CN114107227B - 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 - Google Patents

Abstract

The invention discloses a recombinant turkey herpesvirus live vector vaccine simultaneously expressing classical strain infectious bursal disease virus VP2 protein and a variant strain infectious bursal disease virus live vector vaccine, belonging to the field of biological products for livestock. The invention knocks the VP2 protein and LTB fusion expression cassette of the infectious bursal disease virus classical strain into the non-essential region for replication of the US2 virus of the herpesvirus of turkeys, and inserts the VP2 protein and LTB fusion expression cassette of the variant strain into the non-essential region for replication of the US10 virus. The finally obtained recombinant virus can simultaneously and efficiently express LTB-VP2 fusion antigen proteins of IBDV classical strains and variant strains in CEF cells. The vaccine prepared by the invention can improve the antibody level after immunization, improve the uniformity of the antibody after immunization and ensure the immune effect of the vaccine, has the advantages of high efficiency, good safety and lifelong immunization by one-time inoculation, does not cause clinical reaction and pathological damage to chicks, and has the vaccine protection rate of 100 percent.

Description

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

Technical Field

The invention relates to a recombinant turkey herpesvirus live vector vaccine simultaneously expressing classical strain infectious bursal disease virus VP2 protein and belongs to the field of biological products for livestock.

Background

Infectious Bursal Disease Virus (IBDV) mainly infects chicks of 3-6 weeks old, and the infection causes severe damage to immune organs of the chicks, so that the body generates immunosuppression, and finally the chicks die. The high mortality rate caused by Infectious Bursal Disease (IBD) causes serious economic loss in the poultry industry all over the world, and is classified as three Infectious diseases which are harmful to the health of poultry together with newcastle Disease and Marek's Disease of chickens.

There are two serotypes of IBDV, with IBDV being induced predominantly by serotype I. The serotype I IBDV is divided into 4 branches of classical strains, superstrong strains, attenuated strains and variant strains. The risk of atypical IBD caused by the novel IBDV variant is not negligible. Although the mortality rate of the IBDV novel variant strain is low, the immunity of chicken flocks is low due to irreversible bursal injury and severe immunosuppression caused by the bursal injury, the infection probability of other pathogens is increased, the growth of the chicken flocks is influenced, the weight is reduced, the uniformity is poor, the feed conversion ratio is increased, and the like, so that the economic benefit is seriously influenced. On the other hand, the novel IBDV variant will interfere with the immune efficacy of other important vaccines against diseases. It has been reported that the antibody production of the H5/H7 bivalent vaccine against avian influenza is interfered by the infection with the novel IBDV variant. The new IBDV variant strain in China belongs to the IBDV novel variant strain.

There are four viral proteins of serotype I IBDV, VP1, VP2, VP3 and VP4, 4 of which have molecular weights of about 90-92KD, 41-48KD, 32KD and 28-32 KD. Research shows that different monoclonal antibody reaction spectrums exist between the novel IBDV variant strain and the superstrong strain vvIBDV, amino acid mutations at positions 318 and 323 of VP2 are one of the key factors for the novel IBDV variant strain to escape the neutralizing activity of vvIBDV antibody, and chicken flocks of vaccines for partially immunizing vvIBDV still infect the novel IBDV variant strain, which is an important reason for causing the novel IBDV variant strain to be popularized nationwide.

VP2 is the major structural protein of IBDV and is also the major protective antigen. The two hydrophobic groups of the VP2 protein variable region endow IBDV conformation dependence and high hydrophobicity, can induce a host organism to produce neutralizing antibodies, and is also related to the cell tropism, virus toxicity and the like of viruses. The VP2 protein has virulence related epitopes located in the variable region, and amino acids 253, 279 and 284 are reported to be key sites for affecting IBDV virulence. Therefore, how to ensure that the antibody generated by the VP 2-stimulated organism can resist different epidemic strains becomes a challenge to be solved.

Heat-labile enterotoxin (LT) of escherichia coli and Cholera Toxin (CT) of vibrio cholerae are known to be potent mucosal immunoadjuvants, and the B subunit of LT recognizes mainly the receptor ganglioside GM1 of the intestinal mucosal epithelial cell membrane and binds to it to form a complex, which is not toxic per se. Therefore, LTB shows high activity and nontoxicity in the research of being used as a mucosal adjuvant and is widely concerned, and the LTB can effectively promote and improve the specific immune response of organisms. The patent No. CN102688487A is to fuse and express the VP2 protein of chicken IBDV super-virulent virus and the colibacillus LTB protein with mucosal immune adjuvant, and the expressed product is emulsified to prepare the vaccine, so that each vaccine immune group can resist the attack of the virulent virus and has good immunogenicity.

Turkey Herpesvirus (HVT), an alpha herpes virus of Marek's Disease (MDV), is widely used as a live vaccine for preventing Marek's Disease (MD) because of its antigenic association with MDV and because HVT is clearly distinct from MDV and is not pathogenic to chickens. Therefore, how to study the live vaccine of Infectious Bursal Disease (IBD) of chicken by taking Herpes Virus of Turkeys (HVT) as a carrier becomes an important research direction.

Vaccination is one of the main measures to prevent, control and even eliminate infectious bursal disease in chickens. The VP2 protein is expressed by using herpesvirus of turkeys as a vector. The safety is better, the whole life immunity is realized by one-time inoculation, the generated immune response can be distinguished from wild virus infection, and the control and elimination of epidemic diseases are facilitated.

Therefore, the production method for developing the infectious bursal disease virus recombinant turkey herpesvirus live vector vaccine with low production cost, high production efficiency and good vaccine immunization effect has important practical significance.

Disclosure of Invention

The invention aims to provide a recombinant turkey herpesvirus live vector vaccine capable of expressing infectious bursal disease virus classical strain and variant strain VP2 protein simultaneously. The provided vaccine has the advantages of high efficiency, good safety, high antibody uniformity, high protection rate and long immune duration, thereby making up the defects of the prior art.

The first purpose of the invention is to provide a recombinant turkey herpesvirus which simultaneously expresses VP2 gene expression cassettes of an infectious bursal disease virus classical strain and a variant strain.

In one embodiment, the IBDV classical strain VP2 gene expression cassette comprises, in order, a promoter, an LTB gene, an IBDV classical strain VP2 gene and a terminator; the variant strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV variant strain VP2 gene and a terminator.

In one embodiment, the IBDV classical strain VP2 gene expression cassette and the variant strain VP2 gene expression cassette are located in a replication non-essential region of the herpesvirus turkeys, respectively.

In one embodiment, the classical strain VP2 gene expression cassette is located in US2 of turkey herpesvirus, the variant strain VP2 gene expression cassette is located in US10 of turkey herpesvirus, or the classical strain VP2 gene expression cassette is located in US10 of turkey herpesvirus, and the variant strain VP2 gene expression cassette is located in US2 of turkey herpesvirus.

In one embodiment, the nucleotide sequence of the VP2 gene of the IBDV classical strain is shown as SEQ ID NO.2, and the nucleotide sequence of the VP2 gene of the IBDV variant is shown as SEQ ID NO. 4; the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5.

In one embodiment, the promoter comprises the pec promoter and the terminator is a terminator of herpesvirus turkeys.

In one embodiment, the nucleotide sequence of the pec promoter is as shown in SEQ ID NO. 6.

In one embodiment, the amino acid sequence of the classical strain VP2 protein is shown in SEQ ID NO.1, and the amino acid sequence of the variant strain VP2 protein is shown in SEQ ID NO. 3.

In one embodiment, the herpesvirus of turkeys is strain HVT FC 126.

The second purpose of the invention is to provide a preparation method of the recombinant herpesvirus of turkeys, which is to insert an IBDV classical strain VP2 gene expression cassette and a variant strain VP2 gene expression cassette into replication nonessential regions of herpesvirus of turkeys respectively.

In one embodiment, the IBDV classical strain VP2 gene expression cassette comprises, in order, a promoter, an LTB gene, an IBDV classical strain VP2 gene, and a terminator; the variant strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV variant strain VP2 gene and a terminator.

In one embodiment, the IBDV classical strain VP2 gene expression cassette is inserted into the US2 region of herpesvirus turkey, and the IBDV variant strain VP2 gene expression cassette is inserted into the US10 region of herpesvirus turkey;

or, the IBDV classical strain VP2 gene expression cassette is inserted into the US10 region of the turkey herpesvirus, and the IBDV variant strain VP2 gene expression cassette is inserted into the US2 region of the turkey herpesvirus.

In one embodiment, the promoter comprises the pec promoter and the terminator is a terminator of herpesvirus turkeys.

In one embodiment, the nucleotide sequence of the pec promoter is as shown in SEQ ID NO. 6.

In one embodiment, the nucleotide sequence of the VP2 gene of the IBDV classical strain is shown as SEQ ID NO.2, and the nucleotide sequence of the VP2 gene of the IBDV variant is shown as SEQ ID NO. 4; the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5.

In one embodiment, the method comprises the following specific steps:

(1) an expression cassette consisting of a green fluorescent protein (EGFP) gene sequence and a pec promoter and about homologous arms of US2 are cloned to a transfer plasmid to obtain a recombinant plasmid pB-LR-US 2-EGFP;

(2) rescuing viruses, co-transforming the recombinant plasmid in the step (1) and herpesvirus of turkeys genome into host cells to obtain recombinant virus rHVT-EGFP;

(3) a VP2 gene sequence of an infectious bursal disease virus classical strain with an upstream connected with LTB gene and a pec promoter form an expression cassette, and homologous arms around US2 are cloned to a transfer plasmid to obtain a recombinant plasmid pB-LR-US2-LTB-VP 2C;

(4) rescuing viruses, and co-transforming the recombinant plasmid and the recombinant virus rHVT-EGFP genome in the step (3) into host cells to obtain a recombinant virus rHVT-US 2-EGFP;

(5) an expression cassette consisting of a green fluorescent protein (EGFP) gene sequence and a pec promoter and about homologous arms of US10 are cloned to a transfer plasmid to obtain pB-LR-US 10-EGFP;

(6) rescuing viruses, and co-transforming the recombinant plasmid and the recombinant virus rHVT-US2-EGFP genome in the step (5) into host cells to obtain the recombinant virus rHVT-US 10-EGFP;

(7) cloning a VP2 gene sequence of an infectious bursal disease variant with an upstream connected LTB gene and a pec promoter to transfer plasmids with about US10 homologous arms to obtain pB-LR-US10-LTB-VP 2V;

(8) and (3) rescuing the virus, and co-transforming the recombinant plasmid obtained in the step (7) and the recombinant virus rHVT-US10-EGFP genome into a host cell to obtain the recombinant virus rHVT-US2-US10-LTB-VP2C-VP 2V.

In one embodiment, the resulting recombinant virus is purified.

In one embodiment, the sequence of the herpesvirus of turkeys genome is accession number "AF 291866.1" of NCBI.

In one embodiment, the transfer plasmid comprises pBluescript ii KS.

In one embodiment, the host cell comprises a chicken embryo fibroblast.

The third purpose of the invention is to provide a recombinant live vector vaccine of herpesvirus of turkeys, which expresses VP2 gene expression cassettes of both classical and variant strains of infectious bursal disease virus.

In one embodiment, the IBDV classical strain VP2 gene expression cassette comprises, in order, a promoter, an LTB gene, an IBDV classical strain VP2 gene, and a terminator; the variant strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV variant strain VP2 gene and a terminator.

In one embodiment, the nucleotide sequence of the VP2 gene of the IBDV classical strain is shown as SEQ ID NO.2, and the nucleotide sequence of the VP2 gene of the IBDV variant is shown as SEQ ID NO. 4; the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5.

In one embodiment, the promoter comprises the pec promoter and the terminator is a terminator of herpesvirus turkeys.

In one embodiment, the nucleotide sequence of the pec promoter is as shown in SEQ ID NO. 6.

In one embodiment, the expression cassette of the IBDV classical strain VP2 gene and the expression cassette of the variant strain VP2 gene are located in a replication non-essential region of the herpesvirus of turkeys, respectively.

In one embodiment, the classical strain VP2 gene expression cassette is located in US2 of turkey herpesvirus and the variant strain VP2 gene expression cassette is located in US10 of turkey herpesvirus;

or the classical strain VP2 gene expression cassette is positioned in US10 of the herpesvirus of turkeys, and the variant strain VP2 gene expression cassette is positioned in US2 of the herpesvirus of turkeys.

In one embodiment, the herpesvirus of turkeys is strain HVT FC 126.

In one embodiment, the vaccine contains recombinant herpesvirus of turkeys in an amount of greater than or equal to 2.5X 10 ⁵ PFU _/ ml。

In one embodiment, the adjuvant in the recombinant herpesvirus of turkeys live vector vaccine is an adjuvant for lyophilization of skim milk.

The invention also protects the application of the recombinant herpesvirus of turkeys or the preparation method in preparation of infectious bursal disease virus vaccines.

Has the advantages that:

(1) the expressed infectious bursal disease virus recombinant turkey herpesvirus live vector vaccine selects a promoter which is a pec composite promoter and comprises enhancer combined CMV, a selected terminator is a virus terminator and no exogenous sequence is introduced, and LTB fusion expression is selected as a vaccine active adjuvant.

(2) The invention prepares the vaccine by inoculating recombinant virus rHVT-IBDV-US2-US10-LTB-VP2C-VP2V into CEF cells for amplification culture and then preparing the vaccine by using a common freeze-drying protective agent. The vaccine prepared by the invention can improve the antibody level after immunization, improve the uniformity of the antibody after immunization and ensure the immune effect of the vaccine, has the advantages of high efficiency, good safety and lifelong immunization by one-time inoculation, does not cause clinical reaction and pathological damage to chicks, can protect organisms from being attacked by classical strains and variant strains of infectious bursal disease virus to a certain extent by 3 batches of vaccine passive immunization groups, and has the vaccine protection rate of 100 percent.

Drawings

FIG. 1 is a photograph of lesions of herpesvirus turkeys inoculated CEF cells; a: HVT field virus inoculation control; b: the recombinant rHVT-US2-US10-VP2C-VP2V inoculated lesion picture; c: and (5) negative control.

FIG. 2 is a PCR identification of recombinant turkey herpesvirus; m: DL5000 DNA Marker; a: recombinant identification of US 2; b, carrying out recombination identification on US 10; 1: HVT wild virus; 2: recombinant virus rHVT-US2-EGFP containing marker gene; 3: recombinant virus rHVT-US2-US10-VP2C-VP 2V; 4: negative control; 5: HVT wild virus; 6: recombinant virus rHVT-US10-EGFP containing marker gene; 7: recombinant virus rHVT-US2-US10-VP2C-VP 2V; 8: and (5) negative control.

FIG. 3 shows SDS-PAGE detection of recombinant herpesvirus of turkeys expression products; m: pre-dyeing a protein Marker; 1: negative cell control; 2: HVT wild virus control; 3: recombinant virus rHVT-IBDV-US2-US10-LTB-VP2C-VP 2V.

FIG. 4 is a Western Blot to identify rHVT-IBDV-US2-US10-VP2C-VP2V recombinant virus expression products; a, detecting the activity of VP 2C; b, detecting the activity of VP 2V; m: pre-dyeing a protein Marker; 1: a negative cell; 2: HVT wild virus control; 3: recombinant virus rHVT-US2-US10-VP2C-VP 2V.

FIG. 5 is an IBDV antibody response induced after immunization of SPF chickens with recombinant HVT virus; a is the reaction result of classical strain IBDV antibody; b, variant antibody reaction results.

Detailed Description

Experimental materials referred to in the following examples:

strains, strains and plasmids

Turkey herpesvirus: donation by Liaoning Yikang bionts GmbH, NCBI accession number: AF 291866.1.

The classical strain BC6/85 of infectious bursal disease virus was purchased from the institute of veterinary medicine in China and preserved by Youbang biopharmaceutical, Inc., Youbang, Yangzhou.

A variant strain SN18 of infectious bursal disease Virus was isolated and stored by Yobang, Yoppon, Yoghuba, Youngs, Yoghuba.

Chicken fibroblasts: CEF cells, purchased from ATCC, were maintained by Yobang, Youbang, Yangzhou.

Construction of plasmids reference is made to the molecular cloning guide (fourth edition)

Example 1: determination of infectious bursal disease Virus VP2 Gene

The VP2 gene of the classical strain of the infectious bursal disease virus is a subunit vaccine related sequence produced by the company, is fused with LTB by using molecular software through a Kinry online molecular biology software, takes a chicken source as a host, optimizes and obtains a nucleotide sequence and is named as LTB-VP 2C. The gene sequence of VP2 protein of infectious bursal disease virus variant is obtained by separating virus by the technical service department of the company, sequencing and NCBI comparison, and an online codon optimization tool is also adopted, and a nucleotide sequence is obtained by optimizing by taking chicken as a host, and is named as LTB-VP 2V.

Example 2: preparation of recombinant Virus rHVT-US2-VP2C

1. Extracting herpesvirus of turkeys genome: extracting total DNA of herpesvirus of turkeys by a conventional DNA extraction method (NCBI accession number: AF 291866.1);

2. design and synthesis of homologous recombination homologous arm transfer vector: the left and right homology arms are designed according to the published gene sequence of the turkey herpesvirus US2 in GenBank with 1200bp each, and a pec-EGFP expression cassette (consisting of pec promoter, EGFP gene and US2 self terminator) and pec-VP2C (consisting of pec promoter, LTB-VP2C gene and US2 self terminator) are introduced in the middle of the homology arms and synthesized into pBluescript II KS by Nanjing Smiry Biotech Ltd. The recombinant plasmids are named pB-LR-US2-EGFP and pB-LR-US2-LTB-VP 2C;

3. rescue of recombinant turkey herpesvirus: the transfer vector pB-LR-US2-EGFP and the herpesvirus of turkeys genome obtained in step 1 were co-transfected into CEF cells, and the specific operation method was performed with reference to the instruction of calcium phosphate transfection of Thermo Fisher, to obtain recombinant herpesvirus of turkeys rHVT-EGFP generation F1.

4. Purifying the recombinant turkey herpesvirus: marking the fluorescence region of the recombinant virus plaque obtained by transfection, selecting and purifying, namely selecting the marked pathological cells, inoculating fresh secondary CEF cells, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator for 5-7 days. Repeated screening and purification for 4-5 rounds until all plaques show fluorescence, and identification by US2 recombinantThe primers were identified by PCR to confirm that the fully purified recombinant virus rHVT-US2-EGFP was obtained (FIG. 2).

The sequence of the US2 recombination identification primer is as follows:

P1:5’-ACGCAGGTATCATAGGGGTAAT-3’；

P2:5’-TGGTATCGAGTCCACATGCA-3’。

5. extracting herpesvirus of turkeys genome: extracting recombinant herpesvirus of turkeys rHVT-US2-EGFP total DNA containing marker gene by traditional method;

6. rescue of recombinant turkey herpesvirus: the CEF cells were co-transfected with the transfer vector pB-LR-US2-LTB-VP2C and the recombinant herpesvirus of turkeys genome obtained in step 5, and the specific procedures were performed according to the instruction of calcium phosphate transfection of Thermo Fisher, to obtain recombinant herpesvirus of turkeys rHVT-US2-LTB-VP2C of F1 generation.

7. Purifying the recombinant turkey herpesvirus: marking the non-fluorescent region of the plaque of the transfected recombinant virus in the hole of the transfected recombinant virus, and performing plate picking and purification, namely picking the marked pathological change cells, inoculating fresh secondary CEF cells again, placing the cells at 37 ℃ and 5% CO ₂ Culturing in an incubator for 5-7 days. And repeatedly screening and purifying for 4-5 rounds until all plaques do not show fluorescence, and identifying by using a recombinant identification primer of US2 through PCR (polymerase chain reaction) to confirm to obtain the recombinant virus rHVT-US2-LTB-VP 2C.

Example 3: preparation of recombinant Virus rHVT-US2-US10-LTB-VP2C-VP2V

1. Extracting herpesvirus of turkeys genome: extracting the recombinant virus rHVT-US2-LTB-VP2C total DNA in example 2 by using a traditional DNA extraction method;

2. design and synthesis of homologous recombination homologous arm transfer vector: the right and left homology arms are designed according to the published gene sequence of herpesvirus of turkeys US10 in GenBank with 1200bp each, and a pec-EGFP expression cassette (consisting of pec promoter, EGFP gene and US10 self terminator) and pec-LTB-VP2V (consisting of pec promoter, LTB-VP2V gene and US10 self terminator) are introduced in the middle of the homology arms and synthesized into pBluescript II KS by Nanjing Jinsrie Biotech. The recombinant plasmids are named pB-LR-US10-EGFP and pB-LR-US10-LTB-VP 2V;

3. rescue of recombinant turkey herpesvirus: the transfer vector pB-LR-US10-EGFP and the herpesvirus of turkeys genome obtained in step 1 were co-transfected into CEF cells, and the specific operation method was performed with reference to the instruction of calcium phosphate transfection of Thermo Fisher company, to obtain recombinant herpesvirus of turkeys rHVT-US10-EGFP of generation F1.

4. Purifying the recombinant turkey herpesvirus: marking the fluorescence region of the recombinant virus plaque obtained by transfection, selecting and purifying, namely selecting the marked pathological cells, inoculating fresh secondary CEF cells, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator for 5-7 days. And repeatedly screening and purifying for 4-5 rounds until all plaques show fluorescence, and identifying by using a US10 recombinant identification primer through PCR (polymerase chain reaction) to confirm to obtain the recombinant virus rHVT-US 10-EGFP.

The sequence of the US10 recombination identification primer is as follows:

p3:5’-CAGCTCGCCGATCATATGG-3’；

P4:5’-TGTAACATCAGAGCGTGCCT-3’。

5. extracting herpesvirus of turkeys genome: extracting recombinant herpesvirus of turkeys rHVT-US10-EGFP total DNA containing marker gene by traditional method;

6. rescue of recombinant turkey herpesvirus: the CEF cells were co-transfected with the transfer vector pB-LR-US10-LTB-VP2V and the recombinant herpesvirus of turkeys genome obtained in step 5, and the specific procedures were performed according to the instruction of calcium phosphate transfection of Thermo Fisher, to obtain recombinant herpesvirus of turkeys rHVT-US2-US10-LTB-VP2C-VP2V of the F1 generation.

7. Purifying the recombinant turkey herpesvirus: marking the non-fluorescent region of the recombinant virus plaque obtained by transfection, selecting and purifying, namely selecting the marked pathological cells, inoculating fresh secondary CEF cells, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator for 5-7 days. And repeatedly screening and purifying for 4-5 rounds until all plaques show no fluorescence, and identifying by PCR (polymerase chain reaction) by using a recombinant identification primer of US10 to obtain the recombinant virus rHVT-US2-US10-LTB-VP2C-VP 2V.

The viral pathogram of recombinant virus rHVT-US2-US10-LTB-VP2C-VP2V and HVT wild virus infected CEF cell is shown in figure 1.

Example 4: preparation of recombinant Virus rHVT-US2-US10-LTB-VP2C-VP2V

1. And (3) identifying the expressed protein:

(1) the recombinant virus obtained in example 3, rHVT-US2-US10-LTB-VP2C-VP2V, was inoculated into CEF cells at 37 ℃ with 5% CO ₂ Culturing for 3-7 days, collecting culture, crushing, centrifuging, and collecting supernatant to obtain LTB-VP2C and LTB-VP2V proteins;

(2) SDS-PAGE identification: performing SDS-PAGE electrophoresis on the supernatant; after the electrophoresis was finished, it was found that the molecular weight was in agreement with the theoretical size at about the 45kDa position by staining and destaining, indicating successful expression (FIG. 3).

(3) Western Blot identification: taking the gel after SDS-PAGE electrophoresis, directly transferring the gel to an NC membrane by using a BIO-LAB transfer printing device, and after the transfer printing is finished, carrying out Western blot identification according to a conventional method. Using a chicken infectious bursal disease virus positive serum reference substance (1:200) as a primary antibody; rabbit anti-chicken IgG (1:2000) marked by horseradish peroxidase is used as an enzyme-labeled secondary antibody; finally, TMB was used for color development (Biyuntian Biotech institute). The result shows that 1 obvious specific band appears at 45kDa, while the negative control has no specific reaction, which indicates that the recombinant protein can be recognized by the antibody in the positive serum of the infectious bursal virus and has good specificity and reactogenicity (figure 4).

(4) Indirect immunofluorescence assay (IFA): recombinant virus rHVT-US2-US10-LTB-VP2C-VP2V was inoculated to a single layer of CEF cells, cultured until the cytopathic effect (CPE) reached 80%, the cells were washed with PBS, fixed with cold 4% formaldehyde, primary antibody with chicken anti-IBDV positive serum (1: 100) and secondary antibody with rabbit anti-chicken IgG-FITC (1: 500), observed under a fluorescent microscope, and HVT-infected CEF cells were used as a negative control.

Example 5: vaccine preparation

1. Harvesting of the recombinant virus: the recombinant virus obtained in example 3, rHVT-US2-US10-LTB-VP2C-VP2V, was inoculated to CEF cells at 37 ℃ with 5% CO ₂ Culturing for 3-5 days, removing culture medium, adding appropriate amount of pancreatin-EDTA digestive juice, and immediately adding 10% bovine blood equivalent to digestive juice when CEF cell monolayer is loose and net-pulled to close to the falling bottle wallClear culture medium, stop digestion. Collecting cells in a centrifuge tube, centrifuging at 2000rpm/min for 10min, discarding supernatant, and collecting cell precipitate as recombinant virus culture.

2. Inspection of semi-finished product

(1) And (4) sterile inspection: sterility test was performed according to the appendix of the current "Chinese veterinary pharmacopoeia".

(2) And (3) virus content determination: detecting virus content by plaque method, wherein the virus content is ≧ 2.5 × 10 ⁵ PFU/ml。

3. Preparation of live vector vaccine:

and adding a proper amount of SPGA stabilizer into the cell sediment of the semi-finished product after the semi-finished product is qualified through inspection, cracking the cell sediment by using an ultrasonic cracker, shaking the cell sediment uniformly, filtering the cell sediment by using two layers of gauze, subpackaging the obtained product in small bottles, and immediately performing freeze vacuum drying to obtain the recombinant virus vaccine rHVT-US2-US10-LTB-VP2C-VP 2V.

Example 6: vaccine product inspection

1. Traits

Appearance: the vaccine should be cheese sponge-like and the outer package should be qualified;

the preparation formulation is as follows: and (4) powder preparation. (supplementary explanation).

2. And (4) checking the loading quantity: according to the appendix of the current Chinese animal pharmacopoeia, the prescription is met.

3. And (4) sterile inspection: according to the appendix of the current Chinese animal pharmacopoeia, the prescription is met.

4. And (4) safety inspection:

4.1 Experimental methods

4.1.1 rHVT-US2-US10-LTB-VP2C-VP2V safety test of single-dose vaccination of vaccine

40 experimental chickens at 1 day of age were randomly divided into 2 groups of 20 chickens. Wherein group 1 experimental chickens were intraperitoneally inoculated with the recombinant virus vaccine rHVT-US2-US10-LTB-VP2C-VP2V obtained in example 5 at a dose of 2000 Plaque Forming Units (PFU)/one (1 wing); group 2 experimental chickens were vaccinated with the vaccine diluent as a blank control. The groups of chickens were observed daily for clinical symptoms after inoculation. And (3) randomly selecting 5 experimental chickens in each group 2, 4, 8 and 12 weeks after inoculation, weighing the weight, and evaluating the influence of the recombinant virus vaccine on the growth and development of the experimental chickens. The 5 selected chickens in each group were killed, and organs such as bursa of Fabricius, thymus, spleen, liver, glandular stomach and the like were collected, weighed, and observed for the presence of atrophy or swelling symptoms. The collected tissues were observed for pathological changes and examined histopathologically. Extracting the DNA of the organ tissues, and detecting the distribution and proliferation condition of the vaccine strain viruses in the tissues by using a fluorescent quantitative PCR method.

Safety experiments for Single dose repeat Vaccination of 4.1.2 rHVT-US2-US10-LTB-VP2C-VP2V vaccine

40 experimental chickens at 1 day of age were randomly divided into 2 groups of 20 chickens. Wherein the experimental chicken of group 1 is subcutaneously inoculated with recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V at the neck part with the immune dose of 2000 PFU/chicken; group 2 experimental chickens were vaccinated with the diluent as a blank control and 2 weeks after immunization were repeated with the same method and dose. The clinical symptoms of 2 groups of chickens were observed daily after inoculation. And (3) randomly selecting 5 experimental chickens in each group 2, 4, 8 and 12 weeks after inoculation, weighing the weight, and evaluating the influence of the recombinant virus vaccine on the growth and development of the experimental chickens. The 5 selected chickens in each group were killed, and organs such as bursa of Fabricius, thymus, spleen, liver, glandular stomach and the like were collected, weighed, and observed for the presence of atrophy or swelling symptoms. The collected tissues were observed for gross lesions and examined for histopathology.

4.1.3 rHVT-US2-US10-LTB-VP2C-VP2V vaccine safety experiment for large dose vaccination

40 experimental chickens at 1 day of age were randomly divided into 2 groups of 20 chickens. Wherein the experimental chickens of group 1 were intraperitoneally inoculated with the recombinant virus vaccine rHVT-US2-US10-LTB-VP2C-VP2V at 20000 PFUs per maximum dose (equivalent to 10 immunization doses); group 2 experimental chickens were vaccinated with dilutions as a blank control. The groups of chickens were observed daily for clinical symptoms after inoculation. And randomly selecting 5 experimental chickens in each group 2, 4, 8 and 12 weeks after inoculation, weighing the weights, and evaluating the influence of the recombinant virus vaccine on the growth and development of the experimental chickens. The 5 chickens selected from each group were killed, collected in the bursa of Fabricius, thymus, spleen, liver, glandular stomach and other organs, weighed, and observed for atrophy or swelling. The collected tissues were observed for gross lesions and examined for histopathology.

4.2 results of the experiment

4.2.1 rHVT-IBDV-US2-US10-LTB-VP2C-VP2V vaccine Single dose safety experiment for the best vaccination

After the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is inoculated to experimental chickens of 1 day old at 2000 PFU/immunization dose, the experimental chickens have good mental status, good drinking and eating status, normal weight development and no clinical symptoms. At 2, 4, 8 and 12 weeks after immunization, the breeding hens were dissected and counted for major organ indices (bursa of fabricius, thymus, liver, spleen and glandular stomach). The results show that after the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is used for immunization, the organs of experimental chickens develop normally without phenomena of atrophy and swelling. Histopathological detection is carried out on the collected tissues, and the result shows that after the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is immunized, the outline of bursa of Fabricius of the experimental chicken is clear, lymphocytes are not reduced, and no obvious pathological change exists; other tissues are also intact and have no obvious pathological changes. The genome DNA of the tissues is extracted and subjected to fluorescent quantitative PCR, and the result shows that the vaccine strain virus rHVT-US2-US10-LTB-VP2C-VP2V is distributed in the bursa of Fabricius, thymus, liver, spleen and glandular stomach of the immunized chicken, and the virus load has no significant difference among the tissues. The result shows that the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is not pathogenic to chicken and is safe to chicken.

4.2.2 analysis of the safety of chickens with a Single dose of repeated Vaccination of rHVT-IBDV-US2-US10-LTB-VP2C-VP2V vaccine

Recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V was inoculated with 2000 PFU/immunization dose 1 day old laboratory chicken and then 2 weeks after inoculation with the same dose and route for repeat inoculation. After repeated inoculation, the experimental chicken has good mental status, normal drinking and eating, normal weight development and no clinical symptoms. At 2, 4, 8 and 12 weeks after immunization, the breeding hens are subjected to cesarean examination, the ratio of the main organs (bursa of fabricius, thymus, liver, spleen and glandular stomach) to the body weight is counted, and the organ body weight ratio is calculated to evaluate whether each organ has atrophy or swelling. The result shows that after the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is repeatedly immunized, all organs of the experimental chicken develop normally, and have no obvious difference with a control group, and no phenomena of atrophy and swelling exist. Histopathological detection is carried out on the collected tissues, and the result shows that after the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is repeatedly immunized, the outline of bursa of Fabricius of the experimental chicken is clear, lymphocytes are not reduced, and no obvious pathological change exists; other tissues are also intact and have no obvious pathological changes. The results show that the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is safe to target animals, and has no pathogenicity to target animals.

4.2.3 safety analysis of chickens by high dose vaccination with rHVT-IBDV-US2-US10-LTB-VP2C-VP2V vaccine

To further evaluate the toxicological response of the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V to target chickens, experimental chickens were immunized with the vaccine strain virus at 20000 PFUs per maximal dose (equivalent to 10 immunization doses). The results show that after the inoculation of large dose, the experimental chicken does not have any adverse clinical reaction, the drinking water intake and the growth and development conditions are good, and the weight growth is normal. After the experimental chicken is subjected to a cesarean examination, the organs of the immunized chicken are found to be normal in development, no pathological changes can be seen, and no phenomena of atrophy and swelling exist. Histopathological detection is carried out on the collected tissues, and the result shows that after high-dose immunization, bursa of Fabricius, thymus, liver, spleen and glandular stomach of the experimental chicken are intact, have no obvious difference with a control group, and have no obvious pathological change. The results show that even if the recombinant vaccine rHVT-US2-US10-LTB-VP2C-VP2V is used for inoculating target animal chickens with 10 times of immune dose, the recombinant vaccine does not cause clinical reaction and pathological damage to experimental chickens, and further shows that the vaccine strain virus is safe and reliable for the target animal chickens.

5. And (3) testing the efficacy:

rHVT-US2-US10-LTB-VP2C-VP2V recombinant vaccine, batch No. rHVT-US2-US10-LTB-VP2C-VP2V-001P, rHVT-US2-US10-LTB-VP2C-VP2V-002P, rHVT-US2-US10-LTB-VP2C-VP 2V-VP 63003P 3 in total. The check strain is classical strain BC6/85 and variant strain SN-18.

5.1 Experimental methods

5.1.1 grouping and immunization

100 SPF chickens 1 day old were randomly divided into 5 groups. Optionally, three groups of rHVT-US2-US10-LTB-VP2C-VP2V-001P, rHVT-US2-US10-LTB-VP2C-VP2V-002P, rHVT-US2-US10-LTB-VP2C-VP2V-003P batch vaccines are respectively immunized, the immunizing dose is 2500 PFU/unit, the 4 th group is inoculated with rHVT-VP2 (chicken infectious bursal disease virus and herpesvirus vector live vaccine rHVT-013-69 strain purchased from Meiria animal health Limited company), the immunizing dose is 2500 PFU/unit, the 5 th group is a non-immune control group, and the chickens in each experimental group are respectively marked.

5.1.2 IBDV antibody detection

Blood is collected one by one each week after immunization of the chickens in each experimental group until 28 days after immunization, serum is separated, and the indirect ELISA method is adopted to detect serum samples according to the instruction of the IBDV antibody detection kit.

5.1.3 challenge protection experiment

After immunization for 28 days, a virus challenge protection experiment was performed with a virulent strain BC6/85 (purchased from China veterinary medicine institute) and a variant SN18 (isolated and prepared from Youbang, Yongbo, Yangzhou) of chicken infectious bursal disease virus at a dose of 10 ⁵ EID ₅₀ . After the challenge, the clinical symptoms of the chickens were observed. Killing experimental chickens 12 days after the challenge, collecting bursa of Fabricius, and counting bursa weight ratio (F/B) and Bursa Index (BBIX), wherein F/B is (bursa of Fabricius weight/body weight) x 1000; BBIX ═ ratio of bursa weight of experimental group chicken/cyst ratio of blank control group chicken; when BBIX is less than 0.7, the bursal atrophy is judged; when BBIX is greater than 0.7, the bursa of Fabricius is judged to be normal. Pathological sections are made of collected bursa of Fabricius tissues, histopathological observation is carried out, and lesion scores (HBLS) are counted. Statistical methods for HBLS are as follows: 0, no lesion; 1, mild lesions; 2, sporadic or partial follicular lesions; 3, less than or equal to 50% of the follicular development lesions; 4, 50% -75% of the follicles are diseased; 5, 75% -100% of the follicles are diseased. When the HBLS is unreal, judging the HBLS is protection; when HBLS > 1, the protection is judged to be unprotected.

5.2 results of the experiment

5.2.1 serum antibody detection

At week 1 and week 2 after immunization, the antibody titers were not high in all groups; on day 21 after immunization, the antibody titers of the classical IBDV antibody and the variant IBDV antibody in the experimental group are obviously increased and reach peak values, wherein the antibody titer of the rHVT-IBDV-US2-US10-LTB-VP2C-VP2V immune group reaches 5.7 multiplied by 10 ³ The antibodies of the non-immune control group are negative, the detection of the variant IBDV antibody of the commercial vaccine is negative, and the specific result is shown in FIG. 5.

5.2.2 challenge protection test results

Food intake, water drinking and mental status were all normal 4 days after challenge, and the patients were observed continuously on day 12, and all of the non-immune control groups died 12 days later. Control seedlings were almost unprotected against the variants. After 12 days of toxin attacking, all chickens were dissected, and it was found that the leg muscles of the sick chickens had bleeding spots, the bursa of Fabricius had swollen to different degrees, and yellow jelly-like exudates were present on the surface. The challenge protection results show that all 3 vaccine passive immunization groups can protect the organism from being attacked by two IBDV strains to a certain extent, the protection rate can reach more than 100 percent, and the challenge protection results are shown in Table 1.

Passive immunization for fighting poison of chicks of 11 days old in table

The experimental results show that the 3 batches of vaccines have good immune effect on healthy susceptible chickens, so that the serum of the chicks contains high-level HVT recombinant viruses when the chicks are hatched, the antibody containing the LTB active adjuvant and the protection effect are superior to those of the commercial vaccines, and the vaccines can effectively resist the attack of infectious bursal disease viruses.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Youbang, Yangzhou biopharmaceutical Co Ltd

<120> a recombinant turkey herpesvirus expressing both classical strain infectious bursal disease virus VP2 protein and variant strain infectious bursal disease virus

Live vector vaccine

<130> BAA211303B

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 452

<212> PRT

<213> Artificial sequence

<400> 1

Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg

1 5 10 15

Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr

20 25 30

Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr

35 40 45

Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro

50 55 60

Gly Ser Ile Val Gly Ala His Tyr Thr Leu Gln Ser Asn Gly Asn Tyr

65 70 75 80

Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr

85 90 95

Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr

100 105 110

Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr

115 120 125

Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu

130 135 140

Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val

145 150 155 160

Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly

165 170 175

Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Ile Gly Leu Asp Pro Lys

180 185 190

Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile

195 200 205

Thr Ala Ala Asp Asp Tyr Gln Phe Ser Ser Gln Tyr Gln Ala Gly Gly

210 215 220

Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu

225 230 235 240

Ser Ile Gly Gly Glu Leu Val Phe Gln Thr Ser Val Gln Gly Leu Ile

245 250 255

Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile

260 265 270

Thr Arg Ala Val Ala Ala Asp Asn Gly Leu Thr Ala Gly Thr Asp Asn

275 280 285

Leu Met Pro Phe Asn Ile Val Ile Pro Thr Ser Glu Ile Thr Gln Pro

290 295 300

Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Gly Gly Gln

305 310 315 320

Ala Gly Asp Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr

325 330 335

Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val

340 345 350

Ala Tyr Glu Arg Val Ala Thr Gly Ser Val Val Thr Val Ala Gly Val

355 360 365

Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val

370 375 380

Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu

385 390 395 400

Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr

405 410 415

Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu

420 425 430

Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile

435 440 445

Arg Ala Leu Arg

450

<210> 2

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<212> DNA

<213> Artificial sequence

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atgactaatt tgcaggatca aactcagcag attgtaccct tcattcgatc tttgcttatg 60

cctactacag gaccagctag tatccctgat gacacactgg agaagcacac actccgttca 120

gaaacaagca cctacaacct cacggttgga gacaccggca gtggcttgat agttttcttc 180

cccggcttcc cgggcagcat tgttggggct cattatacgc tacagagcaa tgggaactac 240

aagtttgacc agatgctgct gacggcccag aaccttccag cgtcgtataa ctactgccgg 300

ctggtttccc gctcccttac tgtcaggagc agcacattac caggtggcgt ttatgcactg 360

aatggtacca ttaatgccgt gacgtttcaa ggcagtttgt ctgagctgac tgatgtgtcc 420

tacaatggac tgatgtctgc aaccgccaac atcaatgaca agatcggcaa tgtgctggtg 480

ggagaaggag tcaccgtact gtcgctgccg accagctatg atttaggcta cgtgcgactg 540

ggggatccta tacctgcaat tggtctggac cccaaaatgg tggctacctg tgattcaagt 600

gatcggcccc gcgtgtacac catcacagca gcagatgatt accagttcag ctctcagtac 660

caagcaggcg gggtcactat aactctcttt tcagccaata ttgatgctat aaccagtctt 720

tcaattgggg gagagctcgt cttccagaca tccgtgcagg gcttgattct tggggccacc 780

atctacctga ttggctttga tgggacagct gtgatcacga gggctgttgc tgccgacaac 840

ggtcttactg cgggaacaga taacctgatg ccattcaaca tagtcatacc cacatcagag 900

atcacccagc ccatcacctc tataaaacta gaaattgtaa cttccaagag tggtgggcaa 960

gctggggacc aaatgagctg gagtgcgtct ggttccttag cagtgacgat ccacggtgga 1020

aattatccag gtgctttaag acctgttact ttggttgcat atgagagggt ggctactggg 1080

tcagtggtga ctgtcgccgg ggtctccaac tttgaactca tcccaaaccc tgaattggcc 1140

aaaaatctag taacagaata tggaagattt gatccgggag ccatgaacta taccaagttg 1200

attctgtccg agagagaccg tctaggcatc aaaactgtgt ggccaacacg ggagtacaca 1260

gacttcagag aatacttcat ggaggtggca gacctgaaca gccctctgaa gattgctgga 1320

gcatttggat ttaaagacat catccgcgcg ctcaggtaa 1359

<210> 3

<211> 452

<212> PRT

<213> Artificial sequence

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Met Thr Asn Leu Gln Asp Gln Thr Gln Gln Ile Val Pro Phe Ile Arg

1 5 10 15

Ser Leu Leu Met Pro Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr

20 25 30

Leu Glu Lys His Thr Leu Arg Ser Glu Thr Ser Thr Tyr Asn Leu Thr

35 40 45

Val Gly Asp Thr Gly Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro

50 55 60

Gly Ser Ile Val Gly Ala His Tyr Ile Leu Gln Ser Asp Gly Ser Tyr

65 70 75 80

Lys Phe Asp Gln Met Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr

85 90 95

Asn Tyr Cys Arg Leu Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr

100 105 110

Leu Pro Gly Gly Val Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr

115 120 125

Phe Gln Gly Ser Leu Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu

130 135 140

Met Ser Ala Thr Ala Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val

145 150 155 160

Gly Glu Gly Val Thr Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly

165 170 175

Tyr Val Arg Leu Gly Asp Pro Ile Pro Ala Val Gly Leu Asp Pro Lys

180 185 190

Met Val Ala Thr Cys Asp Ser Ser Asp Arg Pro Arg Val Tyr Thr Ile

195 200 205

Thr Ala Ala Asp Asn Tyr Gln Phe Ser Ser Gln Tyr Lys Thr Gly Gly

210 215 220

Val Thr Ile Thr Leu Phe Ser Ala Asn Ile Asp Ala Ile Thr Ser Leu

225 230 235 240

Ser Val Gly Gly Glu Leu Val Phe Lys Thr Ser Ile Gln Asn Leu Val

245 250 255

Leu Gly Ala Thr Ile Tyr Leu Ile Gly Phe Asp Gly Thr Ala Val Ile

260 265 270

Thr Arg Ala Val Ala Ala Asn Asn Gly Leu Thr Ala Gly Ile Asp Asn

275 280 285

Leu Met Pro Phe Asn Leu Val Ile Pro Thr Ser Glu Ile Thr Gln Pro

290 295 300

Ile Thr Ser Ile Lys Leu Glu Ile Val Thr Ser Lys Ser Asp Gly Gln

305 310 315 320

Ala Gly Glu Gln Met Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr

325 330 335

Ile His Gly Gly Asn Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val

340 345 350

Ala Tyr Glu Arg Val Ala Lys Gly Ser Val Val Thr Val Ala Gly Val

355 360 365

Ser Asn Phe Glu Leu Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val

370 375 380

Thr Glu Tyr Gly Arg Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu

385 390 395 400

Ile Leu Ser Glu Arg Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr

405 410 415

Arg Glu Tyr Thr Asp Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu

420 425 430

Asn Ser Pro Leu Lys Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile

435 440 445

Arg Ala Ile Arg

450

<210> 4

<211> 1359

<212> DNA

<213> Artificial sequence

<400> 4

atgacaaatc ttcaagacca gacgcagcag attgtacctt tcatccgatc gctgctcatg 60

ccgaccactg gtccagctag cataccagat gatacactgg agaagcacac tctgcgttct 120

gaaacctcaa cttacaatct cacggtgggc gatactggca gcggactcat tgtgttcttc 180

cccggctttc ctggctccat tgttggggct cactacatcc tgcagtcaga cgggagttac 240

aagtttgacc aaatgctgtt aacagcgcaa aacctgcctg catcctacaa ctactgcaga 300

ttggtctccc gcagcttgac cgtaaggagt agtacacttc caggaggggt gtatgcactc 360

aatggcacca tcaatgcagt gaccttccag ggatccctca gtgagcttac tgatgtgtcg 420

tataatggtc tgatgtctgc taccgccaat ataaatgaca aaattggcaa tgttcttgtt 480

ggtgaaggag tgacggtgct gtcattgccc acctcttatg acctgggata tgtccggctg 540

ggggacccca taccagcggt aggtctagat cctaaaatgg tggcaacatg tgattcatct 600

gacaggcctc gagtgtacac cattaccgcc gccgacaact accagttcag cagccagtac 660

aaaacaggcg gcgtgaccat cacactcttt tcagcaaaca ttgatgctat aacttcctta 720

tctgtggggg gagaacttgt cttcaagaca agcatccaga acctggtctt gggtgcaact 780

atctacctga ttggttttga tggcacagct gtcataacaa gagctgttgc tgccaacaac 840

gggcttaccg ccggcattga caacttaatg cccttcaact tagttatccc tacgtcagaa 900

attactcaac ctatcacttc tattaaacta gaaattgtta cttccaagtc tgatgggcag 960

gccggagagc agatgagctg gagtgcatct ggcagtctgg ctgtcactat acatggggga 1020

aattacccag gagcgctgcg cccggtgacc ctggtggcgt atgagcgggt ggccaaagga 1080

agcgtagtaa cagtcgcagg ggtctccaac tttgaactga tacccaatcc agaactagca 1140

aaaaatctgg ttacagaata tggaagattt gatccaggag ccatgaacta taccaagctg 1200

atcctctcag agagggaccg cctcggtatc aagactgtgt ggcccactcg ggagtacaca 1260

gacttcaggg agtatttcat ggaggtggct gatttgaact ccccgttgaa gattgcaggg 1320

gcttttggtt ttaaagatat catccgtgcc atcagataa 1359

<210> 5

<211> 372

<212> DNA

<213> Artificial sequence

<400> 5

atgaacaagg tgaagtgcta tgtactgttt actgcactac tgtcttcact ttatgcacat 60

ggcgcgcccc agaccataac agagctctgc agtgagtatc ggaatactca aatatacacc 120

atcaatgaca aaattttgag ttacacagaa tccatggctg ggaaaaggga gatggtcatc 180

atcactttca agtctggtga aaccttccag gtggaagttc caggatccca gcacatcgat 240

tcacagaaga aagccattga gagaatgaaa gacacgttac gcatcaccta cctgactgaa 300

acaaagattg ataaactctg tgtgtggaac aacaaaacac ctaacagcat tgctgccata 360

agcatgaaga at 372

<210> 6

<211> 581

<212> DNA

<213> Artificial sequence

<400> 6

gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 60

acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 120

tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 180

agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 240

atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 300

atgggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc cctccccacc 360

cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg cggggggggg 420

gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag 480

gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc 540

ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc g 581

Claims (4)

1. A recombinant herpesvirus of turkeys is characterized in that the recombinant herpesvirus of turkeys expresses VP2 gene expression cassettes of both classical and variant strains of infectious bursal disease virus IBDV; the IBDV classical strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV classical strain VP2 gene and a terminator; the variant strain VP2 gene expression box sequentially comprises a promoter, an LTB gene, an IBDV variant strain VP2 gene and a terminator;

inserting an IBDV classical strain VP2 gene expression cassette into a US2 region of the herpesvirus of turkeys, and inserting an IBDV variant strain VP2 gene expression cassette into a US10 region of the herpesvirus of turkeys;

or, the IBDV classical strain VP2 gene expression cassette is inserted into the US10 region of the turkey herpesvirus, and the IBDV variant strain VP2 gene expression cassette is inserted into the US2 region of the turkey herpesvirus;

the nucleotide sequence of the IBDV classical strain VP2 gene is shown as SEQ ID NO.2, the nucleotide sequence of the IBDV variant strain VP2 gene is shown as SEQ ID NO.4, and the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5;

the promoter is a pec promoter with a nucleotide sequence shown as SEQ ID NO.6, and the terminator is a terminator of herpesvirus of turkeys; the herpesvirus of turkeys is strain HVT FC 126.

2. A method for preparing the recombinant herpesvirus of turkeys of claim 1, comprising inserting an IBDV classical strain VP2 gene expression cassette into the US2 region of the herpesvirus of turkeys, and inserting an IBDV variant strain VP2 gene expression cassette into the US10 region of the herpesvirus of turkeys;

or, inserting the IBDV classical strain VP2 gene expression cassette into the US10 region of the turkey herpesvirus, and inserting the IBDV variant strain VP2 gene expression cassette into the US2 region of the turkey herpesvirus;

the IBDV classical strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV classical strain VP2 gene and a terminator; the variant VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV variant VP2 gene and a terminator;

the promoter is a pec promoter with a nucleotide sequence shown as SEQ ID NO.6, and the terminator is a terminator of herpesvirus of turkeys;

the nucleotide sequence of the classical strain VP2 gene is shown as SEQ ID NO.2, the nucleotide sequence of the variant strain VP2 gene is shown as SEQ ID NO.4, and the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5.

3. A recombinant live vector vaccine of herpesvirus of turkeys is characterized in that the recombinant live vector vaccine of herpesvirus of turkeys expresses VP2 gene expression cassettes of classical strains and variant strains of infectious bursal disease virus IBDV simultaneously; the IBDV classical strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV classical strain VP2 gene and a terminator; the variant strain VP2 gene expression cassette sequentially comprises a promoter, an LTB gene, an IBDV variant strain VP2 gene and a terminator;

the nucleotide sequence of the VP2 gene of the classical strain is shown as SEQ ID NO.2, the nucleotide sequence of the VP2 gene of the variant strain is shown as SEQ ID NO.4, and the nucleotide sequence of the LTB gene is shown as SEQ ID NO. 5;

inserting an IBDV classical strain VP2 gene expression cassette into a US2 region of herpesvirus of turkeys, and inserting an IBDV variant strain VP2 gene expression cassette into a US10 region of herpesvirus of turkeys;

or, the IBDV classical strain VP2 gene expression cassette is inserted into the US10 region of the turkey herpesvirus, and the IBDV variant strain VP2 gene expression cassette is inserted into the US2 region of the turkey herpesvirus;

the promoter is a pec promoter with a nucleotide sequence shown as SEQ ID NO.6, and the terminator is a terminator of herpesvirus of turkeys; the herpesvirus of turkeys is strain HVT FC 126.

4. Use of a recombinant herpesvirus of turkeys as claimed in claim 1 or the method as claimed in claim 2, for the manufacture of an infectious bursal disease virus vaccine.

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