Recombinant HVT co-expressing H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene and its application

Abstract

The present invention discloses a recombinant HVT co-expressing H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene and its application, belonging to the technical field of medicine or veterinary medicine. The recombinant HVT is obtained by inserting an expression cassette containing H9 subtype AIV HA gene and QX type IBV S gene into the 95322-95323 nucleotides of HVT FC126 strain, and inserting an expression cassette containing IBDV classic strong strain VP2 gene into the 112071-112088 nucleotides of HVT genome, and then recombinant HVT vaccine strain H30201 co-expressing H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene is obtained by virus rescue. The present invention also proposes the application of the recombinant HVT vaccine strain in the preparation of H9 subtype avian influenza, chicken infectious bronchitis and chicken infectious bursal disease vaccines.

Description

Technical Field

[0001] The present invention relates to a recombinant turkey herpes virus that co-expresses the HA gene of H9 subtype avian influenza virus, the S gene of infectious bronchitis virus and the VP2 gene of infectious bursal disease virus, and its application in preparing vaccines of H9 subtype avian influenza, chicken infectious bronchitis and chicken infectious bursal disease. The present invention belongs to the technical field of medicine or veterinary medicine. Background Art

[0002] The H9 subtype avian influenza virus (AIV) was first isolated from a turkey flock in the United States in 1966 and is now distributed worldwide. Its widespread prevalence in poultry poses a continuous threat to the global poultry industry and public health security. Since the H9 subtype AIV was first isolated in Guangdong Province in 1992, the disease has been prevalent in my country for more than 30 years and has become one of the most serious pathogens affecting the development of my country's broiler farming industry. AIV belongs to the Orthomyxoviridae family. The HA protein it encodes can recognize specific receptors and mediate the invasion of the virus into host cells. It has strong immunogenicity and is the main host protective antigen of the virus, which can induce the production of neutralizing antibodies. The H9 subtype avian influenza is currently mainly controlled by inactivated vaccines. However, the isolation rate of H9 subtype AIV in my country has increased year by year in recent years, indicating that traditional inactivated vaccine immunization can no longer effectively prevent and control H9 subtype AIV infection. There is an urgent need to develop a safer and more efficient new vaccine.

[0003] Infectious bronchitis (IB) is an acute, highly contagious disease caused by infectious bronchitis virus (IBV), which can cause respiratory, urinary and reproductive system infections in chickens. The disease is prevalent throughout the year, and is particularly severe in winter and spring. Clinically, it is often mixed with H9 subtype AIV, Escherichia coli and mycoplasma, resulting in high morbidity and mortality. The spike protein (S) is the most important structural protein of IBV, which can induce the production of neutralizing antibodies and is the main host protective antigen of IBV. The S protein consists of two subunits, S1 and S2, both of which are glycoproteins. The S1 protein plays a decisive role in the virulence, tissue tropism and serotype of the virus, and can induce humoral and cellular immune responses in the host. At present, the dominant genotype of IBV in my country is the genotype I group 19 subtype (QX type), which is mainly controlled by traditional attenuated live vaccines and inactivated vaccines.

[0004] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal disease virus (IBDV) that mainly harms chicks. The IBDV genome consists of two double-stranded RNA segments, and the VP2 protein encoded by segment A is the main host protective antigen of IBDV. IBDV mainly attacks the bursa of Fabricius, the immune organ of chickens, causing immune dysfunction in infected chickens and reducing the immune effect of vaccines. In 1957, IBD first broke out in Gambro, Delaware, USA, and the pathogen that caused the disease was later called IBDV classical virulent virus. In 1982, my country first reported the prevalence of IBDV classical virulent virus in Beijing, and then this type of strain became widely prevalent in most poultry-raising areas in my country. By the end of the 1980s, IBDV super-virulent strains and antigenic variants appeared in the United States and Europe, further increasing the difficulty of prevention and control of the disease.

[0005] Recombinant live vector vaccines are live vaccines that use genetic engineering technology to construct a virus or bacteria into a vector, and then insert foreign genes into it for expression. Herpesvirus of turkey virus (HVT) is a herpes virus with a large genome and many replication-non-essential genes that can be inserted or replaced by foreign 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 can be used for early immunization of chicks in hatcheries; after vaccination, the virus exists in the chicken's body for a long time, stimulating the body to produce high antibody levels and maintain them for life; HVT vaccine not only has a low production cost, but can also be freeze-dried, making it easy to store and transport. Summary of the invention

[0006] The present invention aims to provide a recombinant turkey herpes virus (HVT) strain which co-expresses the HA gene of avian influenza virus (AIV), the S gene of infectious bronchitis virus (IBV) and the VP2 gene of infectious bursal disease virus (IBDV) and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention discloses a recombinant HVT virus strain that co-expresses H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene. The recombinant turkey herpes virus strain is obtained by inserting an expression cassette CAGW-HA2AS comprising a chicken β-actin promoter, an AIV HA gene and an IBV S gene coding sequence HA2AS, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylic acid sequence between nucleotides 95322 and 95323 of the HVT genome, and simultaneously inserting an expression cassette CMV-IBDVC2 comprising a mouse cytomegalovirus promoter, an IBDV classical virulent strain VP2 gene coding sequence and an SV40 polyadenylic acid sequence between nucleotides 112071 and 112088 of the HVT genome.

[0009] Among them, preferably, the AIV is an H9 subtype strain, the IBV is a QX type strain, the IBDV is a classical strong strain, and the HVT is an FC126 strain, and its GenBank accession number is AF291866.

[0010] Preferably, the AIV HA gene is obtained by optimizing the chicken codon of the coding region of the H9 subtype AIV HA gene, and its nucleotide sequence is shown in SEQ ID NO.1.

[0011] Among them, preferably, the S gene is obtained by mutating the S1-S2 cleavage site in the QX type IBV S protein sequence from RRRR to GSAS, mutating the amino acids at positions 695, 768, 775, 817, 862 and 863 to proline, and replacing the amino acids at positions 1097-1165 at the C-terminus with the T4 fiber protein trimer domain, and optimizing the chicken codon design, and its nucleotide sequence is shown in SEQ ID NO.2.

[0012] Among them, preferably, the AIV HA gene and IBV S gene coding sequence HA2AS is obtained by connecting the AIV HA gene coding sequence and the IBV S gene coding sequence with the porcine teschovirus 2A (P2A) self-cleavage peptide coding sequence, and its nucleotide sequence is shown in SEQ ID NO.3.

[0013] Among them, preferably, the nucleotide sequence of the expression cassette CAGW-HA2AS comprising chicken β-actin promoter, AIV HA gene and IBV S gene coding sequence HA2AS, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence is as shown in SEQ ID NO.4.

[0014] Among them, preferably, the coding sequence of the VP2 gene of the IBDV classical virulent strain is as shown in SEQ ID NO.5.

[0015] Furthermore, the present invention also proposes a method for constructing the recombinant turkey herpes virus strain, comprising the following steps:

[0016] (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0017] The genomic DNA of HVT FC126 strain was extracted and cloned into the pCC1Fos vector. Six recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned the genomic DNA fragments of FC126 strain and could be spliced ​​to cover the complete HVT genome were selected. Among them, H434 contained 1- The nucleotide fragment H481 includes the nucleotide fragment of 31586-69191 of the FC126 genome, the nucleotide fragment H159 includes the nucleotide fragment of 58293-92645 of the FC126 genome, the nucleotide fragment H483 includes the nucleotide fragment of 76940-110712 of the FC126 genome, the nucleotide fragment H361 includes the nucleotide fragment of 103006-134393 of the FC126 genome, and the nucleotide fragment H220 includes the nucleotide fragment of 123621-159160 of the FC126 genome;

[0018] (2) Construction and identification of recombinant cosmids expressing AIV HA gene and IBV S gene

[0019] The target gene AIV HA was synthesized, and the porcine Teschovirus 2A (P2A) self-cleavage peptide sequence was added to its C-terminus to synthesize the target gene IBV S. The target gene HA2AS containing HA and S coding sequences was obtained by fusion PCR amplification, wherein the HA gene and S gene coding regions were connected by the self-cleavage peptide P2A sequence; the target gene HA2AS was cloned into the downstream of the chicken β-actin promoter, and the woodchuck hepatitis virus post-transcriptional regulatory sequence was inserted between the target gene HA2AS and the rabbit β-globulin polyadenylic acid sequence to obtain the recombinant plasmid pCAGW-HA2AS that co-expressed the AIV HA gene and the IBV S gene; the HA2AS gene expression cassette was cloned into the recombinant cosmid H483 between the nucleotides 95322-95323 corresponding to the FC126 genome by the Red / ET recombination method to construct the recombinant cosmid H483-53-HA2AS that co-expressed the HA gene and the S gene;

[0020] (3) Construction and identification of recombinant cosmid expressing IBDV VP2 gene

[0021] The VP2 gene coding sequence IBDVC2 of the classical strong strain of IBDV was synthesized and inserted into the downstream of the mouse cytomegalovirus promoter of the pCMV vector to construct the recombinant plasmid pCMV-IBDVC2 expressing the IBDV VP2 gene; the IBDVC2 gene expression cassette containing the mouse cytomegalovirus promoter, the IBDV VP2 gene coding sequence and the SV40 polyadenylation sequence was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 by the Red / ET recombination method to construct the recombinant cosmid H361-65-IBDVC2 expressing the IBDV VP2 gene;

[0022] (4) Rescue and identification of recombinant HVT co-expressing HA, S and VP2 genes

[0023] The recombinant cosmids H483-53-HA2AS, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the genomic fragment of HVT FC126 strain were extracted using a plasmid extraction kit. The above six recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method. The viruses were harvested after plaque lesions appeared after 4-5 days of culture, and the recombinant virus H30201 with the target gene HA2AS expression cassette inserted between nucleotides 95322-95323 of the HVT genome and the IBDV VP2 gene expression cassette inserted between nucleotides 112071-112088 was rescued.

[0024] Furthermore, the present invention also proposes the use of the recombinant turkey herpesvirus strain in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bronchitis and infectious bursal disease.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention utilizes recombinant cloning technology to insert an expression frame containing H9 subtype AIV HA gene and QX type IBV S gene into the 95322-95323 nucleotides of HVT FC126 strain, and simultaneously inserts an expression frame containing IBDV classic strong strain VP2 gene into the 112071-112088 nucleotides of HVT genome, thereby rescuing and obtaining a recombinant HVT vaccine strain H30201 that co-expresses H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene. Experiments have shown that the recombinant virus H30201 has good replication ability on CEF and good genetic stability; after the recombinant virus H30201 is inoculated into SPF chickens, it can provide immune protection against H9 subtype AIV, QX type IBV strong toxin and IBDV super strong toxin. The present invention provides an effective technical means for simultaneously preventing H9 subtype avian influenza, chicken infectious bronchitis and chicken infectious bursal disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The PCR identification results of the recombinant cosmids H483-53-HA2AS and H361-65-IBDVC2;

[0028] Figure 2 The plaque lesions produced by the recombinant virus H30201 and the parental virus FC126 on CEF;

[0029] Figure 3 The results of PCR identification of the genomic DNA of the recombinant virus H30201 and the parental virus FC126;

[0030] Figure 4 To detect the expression of HA, S and VP2 proteins in CEF infected with the recombinant virus H30201;

[0031] Figure 5 is the replication kinetic curve of the recombinant virus H30201 in CEF cells;

[0032] Figure 6 This is the PCR identification result of the 20th generation recombinant virus H30201 genomic DNA;

[0033] Figure 7 The results are for the detection of HA, S and VP2 proteins expressed by the 20th generation recombinant virus H30201;

[0034] Figure 8 The results of the test for the titer of H9 AIV HI antibody induced by SPF chickens 4 weeks after H30201 inoculation;

[0035] Fig. 9The results of the test for the titer of IBV neutralizing antibodies induced by H30201 inoculated SPF chickens 4 weeks after inoculation;

[0036] Fig.10 These are the results of the test on the IBDV neutralizing antibody titer induced in SPF chickens 4 weeks after H30201 inoculation. DETAILED DESCRIPTION

[0037] The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention.

[0038] Example 1: Construction and identification of recombinant HVT expressing H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene

[0039] 1.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0040] The genomic DNA of HVT FC126 strain (GenBank accession number AF291866) was extracted and cloned according to the CopyControl According to the instructions of Fosmid Library Production Kit, the genomic DNA of FC126 strain was cloned into pCC1Fos vector. The constructed recombinant cosmids were terminally sequenced using primers pCC1 and pCC1R. According to the sequencing results, six recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned genomic DNA fragments of FC126 strain and could be spliced ​​to cover the complete HVT genome were selected. Among them, H434 contains a nucleotide fragment of 1-40394 of the FC126 genome, H481 contains a nucleotide fragment of 31586-69191 of the FC126 genome, H159 contains a nucleotide fragment of 58293-92645 of the FC126 genome, H483 contains a nucleotide fragment of 76940-110712 of the FC126 genome, H361 contains a nucleotide fragment of 103006-134393 of the FC126 genome, and H220 contains a nucleotide fragment of 123621-159160 of the FC126 genome. The above 6 recombinant cosmids cloned with FC126 genomic DNA fragments were extracted, and the 6 recombinant cosmids were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection. The appearance of cytopathic effects can be observed 4-5 days after transfection, that is, the parental virus strain FC126 was rescued.

[0041] 1.2 Construction and identification of recombinant cosmids expressing HA and S genes

[0042] According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in our laboratory, the porcine Teschovirus 2A (P2A) self-cleavage peptide sequence was added to its C-terminus, and the target gene H9HA was synthesized after chicken codon optimization, and its nucleotide sequence is shown in SEQ ID NO. 1. According to the S protein sequence of the QX type IBV SX strain (GenBank accession number OQ189490), its S1-S2 cleavage site RRRR was mutated to GSAS, and the amino acids at positions 695, 768, 775, 817, 862 and 863 were mutated to proline, and the amino acids at positions 1097-1165 at its C-terminus were replaced with the T4 fiber protein trimer domain. After chicken codon optimization, the target gene IBVS6P was synthesized, and its nucleotide sequence is shown in SEQ ID NO. 2. The target gene HA2AS (SEQ ID NO. 3) containing HA and S coding sequences was obtained by fusion PCR amplification using primers H9P1F, H9S6P1R, H9S6P2F and S6P2R in Table 1, wherein the HA gene and S gene coding regions were connected by a self-cleaving peptide P2A coding sequence. The HA2AS coding sequence obtained by PCR amplification was inserted into the downstream of the chicken β-actin promoter of the pCAGGS vector; at the same time, the woodchuck hepatitis virus post-transcriptional regulatory sequence was inserted between HA2AS and the rabbit β-globulin polyadenylic acid sequence to obtain the recombinant plasmid pCAGW-HA2AS that co-expresses the H9 AIV HA gene and the IBV S gene. The recombinant plasmid was sequenced using primers CAGF and CAGR in Table 1, and it was found that the expression cassette sequence containing chicken β-actin promoter-target gene HA2AS-woodchuck hepatitis virus post-transcriptional regulatory sequence-rabbit β-globulin polyadenylic acid sequence in sequence was correct.

[0043] According to the instructions of the Counter Selection BAC Modification Kit, the HA2AS gene expression cassette (shown in SEQ ID NO.4) was cloned into the recombinant cosmid H483 between nucleotides 95322-95323 corresponding to the FC126 genome using the Red / ET recombination method to construct the recombinant cosmid H483-53-HA2AS that co-expresses the HA gene and the S gene. The recombinant cosmid H483-53-HA2AS was identified by PCR using the target gene-specific primer IBVS2046F and the target gene downstream homology arm primer HVT54R in Table 1. The results showed that the amplified PCR fragment of approximately 2721 bp ( Figure 1), which was consistent with expectations; sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The parent cosmid H483 had no target gene insertion sequence, and the PCR result was negative. The above results showed that the recombinant cosmid H483-53-HA2AS was constructed correctly.

[0044] 1.3 Construction and identification of recombinant cosmids expressing IBDV VP2 gene

[0045] According to the VP2 gene sequence of the classical virulent Faragher 52 / 70 strain of IBDV (GenBank accession number HG974565), the target gene IBDVC2 (shown in SEQ ID NO.5) was synthesized and inserted into the pUC57 plasmid to obtain the recombinant plasmid pUC57-IBDVC2. The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVC2 gene was used as a template to obtain the IBDVC2 gene by PCR amplification using the primers IBDVC2F and IBDVC2R in Table 1. The purified PCR product was cloned into the downstream of the mouse cytomegalovirus promoter of the pCMV vector to construct the recombinant plasmid pCMV-IBDVC2 expressing the IBDV VP2 gene.

[0046] According to the instructions of the Counter Selection BAC Modification Kit, the IBDVC2 gene expression cassette 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 IBDV VP2 gene. The recombinant cosmid H361-65-IBDVC2 was identified by PCR using the target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F in Table 1, and the PCR product was sequenced and verified. The results showed that the amplified PCR fragment of about 1800 bp ( Figure 1 ), which was consistent with expectations. Sequencing results showed that the PCR product contained the IBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results indicate that the recombinant cosmid H361-65-IBDVC2 was constructed correctly.

[0047]

[0048] 1.4 Rescue and identification of recombinant HVT co-expressing HA, S, and VP2 genes

[0049] The recombinant cosmids H483-53-HA2AS, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the genomic fragment of the FC126 strain were extracted using a plasmid extraction kit. The above recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method. After 4-5 days of culture and the appearance of plaque lesions, the virus was harvested and the recombinant virus H30201 with the target gene HA2AS expression cassette inserted between nucleotides 95322-95323 of the HVT genome and the IBDV VP2 gene expression cassette inserted between nucleotides 112071-112088 was rescued. The plaque lesions produced on CEF are shown in FIG. Figure 2 shown.

[0050] The recombinant virus genomic DNA was extracted, and the parent virus HVT FC126 genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene specific primer IBVS2046F and the downstream homology arm primer HVT54R in Table 1, and the PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by amplification of the recombinant virus was about 2721 bp in length, which was consistent with the expected size ( Figure 3 ); Sequencing results showed that the PCR product contained the S 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 downstream homology arm primer HVT65F in Table 1 were used to perform PCR identification on the recombinant virus. The results showed that the recombinant virus amplified a PCR fragment of about 1800 bp in length, which was consistent with the expected size ( Figure 3 ); Sequencing results showed that the PCR product contained the IBDV VP2 gene sequence and the downstream homology arm sequence, and the sequence was correct. The parent virus FC126 had no target gene insertion, and the PCR results were all negative. The above results showed that the target genes HA2AS and IBDVC2 were correctly inserted into the genome of the HVT FC126 strain, and the recombinant virus H30201 was correctly constructed.

[0051] Example 2: Analysis of in vitro biological characteristics of recombinant HVT co-expressing H9 subtype AIV HA gene, IBV S gene and IBDV VP2 gene

[0052] 2.1 Detection of HA, S and VP2 proteins expressed by recombinant virus H30201

[0053] The recombinant virus H30201 and the parental virus HVT FC126 were inoculated into CEF cells cultured in 6-well plates, with 3 wells inoculated with each virus, and 3 uninoculated wells were set up as controls. After 3-4 days of culture, plaque lesions appeared and the cells were fixed with anhydrous ethanol. Rabbit polyclonal antibodies against HA protein, IBV chicken positive serum and mouse monoclonal antibodies against VP2 protein were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG, FITC-labeled rabbit anti-chicken IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies. Indirect immunofluorescence assay was used to detect the expression of HA protein, S protein and VP2 protein, and CEF inoculated with the parental virus FC126 was used as a negative control. The results showed that cells infected with the recombinant virus H30201 could react with HA protein polyclonal antibodies, IBV positive serum and VP2 protein monoclonal antibodies, showing green fluorescence signals ( Figure 4 ). No fluorescence was observed in cells infected with the parental virus FC126 strain and in control cells that were not infected. The above results indicate that the recombinant virus H30201 can co-express HA protein, S protein and VP2 protein in infected cells.

[0054] 2.2 Analysis of in vitro replication characteristics of recombinant virus H30201

[0055] The recombinant virus H30201 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 virus in vitro replication kinetic curve was drawn to analyze the in vitro replication characteristics of the recombinant virus H30201 and the parental virus FC126 in CEF. The results showed that the replication titers of the recombinant virus H30201 and the parental virus FC126 reached the highest peak 120 hours after infection, which were 1.76×10 5 PFU / ml and 1.97×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection with CEF was not significantly different from that of the parent virus FC126 (P>0.05) ( Figure 5 ). The above results show that the recombinant virus H30201 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus HVT FC126 strain.

[0056] 2.3 Genetic stability test of recombinant virus H30201

[0057] The 5th generation recombinant virus H30201 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 genome of the recombinant virus H30201. The results showed that the 20th generation recombinant virus H30201 was identified by PCR using the target gene-specific primer IBVS2046F and the target gene downstream homology arm primer HVT54R in Table 1, and a fragment of about 2721 bp could be amplified ( Figure 6 ), the size was consistent with the expectation. The 20th generation recombinant virus genomic DNA was amplified and sequenced using primers CAGF and CAGR in Table 1, and the target gene sequence was found to be correct. The 20th generation recombinant virus was identified by PCR using the target gene primer IBDVC2F and the downstream homology arm primer HVT65F in Table 1, and a fragment of about 1800 bp was amplified ( Figure 6 ), the size was consistent with the expectation. 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 HA, S and VP2 can be stably present in the genome of HVT FC126 strain.

[0058] At the same time, the 20th generation recombinant virus H30201 was inoculated into CEF cells, and an indirect immunofluorescence test was performed using HA protein polyclonal antibodies, IBV positive serum, and VP2 protein monoclonal antibodies to detect the stability of target gene expression. The results showed that after the recombinant virus H30201 was continuously propagated on CEF cells to the 20th generation, the exogenous genes HA, S, and VP2 inserted into the HVT genome could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus H30201 has good genetic stability.

[0059] Example 3: Safety and immunogenicity testing of recombinant virus H30201

[0060] 3.1 Safety testing of recombinant virus H30201

[0061] The fifth-generation recombinant virus H30201 was inoculated into one-day-old SPF chickens at a dose of 4000 PFU / chicken, and the clinical symptoms of the chickens in each group 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 H30201 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the organs such as the bursa of Fabricius, thymus, spleen, and liver were collected, weighed, and observed for atrophy or swelling. The results showed that the recombinant virus H30201 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 H30201 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 H30201 is safe for SPF chickens.

[0062] 3.2 Immunoprotective test of recombinant virus H30201 against H9 subtype AIV

[0063] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30201 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parental virus FC126 at the same dose, and Group 3 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the HI antibody titer of chickens in each group was detected by hemagglutination inhibition test. 28 days after immunization, Groups 1 and 2 were nasally challenged with H9 subtype AIV LC18 strain respectively; Group 3 was not challenged and served as a healthy control group. Five days after the challenge, laryngeal and cloacal swabs were collected from each chicken in each group, mixed and stored in PBS containing double antibodies for virus isolation. The collected cotton swabs were repeatedly frozen and thawed 3 times and then centrifuged. The supernatant was taken and inoculated into 10-day-old SPF chicken embryos through the allantoic cavity. The chicken embryos were placed in an incubator at 37°C. After 96 hours of incubation, the allantoic fluid was collected and the HA titer was determined. A titer of no less than 1:16 was considered positive for virus isolation. Samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0064] The results showed that 28 days after the recombinant virus H30201 was inoculated into one-day-old SPF chicks, the average titer of HI antibody in the test chickens could reach 1:239, while the HI antibody titer in the parent virus FC126 inoculation group was negative ( Figure 8). The above results show that the recombinant virus H30201 induced a good immune response after inoculation of chicks. After the experimental chickens were challenged with H9 subtype AIV, 9 out of 10 chickens in the parental virus FC126 strain inoculation group were positive for virus isolation, and the virus isolation of the experimental chickens in the blank control group without infection was negative. After the recombinant virus H30201 was inoculated with chickens to challenge H9 subtype AIV, all chickens had normal feeding, drinking, mental state, movement, etc., without obvious adverse clinical symptoms, and virus isolation was negative. The protection rate against H9 subtype AIV was 100%.

[0065] 3.3 Immunoprotective test of recombinant virus H30201 against virulent IBV

[0066] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30201 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parental virus FC126 at the same dose, and Group 3 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the titer of IBV neutralizing antibodies was detected. 28 days after immunization, Group 1 and Group 2 were challenged with the QX-type IBV virulent SD strain by nose and eye drops respectively; Group 3 was not challenged and served as a healthy control group. After the challenge, the chickens were observed for 14 days, and the clinical symptoms and deaths of the experimental chickens in each group were recorded; 14 days after the challenge, all experimental chickens were killed and autopsied, and the autopsy lesions were observed and recorded.

[0067] The results showed that 28 days after the recombinant virus H30201 was inoculated into one-day-old SPF chicks, the average titer of IBV neutralizing antibodies in the experimental chickens could reach 1:137, and the IBV neutralizing antibodies in the parent virus FC126 inoculation group were negative ( Fig. 9 ). The above results show that the recombinant virus H30201 induced a good immune response after inoculation of chicks. After the 10 chickens in the parental virus FC126 strain inoculation group were challenged with IBV, 9 chickens showed clinical symptoms such as depression, runny nose, and messy feathers, of which 4 died. The autopsy showed that all 10 chickens had lesions such as kidney enlargement, spotted kidney, laryngeal or tracheal bleeding, and tracheal mucus. After the 10 chickens in the recombinant virus H30201 inoculation group were challenged with IBV, all the test chickens survived during the observation period, of which 9 chickens had normal feeding, drinking, mental state, and movement, without obvious adverse clinical symptoms, and 1 chicken showed symptoms of depression; autopsy found that 9 chickens had no obvious lesions, and 1 chicken had laryngeal bleeding spots and mucus lesions. The test chickens in the blank control group without challenge showed no abnormal clinical symptoms and autopsy lesions. The above results show that the protection rate of H30201 against QX-type IBV is 90%.

[0068] 3.4 Immunoprotective test of recombinant virus H30201 against IBDV super-virulent virus

[0069] Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 chicks in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30201 at a dose of 4000 PFU / chicken, Group 2 was inoculated with the parent virus FC126 at the same dose, and Group 3 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the titer of IBDV neutralizing antibodies was detected. 28 days after immunization, Group 1 and Group 2 were challenged with the IBDV super-virulent HN strain by nose and eye drops respectively; Group 3 was not challenged and served as a healthy control group. After the challenge, the clinical symptoms of the experimental chickens were recorded for 7 days, and the survival was counted; the bursa of Fabricius was necropsied 7 days after the challenge to observe whether there were lesions such as atrophy, hemorrhage, or yellow jelly-like infiltration.

[0070] The results showed that 28 days after the recombinant virus H30201 was inoculated into one-day-old SPF chicks, the average titer of IBDV neutralizing antibodies in the experimental chickens could reach 1:294, while the titer of IBDV neutralizing antibodies in the parental virus FC126 inoculation group was negative ( Fig.10 ). The above results show that the recombinant virus H30201 induced a good immune response after inoculation of chicks. After the parental virus FC126 strain inoculated group was challenged with IBDV super-virulent virus, 8 of the 10 chickens died. Autopsy showed that the bursa of Fabricius of all 10 chickens was atrophied, with streaky hemorrhages or yellow jelly-like infiltration lesions. After the 10 chickens in the recombinant virus H30201 inoculated group were challenged with IBDV super-virulent virus, all of them survived healthily during the observation period; autopsy found that one chicken had atrophy and yellow jelly-like infiltration lesions in the bursa of Fabricius, and the other 9 chickens had no obvious lesions in the bursa of Fabricius. The experimental chickens in the blank control group without infection showed no abnormal clinical symptoms and autopsy lesions. The above results show that the protection rate of the recombinant virus H30201 against IBDV super-virulent virus is 90%.

Claims

1. A recombinant turkey herpes virus (HVT) strain that co-expresses the HA gene of avian influenza virus (AIV), the S gene of infectious bronchitis virus (IBV) and the VP2 gene of infectious bursal disease virus (IBDV), characterized in that: The recombinant turkey herpesvirus strain is obtained by inserting an expression cassette CAGW-HA2AS comprising a chicken β-actin promoter, an AIV HA gene and an IBV S gene coding sequence HA2AS, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence into the 95322-95323 nucleotides of the HVT genome, and simultaneously inserting an expression cassette CMV-IBDVC2 comprising a mouse cytomegalovirus promoter, an IBDV classic virulent strain VP2 gene coding sequence, and an SV40 polyadenylation sequence into the 112071-112088 nucleotides of the HVT genome; wherein the AIV HA gene and IBV S gene coding sequence HA2AS are obtained by connecting the AIV HA gene coding sequence and the IBV S gene coding sequence with a porcine Teschovirus 2A (P2A) self-cleaving peptide coding sequence, and the S gene is obtained by connecting the QX type IBV The S1-S2 cleavage site in the S protein sequence was mutated from RRRR to GSAS, and the amino acids at positions 695, 768, 775, 817, 862 and 863 were mutated to proline, and the amino acids at positions 1097-1165 at the C-terminus were replaced with the T4 fiber protein trimer domain, and the nucleotide sequence was obtained after chicken codon optimization design as shown in SEQ ID NO.

2.

2. The recombinant turkey herpes virus strain according to claim 1, characterized in that The AIV is a H9 subtype strain, the IBV is a QX type strain, the IBDV is a classical strong strain, and the HVT is a FC126 strain, and its GenBank accession number is AF291866.

3. The recombinant turkey herpes virus strain according to claim 1, characterized in that The AIV HA gene is obtained by optimizing the chicken codon of the H9 subtype AIV HA gene coding region, and its nucleotide sequence is shown in SEQ ID NO.

1.

4. The recombinant turkey herpes virus strain according to claim 1, characterized in that The nucleotide sequences of the AIV HA gene and IBV S gene coding sequence HA2AS are shown in SEQ ID NO.

3.

5. The recombinant turkey herpes virus strain according to claim 1, characterized in that The nucleotide sequence of the expression box CAGW-HA2AS comprising chicken β-actin promoter, AIV HA gene and IBV S gene coding sequence HA2AS, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence is shown in SEQ ID NO.

4.

6. The recombinant turkey herpes virus strain according to claim 1, characterized in that The coding sequence of the VP2 gene of the classical virulent strain of IBDV is shown in SEQ ID NO.

5.

7. A method for constructing the recombinant turkey herpes virus strain according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain Extraction of HVT FC126 strain genomic DNA, the FC126 strain genomic DNA was cloned into the pCC1Fos vector in segments, and 6 recombinant cosmids H434, H481, H159, H483, H361, and H220 that cloned FC126 strain genomic DNA fragments and could be spliced ​​to cover the complete HVT genome were selected, among which H434 contained the nucleotide fragment of 1-40394 of the FC126 genome, H481 contained the nucleotide fragment of 31586-69191 of the FC126 genome, H159 contained the nucleotide fragment of 58293-92645 of the FC126 genome, H483 contained the nucleotide fragment of 76940-110712 of the FC126 genome, H361 contained the nucleotide fragment of 103006-134393 of the FC126 genome, and H220 contained the nucleotide fragment of 123621-159160 of the FC126 genome; (2) Construction and identification of recombinant cosmids expressing AIV HA gene and IBV S gene The target gene AIV HA is synthesized, and the porcine Teschovirus 2A (P2A) self-cleavage peptide sequence is added to its C-terminus to synthesize the target gene IBV S. The target gene HA2AS containing HA and S coding sequences is obtained by fusion PCR amplification, wherein the HA gene and the S gene coding regions are connected by the self-cleavage peptide P2A sequence; the target gene HA2AS is cloned into the downstream of the chicken β-actin promoter, and the woodchuck hepatitis virus post-transcriptional regulatory sequence is inserted between the target gene HA2AS and the rabbit β-globulin polyadenylic acid sequence to obtain the recombinant plasmid pCAGW-HA2AS that co-expresses the AIV HA gene and the IBV S gene; the HA2AS gene expression cassette is cloned into the recombinant cosmid H483 corresponding to the 95322-95323 nucleotides of the FC126 genome by the Red / ET recombination method to construct the recombinant cosmid H483-53-HA2AS that co-expresses the HA gene and the S gene; (3) Construction and identification of recombinant cosmid expressing IBDV VP2 gene The VP2 gene coding sequence IBDVC2 of the classical strong strain of IBDV was synthesized and inserted into the downstream of the mouse cytomegalovirus promoter of the pCMV vector to construct the recombinant plasmid pCMV-IBDVC2 expressing the IBDV VP2 gene; the IBDVC2 gene expression cassette containing the mouse cytomegalovirus promoter, the IBDV VP2 gene coding sequence and the SV40 polyadenylation sequence was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 by the Red / ET recombination method to construct the recombinant cosmid H361-65-IBDVC2 expressing the IBDV VP2 gene; (4) Rescue and identification of recombinant HVT co-expressing HA, S, and VP2 genes The recombinant cosmids H483-53-HA2AS, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the HVTFC126 strain genome fragment were extracted using a plasmid extraction kit. The above six recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method. The viruses were harvested after 4-5 days of culture when plaque lesions appeared, and the recombinant virus H30201 with the target gene HA2AS expression cassette inserted between nucleotides 95322-95323 of the HVT genome and the IBDV VP2 gene expression cassette inserted between nucleotides 112071-112088 was rescued.

8. Use of the recombinant turkey herpesvirus strain according to any one of claims 1 to 6 in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bronchitis and infectious bursal disease.

Images