Recombinant herpesvirus turkey for co-expressing IBDV (Infectious Bursal Disease Virus) classical virulent strain, novel variant VP2 gene and NDV F gene and application of recombinant herpesvirus turkey

Abstract

The invention provides a recombinant herpesvirus turkey for co-expressing a classical virulent strain of an infectious bursal disease virus, a novel variant VP2 gene and a Newcastle disease virus F gene as well as a construction method and application of the recombinant herpesvirus turkey. According to the invention, an expression framework containing a chicken infectious bursal disease virus novel variant VP2 gene and a Newcastle disease virus F gene is inserted between 95322-95323 nucleotides of a turkey herpesvirus genome, and at the same time, a chicken infectious bursal disease virus classical virulent strain VP2 gene expression framework is inserted between 112071-112088 nucleotides of the turkey herpesvirus genome; and rescuing to obtain the recombinant turkey herpesvirus vaccine strain H20103 for co-expressing the classical virulent strain of the infectious bursal disease virus, the novel variant VP2 gene and the Newcastle disease virus F gene. The vaccine prepared from the recombinant turkey herpesvirus provided by the invention can provide multiple protection against a chicken infectious bursal disease virus supervirulent strain, a novel variant and a Newcastle disease virus, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering vaccines, and specifically relates to a recombinant turkey herpes virus that co-expresses the VP2 gene of a classical virulent strain and a novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus and an application thereof. Background Art

[0002] Herpesvirus of turkeys (HVT) is a herpesvirus that is non-pathogenic to chickens. It has a large genome and many replication-non-essential genes that can be inserted or replaced by exogenous genes. It is an ideal viral vector for constructing recombinant live vector vaccines. Compared with other viral vectors, HVT has many advantages as a viral vector. It is non-pathogenic to chickens and other animals and is safe to use; the immune effect of HVT vaccine is not interfered by maternal antibodies and is suitable for early immunization of one-day-old chicks in hatcheries; after vaccination, HVT exists in the chicken body for a long time, stimulating the body to produce high antibody levels and maintain them for life, and lifelong immunity can be obtained with one vaccination; HVT vaccine not only has low production costs, but can also be freeze-dried, making it easy to store and transport.

[0003] Researchers at home and abroad have developed a series of recombinant virus vector vaccine strains based on turkey herpes virus HVT vector, such as Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), H9 subtype avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), etc. These vector vaccines effectively overcome the defects of inactivated vaccines and attenuated live vaccines that the immune effects are easily interfered by maternal antibodies, and effectively solve the current situation that the virus is easy to infect early chicks. When recombinant turkey herpes viruses expressing different exogenous genes are inoculated into one-day-old chickens at the same time, there will be a problem of two or more recombinant virus vector vaccines competing with each other in the chicken, resulting in immune failure. Moreover, when different types of exogenous genes are inserted into the same turkey herpes virus vector at the same time, there will be a problem of only having a good immune effect on one of the diseases, or the immune effect on all diseases is not ideal. Therefore, there is an urgent need for a recombinant HVT vector vaccine that can simultaneously express multiple exogenous antigen genes and stably propagate, and provide good immune protection effects.

[0004] Newcastle disease and infectious bursal disease are two acute, highly contagious diseases that seriously endanger my country's poultry industry. Mixed infection of NDV and IBDV often occurs in the field. However, there is currently a lack of a bivalent vaccine that is not interfered by maternal antibodies and is suitable for early immunization of one-day-old chicks in hatcheries for the combined immunization of the above two diseases. Since its first report in my country in 1982, IBDV infection has been widely prevalent in most poultry areas in my country. Since 2017, atypical IBD epidemics have occurred in my country and neighboring countries. The production performance and immune function of sick chickens are low, and autopsy shows that the bursa of Fabricius is severely atrophied. The pathogen causing the epidemic has been identified as a new variant of IBDV. At present, the new variant of IBDV is widely prevalent in my country, but the commercial IBD vaccine used on the market cannot provide good immune protection against the variant strain, which in turn causes severe immunosuppression in infected chickens, becoming a new threat to the healthy development of my country's poultry industry. Summary of the invention

[0005] The object of the present invention is to provide a recombinant turkey herpes virus which co-expresses the VP2 gene of the classical virulent strain and the novel variant strain of infectious bursal disease virus of chickens and the F gene of Newcastle disease virus.

[0006] The present invention also aims to provide a method for constructing a recombinant turkey herpes virus that co-expresses the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus of chickens and the F gene of Newcastle disease virus.

[0007] The present invention also aims to provide an application of a recombinant turkey herpes virus that co-expresses the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus of chickens and the F gene of Newcastle disease virus.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] The invention discloses a recombinant turkey herpes virus which co-expresses the VP2 gene of the classical virulent strain and novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus. The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-F2AA2 comprising a chicken β-actin promoter, the F gene of Newcastle disease virus and the VP2 gene coding sequence F2AA2 of the novel variant of infectious bursal disease virus, a post-transcriptional regulatory sequence of woodchuck hepatitis virus and a rabbit β-globulin polyadenylic acid sequence into the turkey herpes virus genome, and simultaneously inserting an expression frame CMV-IBDVC2 comprising a mouse cytomegalovirus promoter, the VP2 gene coding sequence of the classical virulent strain of infectious bursal disease virus and a SV40 polyadenylic acid sequence into the turkey herpes virus genome.

[0010] Furthermore, the target gene coding sequence F2AV2 is obtained by connecting the Newcastle disease virus F gene coding sequence and the infectious bursal virus new variant VP2 gene coding sequence with the porcine teschovirus 2A self-cleavage peptide coding sequence, and the nucleotide sequence of the coding sequence F2AA2 is shown in SEQ ID NO.1.

[0011] Furthermore, the expression frame CAGW-F2AA2 is obtained by cloning F2AA2 into the chicken β-actin promoter and the rabbit β-globulin polyadenylation sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between F2AA2 and the rabbit β-globulin polyadenylation sequence. The nucleotide sequence of the expression frame CAGW-F2AA2 is shown in SEQID NO.2.

[0012] Furthermore, the expression framework CMV-IBDVC2 is obtained by cloning the VP2 gene of the classical virulent strain of infectious bursal virus of chicken into the mouse cytomegalovirus promoter and SV40 polyadenylation sequence of the pCMV vector.

[0013] Furthermore, the recombinant turkey herpes virus is obtained by co-transfecting the recombinant cosmids H434, H481, H159, H220 containing the genomic DNA fragment of the turkey herpes virus FC126 strain, the recombinant cosmid H483-53-F2AA2 containing the Newcastle disease virus F gene and the VP2 gene expression framework of the new variant of the infectious bursal disease virus of chickens, and the recombinant cosmid H361-65-IBDVC2 containing the VP2 gene expression framework of the classical virulent strain of the infectious bursal disease virus of chickens into chicken embryo fibroblasts, and then rescued by virus;

[0014] The recombinant cosmids H434, H481, H159, and H220 contain nucleotide fragments at positions 1-40394, 31586-69191, 58293-92645, and 123621-159160 of the genome of the turkey herpesvirus FC126 strain, respectively;

[0015] The recombinant cosmid H483-53-F2AA2 is obtained by inserting the expression framework CAGW-F2AA2 between nucleotides 95322-95323 of the HVT genome on the basis of H483;

[0016] The recombinant cosmid H483 comprises a nucleotide fragment of positions 76940-110712 of the genome of strain FC126;

[0017] The recombinant cosmid H361-65-IBDVC2 is obtained by inserting the expression framework CMV-IBDVC2 between nucleotides 112071-112088 of the HVT genome on the basis of H361;

[0018] The recombinant cosmid H361 contains the nucleotide fragment of position 103006-134393 of the genome of the FC126 strain.

[0019] The invention discloses an application of the recombinant turkey herpes virus which co-expresses the VP2 gene of the classical virulent strain and the novel variant strain of infectious bursal disease virus and the F gene of Newcastle disease virus in the preparation of Newcastle disease and infectious bursal disease vaccines.

[0020] The present invention utilizes recombinant cloning technology to insert an expression framework comprising a novel variant VP2 gene of infectious bursal disease virus of chickens and a Newcastle disease virus F gene into the 95322-95323 nucleotides of the FC126 strain genome of turkey herpes virus, and simultaneously inserts an expression framework comprising a classical virulent strain VP2 gene of infectious bursal disease virus of chickens into the 112071-112088 nucleotides of the FC126 strain genome of turkey herpes virus, thereby constructing a recombinant turkey herpes virus vaccine strain H20103 which co-expresses the VP2 genes of the classical virulent strain and novel variant of infectious bursal disease virus of chickens and the F gene of Newcastle disease virus.

[0021] The vaccine prepared by the recombinant virus provided by the present invention can provide multiple protections against super-virulent strains and new variant strains of infectious bursal disease virus of chickens and Newcastle disease virus infection, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 The figure shows the PCR identification results of the recombinant cosmids H483-53-F2AA2 and H361-65-IBDVC2, where M is DL5000 Marker, 1 is H483-53-F2AA2, 2 is H361-65-IBDVC2, and 3 is ddH2O;

[0024] Figure 2 This is a picture of plaque lesions produced by the recombinant virus H20103 on CEF;

[0025] Figure 3The figure is the result of PCR identification of the genomic DNA of the recombinant virus H20103, wherein M is the DL5000 Marker, 1 is the result of F2AA2 gene amplification of H20103, 2 is the result of F2AA2 gene amplification of FC126, 3 is the result of IBDVC2 gene amplification of H20103, and 4 is the result of IBDVC2 gene amplification of FC126;

[0026] Figure 4 The indirect immunofluorescence assay was used to detect the expression of VP2 and F proteins in CEF infected with the recombinant virus H20103;

[0027] Figure 5 This is a graph showing the replication kinetics of the recombinant virus H20103 on CEF cells;

[0028] Figure 6 The PCR test result of the genetic stability of the recombinant virus H20103 is shown in Figure 1, where M is the DL5000 Marker, 1 is the F2AA2 gene amplification result of the 20th generation H20103, 2 is the F2AA2 gene amplification result of FC126, 3 is the IBDVC2 gene amplification result of the 20th generation H20103, and 4 is the IBDVC2 gene amplification result of FC126;

[0029] Figure 7 The figure shows the results of indirect immunofluorescence test of VP2 and F protein expressed by the 20th generation recombinant virus H20103;

[0030] Figure 8 This is the survival curve of chickens immunized with the recombinant virus H20103 after being challenged with the strong NDV virus;

[0031] Fig. 9 This is the survival curve of chickens immunized with the recombinant virus H20103 after being challenged with a very virulent strain of IBDV;

[0032] Fig.10 This is the statistical result of bursal index of chickens immunized with recombinant virus H20103 and challenged with new IBDV variant strain. DETAILED DESCRIPTION

[0033] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0034] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0036] Example 1: Construction and identification of recombinant turkey herpesviruses co-expressing the VP2 gene of the classical virulent IBDV strain and the novel variant strain and the NDV F gene

[0037] 1.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0038] The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector in segments according to the instructions of the CopyControlFosmid Library Production Kit. The constructed recombinant cosmids were terminally sequenced using primers pCC1F and pCC1R (primer sequences are shown in Table 1). According to the sequencing results, six recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned genomic DNA fragments of FC126 strains and could be spliced ​​to cover the complete HVT genome were selected. Among them, H434 contains the nucleotide fragment of the 1st to 40394th position of the FC126 genome, H481 contains the nucleotide fragment of the 31586-69191th position of the FC126 genome, H159 contains the nucleotide fragment of the 58293-92645th position of the FC126 genome, H483 contains the nucleotide fragment of the 76940-110712th position of the FC126 genome, H361 contains the nucleotide fragment of the 103006-134393th position of the FC126 genome, and H220 contains the nucleotide fragment of the 123621-159160th position of the FC126 genome. The GenBank accession number of the HVT FC126 genome sequence is AF291866. The above-mentioned 6 recombinant clays cloned with the genomic DNA fragments of the FC126 strain were extracted, and the 6 recombinant clays were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. Cytopathic effects can be observed 4-5 days after transfection, indicating that the parental virus strain FC126 was rescued.

[0039] 1.2 Construction and identification of recombinant cosmids co-expressing NDV F gene and IBDV novel variant VP2 gene

[0040] According to the F gene coding region sequence of NDV genotype VII strain GM strain (GenBank accession number DQ486859), the target gene NDF was synthesized by chicken codon optimization design, and the porcine Teschen virus 2A (P2A) self-cleavage peptide sequence was added to its C-terminus. According to the VP2 gene sequence of IBDV new variant strain (varIBDV) FJ-18 strain (GenBank accession number OK167034), the target gene IBDA2 was synthesized after chicken codon optimization design. The target gene F2AA2 containing the F gene and VP2 gene coding sequences was obtained by fusion PCR amplification using primers NDFP1F, NDA2P1R, NDA2P2F and A2P2R, as shown in SEQ ID NO.1, wherein the F gene and VP2 gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The purified PCR product was cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector through the EcoRI and ClaI restriction sites; at the same time, in order to enhance the transcription and translation efficiency of the target gene, the woodchuck hepatitis virus post-transcriptional regulatory sequence was inserted between the target gene F2AA2 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-F2AA2 that co-expresses the NDV F gene and the varIBDV VP2 gene. The recombinant plasmid was sequenced with primers CAGF and CAGR, and it was found that the inserted target gene sequence was correct.

[0041] According to the instructions of the Counter Selection BAC Modification Kit, the F2AA2 gene expression framework CAGW-F2AA2 (SEQ ID NO.2) was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid H483 using the Red / ET recombination method to construct the recombinant cosmid H483-53-F2AA2 that co-expresses the NDV F gene and the varIBDV VP2 gene. The target gene primer varIBDVP2F and the target gene downstream homology arm primer HVT54R were used to identify whether the recombinant cosmid H483-53-F2AA2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that a PCR fragment of about 2800 bp was obtained ( Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results showed that the recombinant cosmid H483-53-F2AA2 was constructed correctly.

[0042] 1.3 Construction and identification of recombinant cosmids expressing the VP2 gene of classic IBDV virulent strains

[0043] According to the VP2 gene sequence of the Faragher 52 / 70 strain of IBDV classical virulent strain (cvIBDV) (GenBank accession number HG974565), the target gene IBDVC2 (SEQ ID NO. 3) was synthesized. The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVC2 gene was used as a template, and the IBDVC2 gene was obtained by PCR amplification using primers IBDVC2F and IBDVC2R. The purified PCR product was cloned into the pCMV vector between the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence to construct a recombinant plasmid pCMV-IBDVC2 expressing the cvIBDV VP2 gene.

[0044] According to the instructions of the Counter Selection BAC Modification Kit, the IBDVC2 gene expression framework CMV-IBDVC2 was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 using the Red / ET recombination method to construct the recombinant cosmid H361-65-IBDVC2 expressing the cvIBDV VP2 gene. The target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F were used to identify whether the recombinant cosmid H361-65-IBDVC2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that the PCR fragment of about 1800 bp was obtained ( Figure 1 ), the size was consistent with expectations. Sequencing results showed that the PCR product contained the cvIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results showed that the recombinant cosmid H361-65-IBDVC2 was constructed correctly. The recombinant virus was named recombinant virus H20103.

[0045] Table 1 PCR primers used to construct and identify recombinant cosmids of F gene and VP2 gene

[0046] Primer name Primer sequence number pCC1F SEQ ID NO.4 pCC1R SEQ ID NO.5 NDFP1F SEQ ID NO.6 NDA2P1R SEQ ID NO.7 NDA2P2F SEQ ID NO.8 A2P2R SEQ ID NO.9 varIBDVP2F SEQ ID NO.10 HVT54R SEQ ID NO.11 CAGF SEQ ID NO.12 CAGR SEQ ID NO.13 IBDVC2F SEQ ID NO.14 IBDVC2R SEQ ID NO.15 HVT65F SEQ ID NO.16

[0047] 1.4 Rescue and identification of recombinant HVT co-expressing cvIBDV and varIBDV VP2 genes and NDV F gene

[0048] The recombinant cosmids H483-53-F2AA2, H361-65-IBDVC2 and other parental cosmids H434, H481, H159, and H220 cloned with HVT FC126 strain genomic fragments were extracted using a plasmid extraction kit. The above 6 recombinant cosmids were linearized by SbfI digestion and co-transfected into CEF cells by calcium phosphate transfection method. The viruses were harvested after plaque lesions appeared after 4-5 days of culture. The harvested viruses were serially passaged and preserved in CEF cells, and the recombinant virus H20103 with the varIBDV VP2 gene and NDV F gene expression framework inserted between nucleotides 95322-95323 of the HVT genome and the cvIBDV VP2 gene expression framework inserted between nucleotides 112071-112088 were rescued. The plaque lesions produced by the recombinant virus H20103 on CEF are shown in the figure. Figure 2 shown.

[0049] The recombinant virus genomic DNA was extracted, and the parent virus FC126 genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene specific primer varIBDVP2F and the target gene downstream homology arm primer HVT54R, and the PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by recombinant virus amplification was about 2800 bp in length, which was consistent with the expected size. For details, see Figure 3 1-2. The sequencing results showed that the PCR product contained the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parent virus FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene F2AA2 was correctly inserted into the genome of the HVT FC126 strain.

[0050] The recombinant virus genomic DNA was extracted, and the parent virus FC126 genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F, and the PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by recombinant virus amplification was about 1800 bp in length, which was consistent with the expected size. For details, see Figure 3 3-4. The sequencing results showed that the PCR product contained the cvIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parent virus FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene IBDVC2 was correctly inserted into the genome of the HVT FC126 strain.

[0051] Example 2: Analysis of in vitro biological characteristics of recombinant turkey herpesviruses co-expressing the VP2 gene of the classical virulent IBDV strain and the novel variant strain and the NDV F gene

[0052] 2.1 Detection of recombinant virus H20103 expressing IBDV VP2 protein and NDV F protein

[0053] The recombinant virus H20103 and the parent virus FC126 strain were inoculated into CEF cells, and after plaque lesions appeared after 3-4 days of culture, the cells were fixed with anhydrous ethanol, and the expression of VP2 protein and F protein were detected by indirect immunofluorescence test using VP2 protein monoclonal antibody and NDV positive serum as primary antibodies, FITC-labeled goat anti-mouse IgG and FITC-labeled rabbit anti-chicken IgG as secondary antibodies, and CEF infected with the parent virus FC126 was used as a negative control. The results showed that the cells infected with the recombinant virus H20103 were able to react with the VP2 protein monoclonal antibody and NDV positive serum, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parental HVT virus FC126 strain and in control cells that were not infected. The above results indicate that the recombinant virus H20103 can co-express VP2 protein and F protein in infected cells.

[0054] 2.2 Analysis of in vitro replication characteristics of recombinant virus H20103

[0055] The recombinant virus H20103 and the parental virus FC126 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). After infection, the virus-containing cells were collected every 24 hours until 144 hours after infection. The viruses collected at each time point were inoculated into CEF, the number of plaques in the virus solution at each time point was determined, and the in vitro replication kinetic curve was drawn to analyze the in vitro replication characteristics of the recombinant virus H20103 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus H20103 and the parental virus FC126 reached the highest peak at 120 hours after infection, which were 1.89×10 5 PFU / ml and 2.06×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection of CEF was not significantly different from that of the parental virus (P>0.05)( Figure 5 ). The above results show that the replication characteristics of the recombinant virus H20103 in CEF are consistent with those of the parent virus FC126 strain.

[0056] 2.3 Genetic stability test of recombinant virus H20103

[0057] The 5th generation recombinant virus H20103 was continuously passaged on CEF to the 20th generation, and the genomic DNA of the 20th generation recombinant virus was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the recombinant virus genome. The results showed that the 20th generation recombinant virus was identified by PCR using the target gene specific primer varIBDVP2F and the target gene downstream homology arm primer HVT54R, and a fragment of about 2800 bp could be amplified, which was consistent with the expected size ( Figure 6 1-2). Sequencing revealed that the PCR product contained the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F were used to perform PCR identification on the 20th generation recombinant virus, and a fragment of about 1800 bp could be amplified, which was consistent with the expected size ( Figure 6 3-4). The sequencing results showed that the PCR product contained the cvIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results showed that the target genes VP2 and F can exist stably in the HVT genome.

[0058] At the same time, the 20th generation recombinant virus was inoculated into CEF cells, and an indirect immunofluorescence test was performed using VP2 monoclonal antibody and NDV positive serum to detect the expression stability of the target genes VP2 and F. The results showed that after the recombinant virus H20103 was continuously propagated on CEF cells to the 20th generation, the target genes VP2 and F could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus H20103 has good genetic stability.

[0059] Example 3: Safety and immunogenicity testing of recombinant virus H20103

[0060] 3.1 Safety testing of recombinant virus H20103

[0061] The fifth generation recombinant virus H20103 was inoculated into one-day-old SPF chickens at a dose of 4000 PFU / chicken, and the clinical symptoms of each group of chickens were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their weights were weighed to evaluate the effect of the recombinant virus H20103 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected, weighed, and observed for atrophy or swelling symptoms. The results showed that the recombinant virus H20103 did not cause adverse clinical reactions in the test chickens after inoculation of SPF chickens, and the feeding and drinking of the immunized chickens were normal. 28 days after inoculation, the weighing and autopsy results showed that the weight of the chickens in the recombinant virus H20103 inoculation group was not significantly different from that of the normal uninoculated control group; the test chickens were autopsied, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected. The results showed that the above organs were normal and no obvious clinical lesions were found. The above results show that the recombinant virus H20103 is safe for SPF chickens.

[0062] 3.2 Immunogenicity test of recombinant virus H20101

[0063] 60 one-day-old SPF chicks were randomly divided into two groups, 30 in each group; Group 1 was subcutaneously inoculated with the recombinant virus H20103 at a dose of 4000 PFU / chicken, and Group 2 was inoculated with the parent virus FC126 at the same dose. Another 20 chickens were not immunized as a blank control group. 28 days after immunization, 10 chickens in Group 1 and Group 2 were respectively challenged with the virulent NDV of gene type VII, observed for 14 days after the challenge, recorded the clinical symptoms of the test chickens, and counted the survival of the chickens in each group. Another 10 chickens in each group were challenged with the super-strong strain of IBDV, observed for 7 days after the challenge, recorded the clinical symptoms of the test chickens, and counted the survival; 7 days after the challenge, the bursa of Fabricius was observed for atrophy, hemorrhage, or yellow jelly-like infiltration. The remaining 10 chickens in Group 1 and Group 2 were challenged with the new variant of IBDV, and observed for 7 days after the challenge, and the clinical symptoms of the test chickens were recorded and the survival was counted; the bursal lesions were observed 7 days after the challenge, and the bursal weight ratio (F / B) and bursal index (BBIX) were counted. F / B = (bursal weight / body weight) × 1000, BBIX = bursal weight ratio of chickens in the test group / cyst ratio of chickens in the blank control group; when BBIX < 0.7, it was judged as bursal atrophy; when BBIX ≥ 0.7, it was judged as normal bursal.

[0064] The results showed that all 10 chickens vaccinated with the recombinant virus H20103 survived the observation period after being challenged with NDV, while all 10 chickens in the parental virus FC126 immunization group died after being challenged with strong NDV ( Figure 8 The results of the IBDV super-virulent strain challenge test showed that all 10 chickens inoculated with the recombinant virus H20103 survived the challenge with super-virulent IBDV during the observation period, and there was no obvious lesion in the bursa of Fabricius; 8 out of 10 chickens inoculated with the parental virus FC126 died after the challenge with super-virulent IBDV ( Fig. 9 ), the bursa of Fabricius showed atrophy, streaky hemorrhage or yellow jelly-like infiltration lesions. All the H20103 immunization group survived the observation period after the varIBDV challenge, with no obvious lesions in the bursa of Fabricius and BBIX greater than 0.7; the parent virus FC126 vaccination group showed atrophy lesions in the bursa of Fabricius after the varIBDV challenge ( Fig.10 The above results show that the recombinant virus H20103 has a good immune protection effect against IBDV super-virulent and new variant strains and NDV virulent attacks.

Claims

1. A recombinant turkey herpes virus that co-expresses the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus, characterized in that: The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-F2AA2 comprising a chicken beta-actin promoter, a Newcastle disease virus F gene, a VP2 gene coding sequence F2AA2 of a new variant of infectious bursal disease virus of chickens, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit beta-globulin polyadenylic acid sequence into the turkey herpes virus genome, and simultaneously inserting an expression frame CMV-IBDVC2 comprising a mouse cytomegalovirus promoter, a VP2 gene coding sequence of a classical virulent strain of infectious bursal disease virus of chickens and a SV40 polyadenylic acid sequence into the turkey herpes virus genome.

2. The recombinant turkey herpes virus co-expressing the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus according to claim 1, characterized in that: The coding sequence F2AV2 is obtained by connecting the Newcastle disease virus F gene coding sequence and the infectious bursal virus new variant VP2 gene coding sequence with the porcine teschovirus 2A self-cleavage peptide coding sequence. The nucleotide sequence of the coding sequence F2AA2 is shown in SEQ ID NO.

1.

3. The recombinant turkey herpes virus co-expressing the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus according to claim 1, characterized in that: The expression frame CAGW-F2AA2 is obtained by cloning the target gene coding sequence F2AA2 into the chicken β-actin promoter and the rabbit β-globulin polyadenylic acid sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between F2AA2 and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-F2AA2 is shown in SEQ ID NO.

2.

4. The recombinant turkey herpes virus co-expressing the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus according to claim 1, characterized in that: The expression framework CMV-IBDVC2 is obtained by cloning the VP2 gene of the classical virulent strain of chicken infectious bursal virus into the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence of the pCMV vector.

5. The recombinant turkey herpes virus co-expressing the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus according to claim 1, characterized in that: The recombinant turkey herpes virus is obtained by co-transfecting recombinant cosmids H434, H481, H159, and H220 containing genomic DNA fragments of turkey herpes virus FC126 strain, recombinant cosmid H483-53-F2AA2 containing the F gene of Newcastle disease virus and the VP2 gene expression framework of the new variant of infectious bursal disease virus of chicken, and recombinant cosmid H361-65-IBDVC2 containing the VP2 gene expression framework of the classical virulent strain of infectious bursal disease virus of chicken into chicken embryo fibroblasts, and then rescued by virus; The recombinant cosmids H434, H481, H159, and H220 contain nucleotide fragments at positions 1-40394, 31586-69191, 58293-92645, and 123621-159160 of the genome of the turkey herpesvirus FC126 strain, respectively; The recombinant cosmid H483-53-F2AA2 is obtained by inserting the expression framework CAGW-F2AA2 between nucleotides 95322-95323 of the HVT genome on the basis of H483; The recombinant cosmid H483 comprises a nucleotide fragment of positions 76940-110712 of the genome of strain FC126; The recombinant cosmid H361-65-IBDVC2 is obtained by inserting the expression framework CMV-IBDVC2 between nucleotides 112071-112088 of the HVT genome on the basis of H361; The recombinant cosmid H361 contains the nucleotide fragment of position 103006-134393 of the genome of the FC126 strain.

6. Use of the recombinant turkey herpes virus co-expressing the VP2 gene of the classical virulent strain and the novel variant of infectious bursal disease virus and the F gene of Newcastle disease virus as claimed in claim 1 in the preparation of Newcastle disease and infectious bursal disease vaccines.

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