and right homology arm is respectively through Hind III+Pst I, Kpn It is connected to pUC-18 carrier after I+BamH I digestion, constructs corresponding recombinant plasmid pT-UL56.
(3) homologous recombination
According to Lipofectamine TM2000 transfection reagent box specification, by transfer vector pT-UL56 and recombination duck plague Viral rDEV Δ UL56-RFP transfects duck embryo fibroblasts (DEF).Specific step is as follows:
DEF in six orifice plates is cultivated to when growing up to 70%~90% cell monolayer, being inoculated with suitable recombination duck plague virus RDEV Δ UL56-RFP is adsorbed 1~4 hour；1 μ g transfer vector pT-UL56 is diluted in the opti- without serum and antibiotic In DMEM, making the final volume of mixed liquor is 50 μ L, is incubated at room temperature 5min；Take 2 μ L Lipofectamine 2000 and 48 μ L not Opti-DMEM containing serum and antibiotic is mixed gently, and is incubated at room temperature 5min；50 μ L Lipofectamine 2000 are diluted Liquid is added drop-wise to respectively in 50 μ L plasmid dilutions, and side edged mixes；It is incubated at room temperature 20min.It during this period, will be thin in six orifice plates After born of the same parents gently wash twice with serum-free OPTI-MEM, 0.5mL serum-free OPTI-MEM is added in every hole, by 100 μ L Lipofectamine 2000/DNA complexes drop-wise is added in 6 orifice plates, and gently shaking mixes it uniformly, sets 37 DEG C of cultures 4h is discarded supernatant, and the M199 culture medium of 10% fetal calf serum is added, after setting 37 DEG C of cultures for 24 hours, with the M199 of 1% fetal calf serum Culture medium changes liquid, sets 37 DEG C of 3~5d of culture.
(4) plaque purification of recombinant virus
The recombinant virus tentatively obtained is seeded to the 6 orifice plates DEF cell for having grown up to good cell monolayer, is adsorbed 1 hour Afterwards, adsorption liquid is discarded, 1% low melting-point agarose is spread, continues to cultivate.Inoculation is seen under fluorescence microscope after 48-72 hours Examine, select single non-blooming plaque in 0.5ml M199 nutrient solution, 6 orifice plates DEF cell is inoculated with after freeze thawing 1 time, so into Row plaque purification 3~4 times, obtain the recombination duck plague disease that missing UL56 gene 3 ' holds 60bp and LORF5 gene 5 ' end 2638bp Malicious rDEV Δ UL56.
The building of 2 duck plague virus rDEV Δ UL56-RFP of embodiment
(1) building of transfer vector pT-UL56-RFP
Using PCR method, PCMV IE-EGFP- is expanded from plasmid pEGFP-C1 (being purchased from Clontech company) SV40poly A expression cassette, is cloned into pMD18T-simple carrier.By Nhe I and BamH I double digestion, replaced with RFP EGFP obtains the plasmid pT-RFP of expression red fluorescence.
With Mlu I digestion, electrophoresis recycles RFP expression cassette (1.7kb) (shown in SEQ ID NO.5), is inserted into 1 step of embodiment Suddenly the Mlu I restriction enzyme site for the pT-UL56 recombinant plasmid that (2) obtain, obtains transfer vector pT-UL56-RFP.
The primer that the building of 1 pT-UL56-RFP of table uses
(2) homologous recombination
According to Lipofectamine TM2000 transfection reagent box specification, by transfer vector pT-UL56-RFP and duck plague Viral CVCC AV1221 transfects duck embryo fibroblasts (DEF).Specific step is as follows:
DEF in six orifice plates is cultivated to suitable DEV when growing up to 70%~90% cell monolayer, is inoculated with, and absorption 1~4 is small When；1 μ g transfer vector, which is diluted in, makes the final volume of mixed liquor be 50 μ L in the opti-DMEM without serum and antibiotic, It is incubated at room temperature 5min；The 2 μ L of μ L Lipofectamine 2000 and 48 are taken gently to mix without the opti-DMEM of serum and antibiotic It is even, it is incubated at room temperature 5min；50 μ L Lipofectamine, 2000 dilution is added drop-wise to respectively in 50 μ L plasmid dilutions, side Edged mixes；It is incubated at room temperature 20min.During this period, after the cell in six orifice plates gently being washed twice with serum-free OPTI-MEM, 0.5mL serum-free OPTI-MEM is added in every hole, and 100 μ L Lipofectamine 2000/DNA complexes drop-wises are added to 6 holes In plate, gently shaking mixes it uniformly, sets 37 DEG C of culture 4h, discards supernatant, and the M199 culture medium of 10% fetal calf serum is added, After setting 37 DEG C of cultures for 24 hours, liquid is changed with the M199 culture medium of 1% fetal calf serum, sets 37 DEG C of 3~5d of culture, observed daily, until going out Existing recombinant virus fluorescent spot.
(3) plaque purification of recombinant virus
The recombinant virus tentatively obtained is seeded to the 6 orifice plates DEF cell for having grown up to good cell monolayer, is adsorbed 1 hour Afterwards, adsorption liquid is discarded, 1% low melting-point agarose is spread, continues to cultivate.It is red under the microscope in fluorescence microscopy after inoculation 48 hours Color fluorescence selects single fluorescence plaque in 0.5ml M199 nutrient solution, after freeze thawing 1 time be inoculated with 6 orifice plates DEF cell, so into Row plaque purification 3~4 times, obtain the recombination duck plague virus rDEV Δ UL56-RFP of expression red fluorescent protein.Duck plague disease 3 ' end 60bp and LORF5 gene 5 ' end 2638bp of the UL56 gene of poison strain are replaced by RFP expression cassette.
The fluorescence microscope result of recombinant virus as shown in Figure 1, the insertion site Δ UL56 red fluorescent protein at Function expression.
Embodiment 3 recombinates the animal safety experiment of duck plague virus rDEV Δ UL56-RFP
By 15 4 week old SPF ducks, it is randomly divided into 3 groups, every group 5.1st group of intramuscular injection recombinant virus rDEV Δ UL56- RFP, every 106.0TCID50；2nd group of intramuscular injection duck plague virus CVCC AV1221, every 104.0TCID50；3rd group does not connect Kind, as control.Every group of independent isolated rearing.14d is observed, records condition of morbidity death daily.As a result duck plague virus CVCC AV1221 inoculation group is all dead in 7d, and recombinant virus immune group and not to be inoculated with control group whole within the 14d observation period It is strong to live, illustrate that recombinant virus rDEV Δ UL56-RFP to duck no pathogenicity, can be used for preparing duck plague vaccine.
The Evaluation of Immunogenicity of the recombination of embodiment 4 duck plague virus rDEV Δ UL56-RFP
By 15 4 week old SPF ducks, it is randomly divided into 3 groups, every group 5.1st group of intramuscular injection recombinant virus rDEV Δ UL56- RFP, every 105.0TCID50；2nd group of intramuscular injection commercialization Duck plague live vaccine, every 105.0TCID50；3rd group is not immunized, and makees For control group.Every group of independent isolated rearing.The 14d after immune, venous blood collection, separation serum are measured for neutralize antibody titers, Then leg muscle injection inoculation DEV virulent (CVCC AV1221), every 1000MLD.14d is observed, record morbidity daily is dead Situation attacks malicious control group 4d after attacking poison and starts to fall ill, and 100% is dead in 7d；Recombinant virus rDEV Δ UL56-RFP and quotient Product Duck plague live vaccine immune group is all good for and lives, and realizes 100% immunoprotection；Recombinant virus rDEV Δ UL56-RFP and It is respectively 1:24.5,1:23.3 that Duck plague live vaccine immune group neutralize antibody titers, which are commercialized, and control group neutralize antibody titers are lower than 1:4 illustrates that rDEV Δ UL56-RFP strain has good immunogenicity.
The building of the recombination duck plague virus live vector of embodiment 5
(1) transfer vector constructs
By Nhe I and BamH I double digestion, with Avian Influenza Virus HA Gene, (H9 hypotype is as shown in SEQ ID NO.6, H5 Hypotype is as shown in SEQ ID NO.7), virus hepatitis VP1 gene (as shown in SEQ ID NO.8), parvovirus VP3 gene (as shown in SEQ ID NO.9), duck tembusu virus E protein gene (as shown in SEQ ID NO.10) replace plasmid pT-UL56- RFP gene in RFP obtains the recombinant transfer vector containing foreign antigen genes.
(2) homologous recombination
According to Lipofectamine TM2000 transfection reagent box specification, by recombinant transfer vector and recombination duck plague virus RDEV Δ UL56-RFP transfects duck embryo fibroblasts (DEF).Specific step is as follows:
DEF in six orifice plates is cultivated to when growing up to 70%~90% cell monolayer, being inoculated with suitable recombination duck plague virus RDEV Δ UL56-RFP is adsorbed 1~4 hour；1 μ g recombinant transfer vector is diluted in the opti- without serum and antibiotic In DMEM, making the final volume of mixed liquor is 50 μ L, is incubated at room temperature 5min；Take 2 μ L Lipofectamine 2000 and 48 μ L not Opti-DMEM containing serum and antibiotic is mixed gently, and is incubated at room temperature 5min；50 μ L Lipofectamine 2000 are diluted Liquid is added drop-wise to respectively in 50 μ L plasmid dilutions, and side edged mixes；It is incubated at room temperature 20min.It during this period, will be thin in six orifice plates After born of the same parents gently wash twice with serum-free OPTI-MEM, 0.5mL serum-free OPTI-MEM is added in every hole, by 100 μ L Lipofectamine 2000/DNA complexes drop-wise is added in 6 orifice plates, and gently shaking mixes it uniformly, sets 37 DEG C of cultures 4h is discarded supernatant, and the M199 culture medium of 10% fetal calf serum is added, after setting 37 DEG C of cultures for 24 hours, with the M199 of 1% fetal calf serum Culture medium changes liquid, sets 37 DEG C of 3~5d of culture.
(3) plaque purification of recombinant virus
The recombinant virus tentatively obtained is seeded to the 6 orifice plates DEF cell for having grown up to good cell monolayer, is adsorbed 1 hour Afterwards, adsorption liquid is discarded, 1% low melting-point agarose is spread, continues to cultivate.Inoculation is seen under fluorescence microscope after 48-72 hours Examine, select single non-blooming plaque in 0.5ml M199 nutrient solution, 6 orifice plates DEF cell is inoculated with after freeze thawing 1 time, so into Row plaque purification 3~4 times, obtain expression of influenza virus HA gene, virus hepatitis VP1 gene, parvovirus VP3 base respectively Because of the recombination duck plague virus of the protective antigens of, duck tembusu virus E protein gene.
Embodiment 6 recombinates duck plague virus rDEV Δ UL56-RFP and is used for vaccine preparation
1, vaccine preparation
(1) prepared by cell
Well-developed 9~11 age in days SPF chicken embryo is selected, CEF is made according to conventional trypsin digestion, cultivates 24 hours left sides It is spare that the right side grows up to single layer.
(2) preparation of virus liquid
Production kind poison rDEV Δ UL56-RFP is inoculated with CEF cell monolayer by 0.01%~0.5% poison amount that connects by volume, 37~38 DEG C are cultivated 36~72 hours, when lesion rate harvests virus liquid, -20 DEG C of preservations of freezing up to 75% or more.
(3) inspection of semi-finished product
1. steriling test: every group separately sampled, by version three progress of " Chinese veterinary pharmacopoeia " two 〇 First Five-Year Plan year.
2. viral level measures: guaranteeing every viral level >=10 0.1ml5.0TCID50。
(4) match seedling and packing
Qualified virus liquid will be examined to filter mixing, suitable 5% sucrose degreasing milk stabilizer and antibiotic is added, sufficiently It mixes, quantitative separating.
(5) it is lyophilized
After packing, carried out rapidly by " People's Republic of China's regulations " (version in 2000) annex page 437 Vacuum freezedrying.
2, vaccine test
The present invention relates to related check method by Republic of China Veterinary Pharmacopoeia (in Chinese veterinary pharmacopoeia committee Two 〇 of magnificent people's republic's veterinary drug allusion quotation, mono- 〇 version (three), Chinese agriculture publishing house, 2011, the present invention claims " Chinese veterinary drug Allusion quotation ") method carry out.
(1) character: faint yellow Sponge Porosity agglomerate is easily detached from bottle wall, dissolves rapidly after adding dilution.
(2) it steriling test: tests by " Chinese veterinary pharmacopoeia ".
(3) mycoplasma is examined: being tested by " Chinese veterinary pharmacopoeia ".
(4) exogenous virus is examined: being tested by " Chinese veterinary pharmacopoeia ".
(5) vaccine diagnostic test: is diluted to 100TCID with sterile saline50/ 0.1ml, with the anti-duck plague virus of equivalent Specific serum mixing sets room temperature effect after sixty minutes, and inoculation 5 has grown up to the cell hole (48 porocyte plates) of CEF single layer, often Hole 0.2ml, while virus control group is set, under the conditions of setting 37~38 DEG C, culture observation 120 hours.
(6) with the susceptible duck of 2~4 week old 10, plumage part, every 10 plumage of intramuscular inoculation vaccine safety verification: are indicated by label Part, it observes 14.
(7) efficacy test: following method, which is appointed, selects one.
1. viral level measures: indicating plumage part by label, vaccine is diluted to every 0.1ml containing 1 plumage part, is further continued for making 10 times It is serially diluted, takes 3 acceptable diluent degree, be inoculated with 5 cell holes (96 porocyte culture plates) for having grown up to CEF single layer respectively, often Hole 0.1ml, under the conditions of setting 37~38 DEG C, culture observation 120 hours.According to CPE production, calculated by Reed-Muench method TCID50。
2. being examined with duck: with the susceptible duck of 2~8 week old 10, indicating plumage part, every intramuscular injection vaccine inoculation 1 by label Plumage part, separately set 5 it is not immune compare, the isolated rearing under similarity condition.After 14 days, every duck intramuscular injection 1000MLD duck Pestivirus is virulent (CVCC AV1221), observes 14.
(8) residual moisture measures: being measured by " veterinary drug allusion quotation " prescriptive procedure.
(9) vacuum degree measures: being measured by " veterinary drug allusion quotation " prescriptive procedure.
To the duck plague virus vaccine of preparation carry out character, pure property, safety and immune efficacy inspection, the results showed that, Character, residual moisture and the vacuum degree of the duck plague virus vaccine of preparation meet the regulation of " Chinese veterinary pharmacopoeia ", and without bacterium, The pollution of mycoplasma and exogenous virus；In the experiment of duck plague virus specificity identification, all there is lesion in virus control group, in antibody There is not cytopathy with group；By the susceptible duck of vaccine inoculation, raised through 14 days, it is all strong to live；In vaccine immunity efficacy test, It is virulent 14 days latter to be inoculated with duck plague virus, compares 4 death of duck, immune duck 8 strong work show that the duck plague virus vaccine can be to duck Pestivirus realizes higher immune efficacy.
The preparation of 7 monoclonal antibody of embodiment
The end of UL56 gene 3 ' and LORF5 gene are related to the virulence of duck plague virus, and therefore, those skilled in the art can benefit The monoclonal antibody of anti-duck plague virus is prepared with the end of UL56 gene 3 ' and LORF5 gene.Monoclonal antibody can be used in specific steps The routine techniques of preparation: (1) end of Bacillus coli expression UL56 gene 3 ' or LORF5 gene the preparation of immunogene: are utilized；(2) it moves Object is immune: using the protein immunization BALB/c healthy mice of expression, acquiring mouse spleen；(3) preparation of hybridoma: marrow Oncocyte is merged with splenocyte, screens positive cell strain；(4) it the foundation of hybridoma cell line: repeats step (3), screening obtains The hybridoma cell line of stably excreting monoclonal antibody.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.
Sequence table
<110>China Veterinery Drug Inspection Office
<120>end of duck plague virus UL56 gene 3 ' missing and LORF5 gene deletion mutants and its construction method and application
<130> KHP181113803.6
<160> 14
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aatcgtccac gcagtacctc aagcgcagct atatttgaac gttcggtgga gaatggaaga 180
tttacggtag aaggaagagc taatcgccgc aatgaaacgt tgtatgataa ctcatcagat 240
gaaagtgata ctgaattcat atcattattg acgacggatg gagatgactg gactaatgat 300
cgaagagact gcggtattcc accaccagcg tatgacgaac tatctatcct gtctacctcc 360
gacgacatgc accacgatag aactgaagaa agtgtaacac actgtgaaag tttatcaaga 420
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gccttcttcg gaaccgagga ggaaaaagct gctgcggaag cagcttcgca gcttggaaag 540
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gaacgggcat ctgcaatttt tgtacaggaa tttcgtaatg cgtttgttaa tataacacgc 1320
gaatatatgg gcgaagacaa ttactataga agctataggg gcgatgaggt gaaagaaaag 1380
ggaatagagc aatccgcctt agacccattc ggctacacca cgtacaaacc gcgcaaacat 1440
tcaaaaaata ttattcaaag gcatttaccg tcatgccttc gagaaccgga ggttaacgca 1500
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tcagcctttt cctggtccaa agacccgttt tttgaaatac ccgccaacga tgacgccaga 1800
tatgattctc tcccatcgga gaagtacaat ccagcagaac tgggactaat agtttttgca 1860
ggcactgaaa tgcgtccagt attccgtaca ttttttttag gtggtaggaa tggtaagcgg 1920
aaagccaaga atgctataat aacggaaata gaacgcctat ctgagcagct agttaatgct 1980
ggtccgtcaa acagatgtcc gtatgtaatc gtactaactg aagttgaatc tgatctagta 2040
accggggcca tggcatcttc aaaagcgttg ctaaagttag ggggaatagc cgtttcgcca 2100
gacgcattgt atactatgtt aaaacgactg gtgggagtaa cgacttattc atcaattaag 2160
tgtacatcca acctgagaag ctttgaatcg aggccaacgg tagggactgt cccataccgc 2220
ttcgcattcc agtttggtcg tcgtaagacg gaatgttaca caccgcgcgt gatacattgc 2280
ggagcggttt catttgttcc gctgccgtct gaagaaacgt cggtattagg atcggaggcg 2340
ctaatgaaca ttccagatca agcaacatat gcccgtactc ttacagcttg tcgcggtatt 2400
ccaccagtac atgattattg tttggggctg ttcttcccta taaagggaat ttattacttg 2460
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ttcattagtg atgttgtcat gaactgtaat tatttcaaat atgggttttt gtttcacctt 2640
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gatgactatt ag 2772
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cccgtcatcc gcgaatcctt actcttacac ctatcgatcg ataaagacaa ggtatgatgg 180
ctgaacatgg caacgatagc acacgttaac atgcataacg gccttaatgc tgccattttt 240
tttagcaact agagaagaca attgttccct aatgcctacc atgtgtgggt aactgtgcca 300
gatggtatta tatagccacg agaacaatca tttccgtatc atgacttact ggaatagaaa 360
gtcaccgctt gttgtgtttc tcggaagatg tattgttcta tataactata cgtaggttcg 420
ttttcagtta cgcttctcga aaaggtgcac atttctaatc aaacgactgg ccttttctgc 480
gcggccgcgg ccgggagagg cgttggacca tcccgctcat tgttcaatag ctaaatgact 540
catgtaatta agaccagact cataacagac atgttatctg aacatcacat gcatcacgag 600
ttatatccgt ccggactggt atataaaatg ttgcgagagg gcgagtattc tgatcttacc 660
gtacagtaca aaaacactgt attactaaaa accagtttag tcatctcgtg tcagttaaag 720
gtacttattc tcaagtactc atggaatctc ataatacgta tggtcggtac atagatcata 780
ttaattgcga agtatgggat ttgggtagac agattccaca taatgattta atgggatacg 840
acaacactac agcacagcca aatcgtccac gcagtacctc aagcgcagct atatttgaac 900
gttcggtgga gaatggaaga tttacggtag aaggaagagc taatcgccgc aatgaaacgt 960
tgtatgataa ctcatcagat gaaagtgata ctgaattcat atcattattg acgacggatg 1020
gagatgactg gactaatgat cgaagagact gcggtattcc accaccagcg tatgacgaac 1080
tatctatcct gtctacctcc gacgacatgc accacgatag aactgaagaa agtgtaacac 1140
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acgtagagag gcctccggat tatttcacat cactactgat ccatccgcct atgtatgaag 1260
aaagttcatc cgatcgacga gatgaatctc ctccgccata tgtagaattg ccgcgcatat 1320
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tgatctt 1387
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<213>artificial sequence (Artificial Sequence)
<400> 4
ttcattgttt accgtgtcag cggcgcgaga aactagtggt ggagatacta cgaagggcta 60
ttgaagatgc acagaaatat aaatccactt ctacatggtt taatgtctat agatatacca 120
gagatgacta ttagcattta atataccggt tttaactttt gacgtctttc acattactta 180
aataaaataa atatgattaa acgaatcagt ctcggctctt gactcttgtt ttgagttata 240
gtggggaagc agactatgta aagacagtcg acggactgcc agtgaacgct gaacaagcta 300
ggacaattac gagattgtct agcttgttca gcgtgtttta ttacccaaat accctgttag 360
tagcttgggt ctttactttt tgcgcggaac attgtccaga tatcatctac gtttcaatga 420
aattctccaa atgtaaccaa tgcaactaca aatataccat taacacaata gtgttaactc 480
tacctccaat tcagatcaga ctagagcact tccaggcggt actgttcgat ctttactatt 540
aaaaaatcaa tttcttatgg ttttaataaa acgctttatt acattgtagt gtaacaagac 600
tcatacatta gctttgtgaa ttccacgttt ccgtatttcg gtacataatg cagcggctct 660
agccgatgcc gatacgaccc cctgccaaca actcattgaa aatggttctt tgcaatgaca 720
atgatatcca aggccagtta tgatcagcac ttccggttca tctatcgaag aatccgtttg 780
tacacatgcg aaatatggcg cgttgcatag acgatgctcc gtacgtattc cggcgacggc 840
catcggatcg ttgaaaactc tgcaaagacg tttcacgtcc ccgacgcttg gctcttcttt 900
attgcataaa atgagcccgc agaagaatat atgtgattct gtttccatat tgcattgtat 960
attcattgaa aaaagacgct gttcaaaaaa cgacgcgctc tctagaacaa cctctggtct 1020
tggtgtaaca cagtggtcct tcttcttggg catgtaaaac atctctatcg catatggcgt 1080
cacgatgcga cttacgcgcg gcatagtcct tacacaagtc tcgcctccga gagaatctat 1140
taccgccttc gcgtcggcca ttgttgtcgg tcacgtctac cgcctcgttc ctcttattcc 1200
aaccgccttt ccgacctata taactcggtc aataccgttc gtattagcag catagcatct 1260
tataacgctt catggcacat gcgcaaatca tgttgtctgc acgtacctcc cactaatttg 1320
catgcaatgg gcgttactat ttattagcga ctcatttaaa cacggcccct cagaactgat 1380
cggtgctccg caacgagaca gtcaaagcga taagcaagat g 1421
<210> 5
<211> 1604
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
acgcgttagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga 60
gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg 120
cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg 180
acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca 240
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc 300
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc 360
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc 420
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 480
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 540
gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta 600
gcgctaccgg tcgccaccat ggtgagcaag ggcgaggagg acaacatggc catcatcaag 660
gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc 720
gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc 780
aagggcggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc 840
aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag 900
ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag 960
gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc 1020
ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg 1080
atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac 1140
ggcggccact acgacgccga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg 1200
cccggcgcct acaacgtcaa catcaagctg gacatcacct cccacaacga ggactacacc 1260
atcgtggaac agtacgagcg cgccgagggc cgccactcca ccggcggcat ggacgagctg 1320
tacaagtaat ccggaagatc tctcgaggga tccaccggat ctagataact gatcataatc 1380
agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg 1440
aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat 1500
ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 1560
tctagttgtg gtttgtccaa actcatcaat gtatcttaac gcgt 1604
<210> 6
<211> 1702
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gctagccgcc accatggaga cagtatcact aataactata ctactagtag taacagtaag 60
caatgcagat aaaatctgca tcggctacca atcaacaaac tccacagaaa ctgtggatac 120
gctaacagaa aacaatgtcc ctgtgacaca tgctaaagaa ttgctccaca cagagcacaa 180
tgggatgctg tgtgcaacaa atctgggaca tcctctcatt ctaaacacct gtaccattga 240
aggactgatc tatggcaacc cttcttgtga tcagctgttg ggaggaggaa aatggtccta 300
catcgtcgaa agaccatcgg ccgttaatgg aatgtgttac cccgggaatg tagaaaacct 360
agaggaacta agatcactct ttagttctgc tagttcctac caaagaattc agatctttcc 420
agacacgatc tggaatgtgt cttacaatgg aacaagcaaa gcatgttcag attcattcta 480
cagaagcatg agatggctga ctcaaaagaa caacgcttac cctattcaag acgcccaata 540
cacaaataat agaggaaaga gcattctttt catgtggggc ataaatcacc cacccaccga 600
tactgtacag acaaatttgt acacaaggac cgacacaaca acaagtgtga caacagaaga 660
tataactaga accttcaaac caatgatagg gccaaggccc cttgtcaatg gtcagcaggg 720
gagaattgat tattattggt cggtattaaa accaggtcag acattgcgaa taagatccaa 780
tgggaatcta attgctccat ggtatggaca cattctttca ggagagagcc acggaagaat 840
cctgaagact gatttaaaca gtggtaactg tgtagtgcaa tgtcagactg aaagaggtgg 900
cttaaacacc acattgccgt tccacaatgt cagtaaatat gcatttggga actgcccaaa 960
gtatgttgga gtaaagagtc tcaaactggc agttggtcta agaaatgtgc ctgctagatc 1020
aagtagagga ctatttgggg ctatagctgg tttcatagag ggaggttggt cagggttagt 1080
cgctggttgg tatgggttcc agcattcaaa tgatcaaggg gtaggtatgg ctgcagatag 1140
agagtcaact caaagggcaa ttgacaaaat aacatccaaa gtgaataata tagtcgataa 1200
aatgaacaag cagtatgaaa ttattgatca tgaattcagc gaggttgaaa ctagactcaa 1260
tatgatcaat aataagattg atgatcaaat acaagacata tgggcttata acgcagaatt 1320
gctagtgctg cttgaaaatc agaaaacact cgatgaacat gatgcaaatg tgaacaatct 1380
atataacaaa gtgaagaggg cactgggttc caatgccatg gaagacggga aaggatgttt 1440
tgagctatac cataaatgtg atgatcagtg catggagaca attcggaacg ggacctataa 1500
caggagaaag tataaagagg aagcaaaact agaaagacag aaaatagaag gggtcaagct 1560
ggaatctgaa ggaacttaca aaatcctcac catttattcg actgtcgcct catctcttgt 1620
gattgcaatg gggtttgctg ccttcttgtt ctgggccatg tccaatggat cttgcagatg 1680
caacatatgt atataaggat cc 1702
<210> 7
<211> 1714
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gctagccgcc accatggaga aaatagtgct ccttcttgca atagtcagcc ttgttaaaag 60
tgatcagatt tgcattggtt accatgcaaa caactcgaca gagcaggttg acacaataat 120
ggaaaagaac gtcactgtta cacatgccca agacatactg gaaaagacac acaacgggaa 180
gctctgcgat ctagatggag tgaagcctct gattttaaga gattgtagtg tagctggatg 240
gctcctcgga aacccaatgt gtgacgaatt catcaatgtg ccggaatggt cttacatagt 300
ggagaaggcc aatccagcca atgacctctg ttacccaggg aatttcaacg actatgaaga 360
actgaaacac ctattgagca gaataaacca ttttgagaaa attcagatca tccccaaaga 420
ttcttggtcc gatcatgaag cctcatcagg ggtgagctca gcatgtccat accagggaac 480
gccctccttt ttcagaaatg tggtatggct tatcaaaaag aacaatgcat acccaacaat 540
aaagaaaagc tacaataata ccaaccaaga agatcttttg gtactgtggg ggattcacca 600
tcctaatgat gcggcagagc agacaaggct ctatcaaaac ccaaccacct atatttccgt 660
tgggacatca acactaaacc agagattggt accaaaaata gctactagat ccaaagtaaa 720
cgggcaaagt ggaaggatgg atttcttctg gacaatttta aaaccgaatg atgcaatcaa 780
cttcgagagt aatggaaatt tcattgctcc agaatatgca tacaaaattg tcaagaaagg 840
agactcaaca attatgaaaa gtgaagtgga atatggtaac tgcaacacca agtgtcagac 900
tccaataggg gcgataaact ccagtatgcc attccacaac atacaccctc tcaccatcgg 960
ggaatgcccc aaatatgtga aatcaaacaa attagtcctt gcgactgggc tcagaaatag 1020
ccctcaagga gagactcgag gactatttgg agctatagca ggttttatag agggaggatg 1080
gcagggaatg gtagatggtt ggtatgggta ccaccatagc aatgagcagg ggagtgggta 1140
cgctgcagac aaagaatcca ctcaaaaggc aatagatgga gtcaccaata aggtcaactc 1200
gatcattgac aaaatgaaca ctcagtttga ggccgttgga agggaattta ataacttaga 1260
aaggagaata gagaatttaa acaagaagat ggaagacgga ttcctagatg tctggactta 1320
taatgctgaa cttctggttc tcatggaaaa tgagagaact ctagacttcc atgactcaaa 1380
tgtcaagaac ctttacgaca aggtccgact acagcttagg gataatgcaa aggagctggg 1440
taacggttgt ttcgagttct atcacaaatg tgataatgaa tgtatggaaa gtgtaagaaa 1500
cggaacgtat gactacccgc agtattcaga agaagcaaga ttaaaaagag aggaaataag 1560
tggagtaaaa ttggaatcaa taggaactta ccaaatactg tcaatttatt caacagtggc 1620
gagttcccta gcactggcaa tcatggtggc tggtctatct ttatggatgt gctccaatgg 1680
gtcgttacaa tgcagaattt gcatctaagg atcc 1714
<210> 8
<211> 727
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cgccaccatg ggtgattcta accagttagg ggatgatgag ccggtttgct ttctgaattt 60
tgagacagct aatgtcccaa tacaaggtga atctcacact cttgtcaaac acctgtttgg 120
gaggcaatgg ttagttagga ctgtgcaaca cgcctcaact gtgcaagagc tggacctcca 180
agttccagac agaggacatg cctctctcat ccggttcttt gcctattttt ctggagagat 240
cattcttacc attgtcaata atggcactac accagcaatg gtagcacact cttattctat 300
ggatgacctc acttcagagt atgctgttac agcaatggga ggtgtgatga ttcctgctaa 360
cagtgccaaa aatatttctg tgccattcta ctctgtgaca ccactcaggc caactcgacc 420
aattcctggc acatcagagg caacttttgg cagactgttc atgtggactc aatcaggaag 480
tctttcagtt tttatgggtc tcaaaaagcc agctctcttc tttccactcc ctgctcctac 540
ctccacaaca tcatcacggg gatccaatga tgttattccc acattgaatc agtctgggga 600
tgaagtagat tgtcacttct gcgaaatttg ttctaaaatg aagaggaggt ggaagccaag 660
agggcacttc agattttgct ttagactcaa aacactagca tttgaactca atctggaaat 720
tgaataa 727
<210> 9
<211> 1612
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgccaccatg gcagagggag aaggcggagc tttgggcgac gcttcagggg gtgccgatgg 60
agtgggtaat gcctcgggaa attggcattg cgattcccaa tggatgggaa acacagtcat 120
cacaaagacc accagaacct gggtcctgcc cagctacaac aaccacatct acaaagcgat 180
taccagtgga acctctcaag atgcaaatgt ccagtatgca ggatacagta ccccctgggg 240
gtactttgat ttcaaccgct tccactgcca cttctcccct agagactggc agagacttat 300
caacaaccat tggggaatca gacccaagtc tcttaaattc aagatcttca atgtccaagt 360
caaagaagtc acaacgcagg atcagacgaa gaccattgca aacaatctca cgtcaacaat 420
tcaagtcttt acggatgacg agcatcaact cccgtatgtc ctgggctcgg ctacggaagg 480
caccatgccg ccgttcccgt cggatgtcta tgccctgccg cagtacgggt attgcacaat 540
gcacaccaac cagaacggtg cacgattcaa tgaccggagt gcattctact gcttagaata 600
cttccccagt cagatgctaa gaacaggcaa caactttgag ttcacgtttg actttgaaga 660
agttcctttc cacagcatgt tcgctcattc acaggactta gacaggctga tgaacccctt 720
agtggatcaa tacctctgga atttcaatga ggtagacagc agcagaaatg ctcaatttaa 780
aaaggctgtg aaaggcgctt atggcaccat gggccgcaat tggctgccag gacctaaatt 840
cctggaccag agagttaggg cctatacagg cggaactgat aattatgcaa actggaacat 900
ctggagtaat ggaaacaagg ttaatttgaa ggacaggcag tacctcctgc aacccggacc 960
tgtatcagct actcacacag aagcagaggc ttccagcatc ccagcccaaa atattttagg 1020
tttagctaaa gatccataca gatctggcag cactacagca ggaataagtg atattatggt 1080
cacggacgag caggaagtag cacctacaaa cggcgtaggg tggaaaccat atggcaagac 1140
tgtaacgaat gaacaaaaca ctactacagc tcctacaagt tcagatctgg atgttcttgg 1200
agctttacca ggaatggttt ggcagaacag ggatatatat ctacagggac ctatttgggc 1260
aaaaataccg aagactgatg gtaaattcca tccttctccg aatctcggag gatttggtct 1320
gcacaatcca ccaccgcagg tgttcatcaa gaatacacca gtgcctgcag accctccagt 1380
agaatacgtg caccagaagt ggaattccta cataacccag tactctacgg gccagtgtac 1440
agtagagatg gtgtgggagc tgagaaaaga gaattcaaag agatggaacc cagaaatcca 1500
gttcaccagt aatttcagtg acagaacaag cataatgttt gcacctaatg aaactggtgg 1560
atatgtagaa gatagattaa ttggaaccag atatctaact caaaatctgt aa 1612
<210> 10
<211> 1525
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
cgccaccatg gcgtacagct tcagctgtct ggggatgcag aaccgagact ttgttgaggg 60
agtgaatggt gttgagtgga tcgatgtcgt tctggaagga ggctcatgtg tgaccatcac 120
ggcaaaagac aggccgacca tagacgtcaa gatgatgaac atggaggcta cggaattagc 180
ggttgtgaga tcttactgct atgagccgaa agtgtcggac gtgacgacag aatccagatg 240
cccaaccatg ggagaggctc ataatcccaa ggcaacttat gctgaataca tatgcaaaaa 300
agattttgtg gataggggtt ggggcaatgg ctgcggcttg tttggaaagg ggagcataca 360
gacatgtgcc aagtttgact gcacaaagaa agcagaaggc aggattgtgc agaaggaaaa 420
cgtccagttt gaagttgcag ttttcataca tggttccacg gaagcgagca cctaccacaa 480
ttattcagcc cagcagtcgc tgaaacatgc cgctagattc gttataacgc ccaaaagtcc 540
cgtctacacc gctgagatgg aggattatgg taccgtcaca ctcgaatgtg aaccccgatc 600
tggggttgac atggggcaat tctatgtctt taccatgaac acaaaaagct ggcttgttaa 660
cagagactgg tttcatgatc tcaacttacc atggacaggg tcatcagcgg ggacgtggca 720
aaacaaagag tcattgatag aatttgagga ggcccacgcc accaaacaat cagtggtggc 780
tttggcatca caagaaggag ccctccatgc agcattggcg ggagctattc cagtgaagta 840
ctctggaagc aaattggaaa tgacctcagg tcatcttaaa tgcagggtta aaatgcaggg 900
tttgaagctg aaaggaatga cctacccgat gtgtagcaat acattttccc tagtgaagaa 960
tcctaccgac actgggcatg gcactgtcgt ggtggaattg tcttatgcag gtaccgatgg 1020
gccctgtaga gttcccatat ccatgtcggc agatctgaat gacatgacac cagttggacg 1080
cttgataaca gtcaacccat acgtgtcgac ctcctccacg ggtgccaaga taatggtgga 1140
agtggaacct ccattcgggg attcattcat cttagtagga agtggaaaag gacagatcag 1200
gtaccagtgg catagaagtg ggagcacaat tggaaaagct tttacgtcaa cactcaaagg 1260
agcacaaagg atggttgctt tgggtgacac tgcatgggat tttggctcag ttgggggtgt 1320
actcacctcc attgggaaag gcattcatca ggttttcggc tcagcattta aaagcttatt 1380
tggaggaatg tcatggatta ctcaaggcat gttgggggca ctgctattgt ggatggggct 1440
gaatgcaagg gacagatcca tttctatgac ttttctagcc gtaggaggaa ttttagtctc 1500
cctggcagta aatgtcaatg cctaa 1525
<210> 11
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cggggtaccg tctgctcttc cgccattc 28
<210> 12
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
gcggatccag atctaagatc agaccgccgc cta 33
<210> 13
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gcctgcagac gcgtttcatt gtttaccgtg tc 32
<210> 14
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
cccaagcttc atcttgctta tcgcttt 27

Claims (10)

1. a kind of Duck plague virus strains of attenuation, which is characterized in that the UL56 gene of the Duck plague virus strains inactivates or UL56 Gene and LORF5 gene inactivate simultaneously；Preferably, the inactivation holds 60bp and LORF5 gene 5 ' end for missing UL56 gene 3 ' 2638bp。
2. Duck plague virus strains according to claim 1, which is characterized in that the UL56 gene 3 ' of the Duck plague virus strains LORF5 gene inactivates while the missing of end, and is inserted into foreign gene, it is preferred that the insertion foreign gene is in UL56 gene Foreign gene is inserted into the position of 3 ' end 60bp missings and LORF5 gene 5 ' end 2638bp missing.
3. the construction method of Duck plague virus strains described in claim 1~2 any one, which is characterized in that including walking as follows It is rapid:
(1) building UL56 gene 3 ' end missing and LORF5 gene inactivation or the end of UL56 gene 3 ' missing and LORF5 gene inactivation And it is inserted into the transfer vector of foreign gene；
(2) transfer vector and duck plague virus parent's strain cotransfection obtained step (1) is into host cell, through homologous heavy Group, screening obtain the end of UL56 gene 3 ' missing and LORF5 gene inactivation or the end of UL56 gene 3 ' missing and LORF5 gene inactivation And it is inserted into the Duck plague virus strains of foreign gene in the position.
4. Duck plague virus strains described in claim 1~2 any one are in preparation duck plague vaccine or recombination live vector epidemic disease Application in seedling, the duck plague vaccine are duck plague list seedling or duck plague combined vaccine.
5. a kind of recombination live vector is the strain of duck plague virus described in claim 1~2 any one.
6. a kind of duck plague virus or live recombinant vectors viral vaccine, which is characterized in that including claim 1~2 any one institute Exogenous antigen coding is inserted into the Duck plague virus strains or the Duck plague virus strains described in claim 1~2 any one stated The recombinant virus strain of gene preparation；Preferably, the exogenous antigen encoding gene is Avian Influenza Virus HA Gene, viral Hepatitis VP1 gene, parvovirus VP3 gene or duck tembusu virus E protein gene.
7. duck plague virus UL56 gene or UL56 gene association LORF5 gene in regulation duck plague virus virulence or are prepared in vaccine Using, which is characterized in that the UL56 gene inactivation for preparing duck plague virus in vaccine or UL56 gene and LORF5 gene are simultaneously Inactivation.
8. application according to claim 7, which is characterized in that the sequence of the UL56 gene as shown in SEQ ID NO.1, The sequence of the LORF5 gene is as shown in SEQ ID NO.2.
9. a kind of monoclonal antibody, which is characterized in that its corresponding epitope contain UL56 gene or LORF5 gene coding Amino acid sequence.
10. application of the monoclonal antibody as claimed in claim 9 in preparation duck plague virus detection kit.
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CN109593730A (en) * 2018-12-26 2019-04-09 四川农业大学 The seamless gene-deleted strain DPV CHv- Δ Lorf5 of duck plague virus Lorf5 gene and its construction method
CN109609547A (en) * 2018-12-26 2019-04-12 四川农业大学 The seamless gene-deleted strain CHv-BAC-G- Δ Lorf5 of duck plague virus Lorf5 gene and its construction method

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