CN104031889A - Recombinant turkey herpesvirus vaccine expressing infectious bursal disease virus VP2 protein and application thereof - Google Patents
Recombinant turkey herpesvirus vaccine expressing infectious bursal disease virus VP2 protein and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant turkey herpesvirus vaccine expressing infectious bursal disease virus VP2 protein and application thereof. According to the invention, on the basis of a Fosmid library continuously covering the whole genome of herpesvirus of turkey (HVT), a VP2 gene carrying a Pec composite promoter is inserted into site HVT053 and site HVT054 in the nonessential region of HVT replication by using Red ET homologous recombination technology and Gateway LR clone technology so as to obtain recombinant HVT (rHVT-VP2). The recombinant HVT (rHVT-VP2) can provide a complete protecting force on the infectious bursal disease virus and is applicable to prevention of the infectious bursal disease virus.
Description
Technical field
The invention belongs to molecular biology and biological technical field, be specifically related to a kind of restructuring be infectious the herpes turkey virus vaccine of bursal disease virus VP 2 albumen and the purposes in infection prevention fabricius bursa thereof.
Background technology
Infectious bursal disease (Infectious bursal disease, IBD) be by infections chicken cloacal bursa virus (Infectious bursaldisease virus, IBDV) bird that causes is acute, height contact immunosuppression sexually transmitted disease, can be by causing fabricius bursa damage, immunosuppression and causing that chick high mortality causes huge financial loss [1] to poultry farming.Current commercial infectious bursal disease malicious vaccine alive is divided into weak poison, poisoning and strong malicious three classes.Attenuated live vaccines is to SPF chicken safety, but to there being maternal antibody chicken group's immune protective efficiency effect undesirable.Although it is high a lot of that the immune protective efficiency of mesogenic and strong malicious vaccine is wanted, and damages the fabricius bursa owing to having certain virulence.Meanwhile, due to interference and the IBDV of maternal antibody, traditional vaccine can form the inevitable Blank immunization phase in actual applications.Therefore, fabricius bursa recombinant vaccine is one of study hotspot in recent years safely and effectively.VP2 albumen is the conformation protective antigen of IBDV, and VP2 neutralizing antibody can cause the protection [2] of body for fabricius bursa strong virus attack, and the recombinant live-vector vaccine of construction expression IBDV VP2 gene has potential using value.
HVT Fc-126 strain has been used since 1970 as prototype horse Garrick vaccine always, and this vaccine is without tumorigenicity, protect respond well, can freeze-drying, easy to use, become the vaccine [3-5] that countries in the world are generally used.Herpes turkey virus (HVT) carries external source protective antigen gene aspect as virus vector, has many advantages compared with other virus vector: it uses safety to chicken and the equal no pathogenicity of other animal; After inoculation chicken body, virus forms the viremia that reaches several weeks in chicken body, can stimulate body to produce higher antibody horizontal and remain lifelong, and inoculation once can obtain semelincident immunization; HVT vaccine not only production cost is low, and can freeze-drying, is easy to storage and transport.Meanwhile, the recombinant vaccine taking HVT as carrier is the focus of Chinese scholars research always in the last few years.The protective antigen of Avian pneumo-encephalitis virus (NDV), serum 1 type MDV, infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), avian leukosis virus (ALV) and eimeria tenella disease (Eimeria) all obtains and expresses in HVT carrier; and can resist the infection [6-8,16-19] of lethal dose cause of disease.Thereby the research of recombinant vaccine taking HVT as carrier has broad application prospects.
Summary of the invention
The complete genomic Fosmid of continuous covering herpes turkey virus (HVT) library that this research builds taking laboratory is basis, utilize Red ET homologous recombination technique and Gateway LR clone technology, the VP2 gene that carries Pec combined promoter is inserted into herpes turkey virus Nonessencial region HVT053 and HVT054 site, has obtained the recombinant hvt (rHVT-VP2) of expressing VP2 albumen.Results of animal shows that rHVT-5354VP2 can provide the complete protection for infectious bursa of Fabricius virus, has potential application value.
Concrete, the invention provides the following:
1. express the recombinant herpesvirus of turkeys (HVT) of Infectious bursal disease virus VP2 for one kind, it is characterized in that, the encoding gene of described VP2 albumen inserts the genomic Nonessencial region of HVT.
2. recombinant herpesvirus of turkeys as described in 1, wherein, described Nonessencial region is between HVT genome HVT053 and HVT054.
3. recombinant herpesvirus of turkeys as described in 1 or 2, wherein, the encoding gene of described VP2 albumen is in strong promoter downstream.
4. recombinant herpesvirus of turkeys as described in 3, wherein, described promotor can be combined promoter, preferably Pec promotor, described Pec promotor comprises a cmv enhancer and β-actin promotor.
5. recombinant herpesvirus of turkeys (Meleagrid herpesvirus1) rHVT-5354VP2 who expresses Infectious bursal disease virus VP2, it is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:V201327.
6. a method for the recombinant herpesvirus of turkeys of construction expression Infectious bursal disease virus VP2, comprises the following steps:
1) from the genomic Fosmid of HVT library, filter out and can save viral clay group by cotransfection;
2) in a clay of described clay group, introduce the encoding gene of VP2 albumen;
3) rescue recombinant virus.
7. the method as described in 6, wherein, the whole HVT genome of the common covering of the clay of described clay group.
8. the method as described in 6 or 7, wherein, the encoding gene of described introducing VP2 albumen, for using homologous recombination to introduce, preferably uses Red ET homologous recombination technique and Gateway LR clone technology.
9. the method as described in 6 or 7, wherein, the clay of introducing the encoding gene of VP2 albumen has the genomic Nonessencial region of HVT.
The recombinant virus that described in recombinant virus described in 10.1-5 any one and/or 6-9 any one prepared by method is for the preparation of the purposes of infection prevention bursal disease vaccine.
Detailed Description Of The Invention
Below technical scheme of the present invention is elaborated further.It should be pointed out that each embodiment of the present invention can combine as required by any way.
First aspect of the present invention, provides a kind of recombinant herpesvirus of turkeys (HVT) of expressing Infectious bursal disease virus VP2, and specifically the encoding gene of described VP2 albumen inserts in the genomic Nonessencial region of HVT.
In a specific embodiment, it is HVT genome HVT053 and HVT054 that described Nonessencial region can be, but not limited to, as long as this region is not essential in genomic copying, can be used as insert district.
Due to the existing protection that experimental results show that the expression amount of VP2 on herpes turkey virus (HVT) carrier and bursa of Fabricius in poultry (IBD) be proportionate [7].Therefore, in a concrete embodiment of the present invention, the encoding gene of described VP2 albumen in strong promoter downstream with strengthening to bursa of Fabricius in poultry.It is combined promoter that described strong promoter can be, but not limited to, as long as the suitable startup regulating and controlling sequence that can strengthen downstream target protein all within the scope of the present invention.The preferred Pec promotor of described combined promoter, Pec promotor has comprised a cmv enhancer and β-actin promotor as a kind of novel promotor, and its startup ability in chick embryo fibroblast is three times more than of CMV.
Second aspect of the present invention, provides a kind of method of recombinant herpesvirus of turkeys of construction expression Infectious bursal disease virus VP2, wherein uses fosmid library technology.
Fosmid library technology is a new technology of biology field, is widely used for gene order-checking and the new gene of screening etc.
Therefore,, in a specific embodiments of the present invention, the method for the recombinant herpesvirus of turkeys of described construction expression Infectious bursal disease virus VP2 comprises the following steps:
1) from the genomic Fosmid of HVT library, filter out and can save viral clay group by cotransfection;
2) in a clay of described clay group, introduce the encoding gene of VP2 albumen;
3) rescue recombinant virus.
In a further embodiment, it is described that can cotransfection to save viral clay group be the combination filtering out in clay a large amount of from Fosmid library, these clay groups can cover whole HVT genome area jointly, thereby can after cotransfection cell, there is HVT virus typical cytopathic, i.e. virus rescue.Also all can, for the structure of recombinant virus of the present invention, combine and be not limited to clay concrete in following examples as long as saving the viral clay group of generation.
In another further embodiment, describedly can be, but not limited to adopt the mode of homologous recombination to carry out to the encoding gene of introducing VP2 albumen in a clay, as long as can make VP2 protein expression in virus surface, and any introducing method that does not affect virus rescue all within the scope of the invention.
In preferred embodiments, the encoding gene of described introducing VP2 albumen is for using homologous recombination to introduce, preferably use Red ET homologous recombination technique and Gateway LR clone technology, by the method, the element homology on VP2 protein coding gene and recombinant vectors is replaced.
In further preferred embodiment, described VP2 protein coding gene is under foregoing strong promoter control.
In addition, the 3rd aspect of the present invention, provides recombinant virus prepared by foregoing recombinant virus and/or the described method purposes for the preparation of infection prevention bursal disease vaccine.
In sum; this research is taking HVT reverse genetic operating system as basis; adopt Gateway LR clone technology; IBDV protective antigen gene VP2 is inserted into the Nonessencial region between HVT genome HVT053 and HVT054 gene, has successfully built the recombinant herpesvirus of turkeys of expressing infectious bursal disease virus VP 2 gene.This kind of construction strategy is simplification and the improvement to traditional structure recombinant live-vector vaccine method, save the structure of transfer vector and the tediously long steps such as screening of repeatedly transfectional cell being pressurizeed, for the structure of recombinant live-vector vaccine provides new approaches and novel method.VP2 expressed (comprising HVT) at multiple live vector as the main host protective antigen of IBDV, but all exist generation or the lower problem of induction protection ratio that can not effectively induce neutralizing antibody, existing research comprise comprise SV40 early promoter express the recombinant mdv result of VP2 prove to chicken be safe but can only generating portion protection and time length short [5,6]; When the recombinant hvt (rHVT) of the expression VP2 that comprises CMV promotor carries out primary immune response, can only generating portion protect, the selection of VP2 expression system has vital impact to vaccine protection effect as can be seen here.The existing protection that experimental results show that the expression amount of VP2 on herpes turkey virus (HVT) carrier and bursa of Fabricius in poultry (IBD) be proportionate [7].We have selected Pec promotor thus, and Pec promotor has comprised a cmv enhancer and β-actin promotor as a kind of novel promotor, and its startup ability in chick embryo fibroblast is three times more than of CMV.The result of study of this experiment shows that inoculating 1 age in days chicken with the rHVT-VP2 of 5000PFU dosage can provide the complete protection for infectious bursal disease.
Brief description of the drawings
The building process of Fig. 1 .pEntr-VP2.
With primer VP2CU and VP2CD amplification VP2, the VP2 gene and the PCAGGS plasmid that utilize EcoR I and Bgl II that amplification is obtained are connected to form PCAGG-VP2, secondly, utilize Sal I and Hind III that VP2 is expressed to framework Pec-VP2-POLYA cuts and is connected with pEntrMCS (Invitrogen) and form pEntr-VP2.
Fig. 2 .pFosC DEST plasmid map
Fig. 3 .pFosCVP2 plasmid map
Fig. 4. the rescue schematic diagram of recombinant virus rHVT-5354VP2.
Fig. 5. the PCR qualification result of recombinant virus rHVT-5354VP2.
M:Marker
1:rHVT5354VP2
2:HVT
Fig. 6. the immunoblotting qualification qualification result of recombinant virus rHVT5354VP2.
M:Marker
1:rHVT5354VP2
2:HVT
3:CEF
Fig. 7. the indirect immunofluorescence qualification result of recombinant virus rHVT5354VP2.
A:rHVT5354VP2
B:HVT
Embodiment
Material method
Material
HVT be FC126 strain purchased from China Veterinery Drug Inspection Office, IBDV HLJ-0504 strain, is provided by fowl transmissible disease research department of Harbin Veterinary Medicine Inst., China Academy of Agriculture.9～10 age in days SPF chicken embryos are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center.PCAGGs[20], pENTR (purchased from invitrogen company) plasmid preserved by this laboratory.Reverse transcription test kit, LR Clonase II enzyme, One Shot ccdBSurvival2T1R competent cells are all purchased from invitrogen company, phusion high-fidelity enzyme is purchased from NEB company, EcoRI, BglII, HindIII, SalI endonuclease are purchased from NEB company, CounterSelectionBAC modification kit is purchased from Gene Bridge company, and the high copy of EPI300-T1cells and fosmid thereof inductor is all purchased from Epicentre company.
Method
The extraction of 1HVT genomic dna
With 9-10 age in days SPF chicken embryo, carry out primary and inferior preparation and cultivation for chick embryo fibroblast (CEF) according to ordinary method.24h cell grows up to after individual layer, by 1 × 10
-4pFU/ cell inoculation HVT, after inoculation 72h, in the time that 80% pathology appears in cell, collects cell.Method as described in Morgan etc. is prepared viral DNA [16].
2HVT genome Fosmid library construction
The viral DNA random shearing of with physical method being prepared by aforesaid method is into about the fragment of 30-45Kb size.Glue reclaims the DNA fragmentation of 30-45Kb, reclaims product and carries out end modified.End modified reaction system (all reagent is all purchased from Takara company): 10 × End-Repair Buffer8ul, 2.5mM dNTP Mix8ul, 10mM ATP8ul, End-Repairenzyme Mix4ul, concentration is the shearing DNA20ug of 0.5ug/ul, and last sterilized water is supplied 80ul.Incubated at room 45 minutes, hatches 10 minutes deactivation End-Repair enzyme (Takara) for 70 DEG C.The refining DNA that has modified end, adds Sbf I joint.DNA fragmentation after reclaiming is connected on pCC1FOS (Epicentre) carrier, and 4 DEG C are spent the night.Connecting fluid is packed reaction, and packaged mixed liquid carries out 10 times of gradient dilutions, gets respectively 10
-2, 10
-4, 10
-5, 10
-6four dilution diluent 10 μ l infect 100 μ l EPI300 bacterial strains, and this bacterium is applied to the LB flat board containing 12.5 μ g/ml paraxin, 37 DEG C of incubated overnight, and statistics colony counts, and calculate its titre, result is 4.2x104cfu/lib.Successfully build the fosmid library of HVT.
The screening of 3 rescue rHVT virus clays
After library construction success, 288 clones of picking extract clay, use alkaline lysis method of extracting clay, send the precious biotech firm in Dalian to check order to the HVT DNA fragmentation end inserting in pCC1Fos, and sequencing primer sequence is as follows:
FP：5’-GGATGTGCTGCAAGGCGATTAAGTTGG-3’(SEQ?ID?NO：1)
RP：5’-CTCGTATGTTGTGTGGAATTGTGAGC-3’(SEQ?ID?NO：2)
Through the analysis of end sequencing, obtain altogether Insert Fragment two ends and be all connected with 246 of the clones of complete Fse I-Sbf I joint.From these 246 clones, choose many groups for saving the 5 clay combinations of HVT.Fse I-Sbf I joint is all contained at the HVT DNA fragmentation two ends of wherein cloning in every group, can be overlapped, and can splice the full HVT genome of covering.
Extract the DNA of selected clay with the middle amount extraction test kit of Qiagen company.Selected clay is carried out to linearization process with Fse I, Sbf I restriction endonuclease (all purchased from New England Biolabs), reaction conditions is as follows: Sbf I restriction endonuclease 20U, and clay 5 μ g, 37 DEG C act on 2 hours, the extracting of phenol/chloroform, isopropanol precipitating is prepared transfection HVT DNA.With reference to the calcium phosphate method of Reddy SM (2002) by 5 sections of HVT DNA cotransfections time for chick embryo fibroblast (CEF), through repeatedly repeating, wherein there is 1 group of 5 clay combination (called after pFosA respectively, pFosB, pFosC, pFosD, pFosE, its length and the position in genome are as shown in table 1) there is HVT virus typical cytopathic after transfection 6-8 days, gather in the crops this and organize the virus of 5 clay cotransfections rescues, called after rHVT.
Table 1
| Clay title | Base quantity | Position in genome |
| pFosA | 41319bp | 10162-51480 |
| pFosB | 32726bp | 39389-74114 |
| pFosC | 44754bp | 67199-111952 |
| pFosD | 39360bp | 108578-147937 |
?
| pFosE | 37970bp | 143007-21816 |
Note: reference sequences is: Meleagrid herpesvirus1, complete genome (159160bp), ncbi database is numbered: NC_002641
4 express the structure of infectious bursal disease virus VP 2 gene recombinant herpesvirus of turkeys
For the recombinant virus of construction expression infectious bursal disease virus VP 2 gene, first pcr amplification VP2 gene (sequence as shown in SEQ ID NO:3, corresponding VP2 Argine Monohydrochloride sequence is as shown in SEQ ID NO:4), amplimer is as follows:
VP2CU:ATG?GAA?TTC?GCC?GCC?ACC?ATG?ACA?AAC?CTG?CAA?GAT?CAA?ACC(SEQ?ID?NO：5)
VP2CD:GCG?AGA?TCT?TTA?CAG?CTA?TCC?TCC?TTA?AGG?CCC?GA(SEQ?ID?NO：6)
The VP2 gene and the PCAGGS plasmid that utilize EcoR I and Bgl II that amplification is obtained are connected to form PCAGG-VP2, secondly, utilize Sal I and Hind III that VP2 is expressed to framework Pec-VP2-POLYA cuts and is connected with pEntr MCS (Invitrogen) and form pEntr-VP2 (building process is shown in Fig. 1).
The structure of 5pFosC DEST
Choose the FosC clay that comprises HVT the 53rd and No. 54 genes as insertion objects, utilize the method for homologous recombination to be inserted into (HVT95318-95319) between its HVT comprising the 53rd and No. 54 genes attR1-Kan-ccdB-attR2 element and form pFosCDEST, the primer is as follows:
ET5354F:5’-cgcgattattatgaagtctacgcctctgtcctctctaatgcgatgagtaaCAGCTCT?GGGGATCATAAAAC-3’(SEQ?ID?NO：7)
ET5354R:5’-tggtgtgattggataaataaaacacagtaaccgttagaggtgcgtttttatCGTACC?AATTGTTGAGGTTCC-3’(SEQ?ID?NO：8)
Corresponding plasmid map is shown in Fig. 2.
The structure of 6pFosCVP2
PEntr-VP2 and pFosC DEST are blended in, under the effect of the LR of Invitrogen company recombinase, make VP2 express framework Pec-VP2-POLYA and be inserted into (particular location is between HVT genome 95318-95319 base) between the HVT the 53rd and No. 54 genes that pFosC clay comprises and form pFosCVP2 (referring to Fig. 3).
The rescue of 7 recombinant virus rHVT-5354VP2
With middle extraction reagent kit extraction pFosA, pFosB, pFosD, pFosE and five cosmid DNAs of Qiagen company.With Sbf I restriction endonuclease (purchased from New England Biolabs company), by clay linearization process used, its reaction conditions is as follows: Sbf I restriction endonuclease 60U, clay 10 μ g, 37 DEG C act on 2 hours, the extracting of phenol/chloroform, ethanol precipitation, preparation transfection HVT DNA.Method with reference to Reddy SM (2002) is rescued five clay cotransfections to obtain recombinant virus (Fig. 4 is shown in by rescue schematic diagram) for chick embryo fibroblast CEF respectively, the herpetoviridae α Herpesvirus herpes turkey virus rHVT-5354VP2Meleagrid herpesvirus1 obtaining has been preserved in Chinese Typical Representative culture collection center on July 11st, 2013, Wuhan University of Wuhan City of Hubei China province, preserving number is CCTCC NO:V201327.
The immunoblotting qualification of 8 recombinant virus rHVT-5354VP2
Ordinary method is prepared CEF cell, with 0.1MOI inoculation recombinant virus rHVT-5354VP2, in the time that appearring in 80% cell, pathology removes supernatant, harvested cell carries out SDS-PAGE electrophoresis, first use after wetted with methanol pvdf membrane (purchased from Millipore Corp.), with transferring film immersion vacuolar membrane and filter paper 10 minutes, carry out transfer printing by semidrying, transferring film condition is voltage 15V, 15 minutes.Wash film 3 times, each 5 minutes with PBS.Film is positioned in plate, add 5% skimming milk confining liquid, sway after 1 hour in 37 DEG C, add with confining liquid by the anti-IBDV serum of the chicken of the dilution proportion of 1:200 (fowl transmissible disease research department of Harbin Veterinary Medicine Inst., China Academy of Agriculture prepares and preserves) (as primary antibodie), room temperature is swayed 1 hour, wash film 3 times, each 5 minutes with PBST; Add with confining liquid by the anti-chicken igg antibody of the Infrared fluorescence mark of the dilution proportion of 1:5000 (purchased from Sigma company) (anti-as two), room temperature is swayed 1 hour, washes film 3 times, each 5 minutes with PBST; Finally add a little PBS, in Odyssey's scanner, take pictures.
The indirect immunofluorescence qualification of 9 recombinant virus rHVT-5354VP2
Ordinary method is prepared CEF cell, with 0.1MOI inoculation recombinant virus rHVT-5354VP2, in the time there is cytopathy, remove supernatant, wash 2 times with PBS, with 4% paraformaldehyde fixed cell 20 minutes under room temperature condition, PBS washes 3 times, adds and uses 1%BSA confining liquid by the anti-IBDV serum of the chicken of the dilution proportion of 1:200 (fowl transmissible disease research department of Harbin Veterinary Medicine Inst., China Academy of Agriculture prepares and preserves), and 37 DEG C act on 1 hour.Wash 5 times each 3 minutes with PBST.With the goat-anti chicken igg antibody (purchased from Millipore Corp.) of green fluorescence (FITC) mark 37 DEG C of effects 1 hour.Wash 5 times with PBS, at fluorescence microscopy Microscopic observation and take pictures.
10 experimentation on animalies
80 1 age in days SPF chickens (being provided by Harbin veterinary institute Animal House) are divided into 4 groups, 20 every group at random.I group and II group, respectively with 5000PFU dosage immunity rHVT-5354VP2; III group immunity HVT; IV group in contrast.After immunity, take a blood sample weekly and separation of serum.Immunity is rear attacks by collunarium and eyedripping way with Very virulent IBDV strain HLJ0504 for the 3rd week and the 5th week, attacks the rear death condition of observing of poison, attacks rear the cuing open for the 5th day of poison and kills the damage of observing the fabricius bursa.
Embodiment 1
The PCR qualification of recombinant virus rHVT-5354VP2
After recombinant virus is passed to 20 generations continuously, extract total DNA, with primer amplified VP2 gene, result as shown in Figure 5.Recombinant virus sample lane occurs that specific size is about the band of 1.4kb, and parent's strain HVT is without this band, illustrate thus VP2 gene can be in HVT genome stable existence.
Embodiment 2
The immunoblotting qualification of recombinant virus rHVT-5354VP2
After recombinant virus is passed to 20 generations continuously, extract total protein of cell, carry out westernblot, with the expression of detection of specific antibody VP2 gene, result as shown in Figure 6.Recombinant virus sample lane occurs that specific size is about 48ku object band, and parent's strain HVT and CEF swimming lane are without this band.Illustrate thus VP2 gene can be in HVT genome stable existence and express VP2 antigen.Embodiment 3
The indirect immunofluorescence qualification of recombinant virus rHVT-5354VP2
After recombinant virus is passed to 20 generations continuously, carry out immunofluorescence detection, with the expression of detection of specific antibody VP2 gene, result as shown in Figure 7.There is specific fluorescent signal in recombinant virus-infected cell, parent's strain HVT, without this signal, illustrates that VP2 gene can give full expression in the reproduction process of recombinant virus thus.
Embodiment 4
The immunoprotection that recombinant virus rHVT-5354VP2 induction produces
With rHVT-VP2 with 5000PFU dosage immunity 1 age in days SPF chicken (being provided by Harbin veterinary institute Animal House).Immunity is rear attacks with Very virulent IBDV strain HLJ-0504 for the 3rd week and the 5th week, attacks the rear death condition of observing of poison, attacks rear the cuing open for the 5th day of poison and kills the damage of observing the fabricius bursa.Result shows that rHVT-VP2 immune group chicken attacks after poison 100% survival and do not occur fabricius bursa damage, and HVT immune group and control group chicken are all dead and occur fabricius bursa atrophys (in table 2) in various degree.
Table 2 is attacked malicious Protection result
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Claims (10)
1. express the recombinant herpesvirus of turkeys (HVT) of Infectious bursal disease virus VP2 for one kind, it is characterized in that, the encoding gene of described VP2 albumen inserts the genomic Nonessencial region of HVT, the encoding gene of described VP2 albumen is as shown in SEQ ID NO.3, and corresponding aminoacid sequence is as shown in SEQID NO.4.
2. recombinant herpesvirus of turkeys as claimed in claim 1, wherein, described Nonessencial region is between HVT genome HVT053 and HVT054.
3. recombinant herpesvirus of turkeys as claimed in claim 1 or 2, wherein, the encoding gene of described VP2 albumen is in strong promoter downstream.
4. recombinant herpesvirus of turkeys as claimed in claim 3, wherein, described promotor can be combined promoter, preferably Pec promotor, described Pec promotor comprises a cmv enhancer and β-actin promotor.
5. recombinant herpesvirus of turkeys (Meleagrid herpesvirus1) rHVT-5354VP2 who expresses Infectious bursal disease virus VP2, it is preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCC NO:V201327.
6. a method for the recombinant herpesvirus of turkeys of construction expression Infectious bursal disease virus VP2, comprises the following steps:
1) from the genomic Fosmid of HVT library, filter out and can save viral clay group by cotransfection;
2) in a clay of described clay group, introduce the encoding gene of VP2 albumen;
3) rescue recombinant virus.
7. method as claimed in claim 6, wherein, the whole HVT genome of the common covering of the clay of described clay group.
8. the method as described in claim 6 or 7, wherein, the encoding gene of described introducing VP2 albumen, for using homologous recombination to introduce, preferably uses Red ET homologous recombination technique and Gateway LR clone technology.
9. the method as described in claim 6 or 7, wherein, the clay of introducing the encoding gene of VP2 albumen has the genomic Nonessencial region of HVT.
10. the recombinant virus that described in the recombinant virus described in claim 1-5 any one and/or claim 6-9 any one prepared by method is for the preparation of the purposes of infection prevention bursal disease vaccine.
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| CN105695423A (en) * | 2016-03-15 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Recombined chicken Marek's disease virus vaccine strain for expressing infectious bursaldisease virus VP2 gene and construction method and application of recombined chicken Marek's disease virus vaccine strain |
| CN105695422A (en) * | 2016-03-15 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain |
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| CN114107227A (en) * | 2021-11-04 | 2022-03-01 | 扬州优邦生物药品有限公司 | Recombinant turkey herpesvirus live vector vaccine for simultaneously expressing classical strain infectious bursal disease virus VP2 protein and variant strain infectious bursal disease virus VP2 protein |
| CN116083375A (en) * | 2022-08-30 | 2023-05-09 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant turkey herpesvirus expressing infectious bursal disease virus VP2 gene, construction method and application thereof |
| CN117551699A (en) * | 2023-12-28 | 2024-02-13 | 天津瑞普生物技术股份有限公司 | Construction method of recombinant turkey herpesvirus rHVT-HA-VP2 |
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| CN105695422A (en) * | 2016-03-15 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Recombined chicken Marek's disease virus vaccine strain for expressing Gag and Env genes of avian leukosis virus subgroup J and construction method and application of recombined chicken Marek's disease virus vaccine strain |
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| CN111909965A (en) * | 2020-08-06 | 2020-11-10 | 中国兽医药品监察所 | Construction and application of recombinant virus vector for expressing infectious bursal disease virus VP2 protein |
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