Recombinant herpesvirus turkey for co-expressing IBDV supervirulent strain, novel variant VP2 gene and H9 subtype AIV HA gene and application of recombinant herpesvirus turkey

Abstract

The invention provides a recombinant herpesvirus turkey for co-expressing a supervirulent strain of an infectious bursal disease virus, a novel variant VP2 gene and an H9 subtype avian influenza virus HA gene as well as a construction method and application of the recombinant herpesvirus turkey. The method comprises the following steps: inserting an expression framework containing a chicken infectious bursal disease virus novel variant VP2 gene and an H9 subtype avian influenza virus HA gene between 95322-95323 nucleotides of a turkey herpesvirus genome; meanwhile, a chicken infectious bursal disease virus super virulent strain VP2 gene expression framework is inserted between the 112071-112088 site nucleotides of a turkey herpesvirus genome, and a recombinant turkey herpesvirus vaccine strain H20401 which co-expresses the chicken infectious bursal disease virus super virulent strain, the novel variant VP2 gene and the H9 subtype avian influenza virus HA gene is obtained through rescue. 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 an avian influenza 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 super-virulent strain and a novel variant of infectious bursal disease virus of chickens and the HA gene of H9 subtype avian influenza virus and an application thereof. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal virus (IBDV) that mainly harms chicks. It is listed as an "important disease affecting the socio-economic situation" by the World Organization for Animal Health. At present, the super virulent strain of IBDV (vvIBDV) and the new variant strain (varIBDV) are the two dominant strains of IBDV in my country, but there is a lack of commercial IBD vaccines that can prevent both strains on the market. The H9 subtype avian influenza virus (AIV) is another pathogen that seriously harms China's poultry industry. The isolation rate of this virus 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] Researchers at home and abroad have developed a series of recombinant virus vector vaccine strains based on turkey herpes virus (HVT) vectors, 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. However, when the prior art simultaneously inoculates one-day-old chickens with recombinant turkey herpes viruses that express different exogenous genes separately, there will be a problem of two or more recombinant virus vector vaccines competing with each other in the chicken, thereby causing immune failure, and when different types of exogenous genes are simultaneously inserted into the same turkey herpes virus vector, there will be a problem of only having a good immune effect on one of the diseases, or the result of unsatisfactory immune effects on all diseases. 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. Summary of the invention

[0004] The object of the present invention is to provide a recombinant turkey herpes virus which co-expresses the VP2 gene of the super-virulent strain and the novel variant of infectious bursal disease virus of chickens and the HA gene of the H9 subtype avian influenza virus.

[0005] The present invention also aims to provide a method for constructing a recombinant turkey herpes virus that co-expresses the VP2 gene of the super-virulent strain and the new variant of infectious bursal disease virus of chickens and the HA gene of the H9 subtype avian influenza virus.

[0006] The present invention also aims to provide an application of a recombinant turkey herpes virus that co-expresses the VP2 gene of the super-virulent strain and the novel variant of infectious bursal disease virus of chickens and the HA gene of the H9 subtype avian influenza virus.

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

[0008] A recombinant turkey herpes virus co-expressing the VP2 gene of a very virulent strain and a new variant of infectious bursal disease virus and the HA gene of H9 subtype avian influenza virus. The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-HAA2 comprising a chicken β-actin promoter, the HA gene of H9 subtype avian influenza virus and the VP2 gene coding sequence HAA2 of a new variant of infectious bursal disease virus, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylic acid sequence into the turkey herpes virus genome, and simultaneously inserting an expression frame CMV-IBDV2 comprising a mouse cytomegalovirus promoter, the VP2 gene coding sequence of a very virulent strain of infectious bursal disease virus and an SV40 polyadenylic acid sequence into the turkey herpes virus genome.

[0009] Furthermore, the coding sequence HAA2 is obtained by connecting the H9 subtype avian influenza virus HA 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 HAA2 is shown in SEQ ID NO.1.

[0010] Furthermore, the expression frame CAGW-HAA2 is obtained by cloning the target gene coding sequence HAA2 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 HAA2 and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-HAA2 is shown in SEQ ID NO.2.

[0011] Furthermore, the expression framework CMV-IBDVC2 is obtained by cloning the VP2 gene of the super virulent strain of chicken infectious bursal virus into the mouse cytomegalovirus promoter and SV40 polyadenylation sequence of the pCMV vector.

[0012] Furthermore, the recombinant turkey herpes virus is obtained by co-transfecting recombinant cosmids HVT01, HVT02, HVT03, and HVT06 containing genomic DNA fragments of turkey herpes virus FC126 strain, recombinant cosmid HVT04-53-HAA2 containing the HA gene of H9 subtype avian influenza virus and the VP2 gene expression framework of a new variant of infectious bursal disease virus of chicken, and recombinant cosmid HVT05-65-IBDV2 containing the VP2 gene expression framework of a very virulent strain of infectious bursal disease virus of chicken into chicken embryo fibroblasts, and then rescued by virus; the recombinant cosmids HVT01, HVT02, HVT03, and HVT06 contain nucleotide fragments at positions 1-30189, positions 21572-61476, positions 50871-90478, and positions 129602-159160 of the turkey herpes virus FC126 strain genome, respectively.

[0013] Furthermore, the recombinant cosmid HVT04-53-HAA2 is obtained by inserting the expression framework CAGW-HAA2 between nucleotides 95322-95323 of the HVT genome on the basis of HVT04; the recombinant cosmid HVT04 contains a nucleotide fragment of nucleotides 73762-108139 of the FC126 strain genome.

[0014] Furthermore, the recombinant cosmid HVT05-65-IBDV2 is obtained by inserting the expression framework CMV-IBDV2 between nucleotides 112071-112088 of the HVT genome on the basis of HVT05; the recombinant cosmid HVT05 contains a nucleotide fragment of nucleotides 96776-135815 of the FC126 strain genome.

[0015] A recombinant turkey herpes virus that co-expresses the VP2 gene of a super-virulent strain and a novel variant of infectious bursal disease virus of chickens and the HA gene of H9 subtype avian influenza virus is used in the preparation of H9 subtype avian influenza and infectious bursal disease vaccines of chickens.

[0016] The present invention utilizes recombinant cloning technology to insert an expression framework comprising a novel variant VP2 gene of infectious bursal disease virus of chicken and an HA gene of H9 subtype avian influenza virus into the 95322-95323 nucleotides of a turkey herpes virus genome, and simultaneously inserts an expression framework comprising a super virulent strain VP2 gene of infectious bursal disease virus of chicken into the 112071-112088 nucleotides of a turkey herpes virus genome, thereby constructing a recombinant turkey herpes virus vaccine strain H20401 which co-expresses the VP2 genes of the super virulent strain and novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus.

[0017] 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 H9 subtype avian influenza virus infection, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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:

[0019] Figure 1 The figure shows the PCR identification results of the recombinant cosmids HVT04-53-HAA2 and HVT05-65-IBDV2, where M is DL5000 Marker, 1 is HVT04, 2 is HVT05, 3 is HVT04-53-HAA2, and 4 is HVT05-65-IBDV2;

[0020] Figure 2 This is a picture of plaque lesions produced by the recombinant virus H20401 on CEF;

[0021] Figure 3 The figure is the PCR identification result of the recombinant virus H20401 genomic DNA, wherein M is DL5000 Marker, 1 is the HAA2 gene amplification result of H20401, 2 is the HAA2 gene amplification result of FC126, 3 is the IBDV2 gene amplification result of H20401, and 4 is the IBDV2 gene amplification result of FC126;

[0022] Figure 4 The indirect immunofluorescence assay was used to detect the expression of VP2 and HA proteins in CEF infected with the recombinant virus H20401;

[0023] Figure 5 This is a replication kinetic curve of the recombinant virus H20401 on CEF cells;

[0024] Figure 6 The PCR identification results of the 20th generation recombinant virus H20401 genomic DNA, where M is DL5000 Marker, 1 is H20401-F20-HAA2, 2 is H20401-F20-IBDV2, and 3 and 4 are FC126;

[0025] Figure 7 The indirect immunofluorescence test results of the 20th generation recombinant virus H20401 expressing VP2 and HA proteins are shown;

[0026] Figure 8 This is the result of the test of the titer of H9 subtype AIV HI antibody induced in SPF chickens 4 weeks after inoculation of recombinant virus H20401;

[0027] Fig. 9 This is the survival curve of the recombinant virus H20401 after inoculation of chickens with IBDV super-virulent strains;

[0028] Fig.10 This is the statistical result of the bursal index of chickens challenged with the new variant strain of IBDV after inoculation of the recombinant virus H20401. DETAILED DESCRIPTION

[0029] 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.

[0030] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0031] 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.

[0032] Example 1: Construction and identification of a recombinant turkey herpes virus co-expressing the VP2 gene of an IBDV super-virulent strain and a novel variant and the AIV HA gene

[0033] 1.1 Establishment of the multi-fragment cosmid rescue system of HVT FC126 strain

[0034] 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 (Table 1). According to the sequencing results, six recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that cloned genomic DNA fragments of FC126 strains and could be spliced ​​to cover the complete HVT genome were selected. Wherein, HVT01 comprises the nucleotide fragment of 1-30189 position of FC126 genome, HVT02 comprises the nucleotide fragment of 21572-61476 position of FC126 genome, HVT03 comprises the nucleotide fragment of 50871-90478 position of FC126 genome, HVT04 comprises the nucleotide fragment of 73762-108139 position of FC126 genome, HVT05 comprises the nucleotide fragment of 96776-135815 position of FC126 genome, and HVT06 comprises the nucleotide fragment of 129602-159160 position of FC126 genome. The GenBank accession number of the described HVT FC126 strain genome sequence is AF291866. The above-mentioned 6 recombinant clays cloned with FC126 genomic DNA fragments are extracted, and the 6 recombinant clays are 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.

[0035] 1.2 Construction and identification of recombinant cosmids co-expressing the HA gene of H9 subtype AIV and the VP2 gene of the novel variant of IBDV

[0036] According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in this laboratory, the porcine Teschen virus 2A (P2A) self-cleavage peptide sequence was added to its 3' end, and the target gene H9HA was synthesized after chicken codon optimization. According to the VP2 gene sequence of the varIBDV FJ-18 strain (GenBank accession number OK167034), the target gene IBDA2 was synthesized after chicken codon optimization design. The target gene HAA2 containing the HA gene and VP2 gene coding sequences was obtained by fusion PCR amplification using primers H9P1F, H9A2P1R, H9A2P2F and A2P2R. The sequence is shown in SEQ ID NO.1, in which the HA 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 HAA2 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-HAA2 that co-expresses the AIV HA gene and varIBDV VP2 gene. The recombinant plasmid was sequenced using primers CAGF and CAGR, and it was found that the cloned target gene expression cassette sequence was correct.

[0037] According to the instructions of the Counter Selection BAC Modification Kit, the HAA2 gene expression framework CAGW-HAA2 (sequence shown in SEQ ID NO.2) was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 using the Red / ET recombination method to construct the recombinant cosmid HVT04-53-HAA2 that co-expresses the AIV HA gene and the varIBDV VP2 gene. The target gene primer H9A2P2F and the target gene downstream homology arm primer HVT54R were used to identify whether the recombinant cosmid HVT04-53-HAA2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that a PCR fragment of approximately 2768 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 HVT04-53-HAA2 was constructed correctly.

[0038] 1.3 Construction and identification of recombinant cosmids expressing the vvIBDV VP2 gene

[0039] According to the VP2 gene sequence of the vvIBDV HLJ-0504 strain (GenBank accession number GQ451330), the target gene IBDV2 (SEQ ID NO. 3) was synthesized. The recombinant plasmid pUC57-IBDV2 cloned with the IBDV2 gene was used as a template, and the IBDV2 gene was obtained by PCR amplification using primers IBDV2F and IBDV2R. The purified PCR product was cloned into the pCMV vector between the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence to construct the recombinant plasmid pCMV-IBDV2 expressing the vvIBDV VP2 gene.

[0040] According to the instructions of the Counter Selection BAC Modification Kit, the IBDV2 gene expression framework CMV-IBDV2 was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 using the Red / ET recombination method to construct the recombinant cosmid HVT05-65-IBDV2 expressing the vvIBDV VP2 gene. The target gene primer IBDV2F and the target gene downstream homology arm primer HVT65F were used to identify whether the recombinant cosmid HVT05-65-IBDV2 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 1800 bp was obtained ( Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the vvIBDV 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 HVT05-65-IBDV2 was constructed correctly.

[0041] Table 1 PCR primers used to construct and identify recombinant cosmids of HA gene and VP2 gene

[0042]

[0043]

[0044] 1.4 Rescue and identification of recombinant HVT co-expressing vvIBDV VP2 gene, varIBDV VP2 gene and H9 subtype AIV HA gene

[0045] The recombinant cosmids HVT04-53-HAA2, HVT05-65-IBDV2 and other parental cosmids HVT01, HVT02, HVT03, and HVT06 cloned with HVT FC126 strain genomic fragments were extracted using a plasmid extraction kit. The above 6 recombinant cosmids were co-transfected into CEF cells by calcium phosphate transfection, and 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 viruses with the varIBDV VP2 gene and AIV HA gene expression framework inserted between nucleotides 95322-95323 of the HVT genome and the vvIBDVVP2 gene expression framework inserted between nucleotides 112071-112088 were rescued and named H20401. The plaque lesions produced by the recombinant virus H20401 on CEF are shown in the figure. Figure 2 shown.

[0046] 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 H9A2P2F 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 2768bp in length, which was consistent with the expected size ( Figure 3 ). 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 HAA2 was correctly inserted into the genome of the HVT FC126 strain.

[0047] 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 IBDV2F 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 1800bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the vvIBDV 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 IBDV2 was correctly inserted into the genome of the HVT FC126 strain.

[0048] Example 2: Analysis of in vitro biological characteristics of recombinant turkey herpesvirus co-expressing the VP2 gene of IBDV super-virulent strain and novel variant and the HA gene of H9 subtype AIV

[0049] 2.1 Detection of recombinant virus H20401 expressing IBDV VP2 protein and AIV HA protein

[0050] The recombinant virus H20401 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 HA protein was detected by indirect immunofluorescence test using VP2 protein monoclonal antibody and HA protein polyclonal antibody as primary antibodies, FITC-labeled goat anti-mouse IgG and FITC-labeled goat anti-rabbit 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 H20401 were able to react with the VP2 protein monoclonal antibody and the HA protein polyclonal antibody, 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 H20401 can co-express VP2 protein and HA protein in infected cells.

[0051] 2.2 Analysis of in vitro replication characteristics of recombinant virus H20401

[0052] The recombinant virus H20401 and the parental virus FC126 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). The virus-containing cells were collected every 24 hours after infection 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 H20401 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus H20401 and the parental virus FC126 reached the highest peak at 120 hours after infection, which were 1.94×10 5 PFU / ml and 2.11×10 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 recombinant virus H20401 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus FC126 strain.

[0053] 2.3 Genetic stability test of recombinant virus H20401

[0054] The 5th generation recombinant virus H20401 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 H9A2P2F and the target gene downstream homology arm primer HVT54R, and a fragment of about 2768 bp could be amplified ( Figure 6 ), the size was consistent with expectations. The 20th generation recombinant virus genomic DNA was amplified using primers CAGF and CAGR and the PCR products were sequenced, and it was found that the sequence of the target gene HAA2 inserted into the HVT genome was correct. The 20th generation recombinant virus was identified by PCR using the target gene primer IBDV2F and the target gene downstream homology arm primer HVT65F, and a fragment of about 1800 bp could be amplified ( Figure 6 ), the size was consistent with expectations. The sequencing results showed that the PCR product contained the vvIBDV 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 HAA2 and vvIBDV VP2 inserted into the HVT gene can exist stably in the HVT genome.

[0055] 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 HA protein polyclonal antibody to detect the expression stability of the target genes VP2 and HA. The results showed that after the recombinant virus H20401 was continuously propagated on CEF cells to the 20th generation, the target genes VP2 and HA could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus H20401 has good genetic stability.

[0056] Example 3: Safety and immunogenicity testing of recombinant virus H20401

[0057] 3.1 Safety testing of recombinant virus H20401

[0058] The fifth generation recombinant virus H20401 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 H20401 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 H20401 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 H20401 inoculation group was no 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 H20401 is safe for SPF chickens.

[0059] 3.2 Immunoprotective test of recombinant virus H20401 against H9 subtype AIV

[0060] 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 H20401 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. 28 days after immunization, Groups 1 and 2 were nasally challenged with the 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 three 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 for incubation. After 96 hours of incubation, the allantoic fluid was collected and the HA titer was determined. The titer was not less than 1:16 and was judged as positive for virus isolation. The samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0061] The results showed that 28 days after the recombinant virus H20401 was inoculated into one-day-old SPF chickens, the average HI antibody titer could reach 1:362 ( Figure 8 ), the HI antibody titer of the parental virus FC126 inoculation group was negative. The above results show that the recombinant virus H20401 induced a good immune response after inoculation of chicks. After the experimental chickens were challenged with H9 subtype AIV, the virus isolation of 10 chickens in the parental virus FC126 strain inoculation group was positive, and the virus isolation of the unchallenged blank control group was negative, and the challenge test was established. After the recombinant virus H20401 inoculated chickens were challenged with H9 subtype AIV, all chickens' eating, drinking, mental state, movement, etc. were normal, with no obvious adverse clinical symptoms, virus isolation was negative, and the challenge protection rate was 100%.

[0062] 3.3 Immunoprotective test of recombinant virus H20401 against IBDV super-virulent strains and new variants

[0063] 60 one-day-old SPF chicks were randomly divided into 3 groups, 20 in each group; Group 1 was subcutaneously inoculated with the recombinant virus H20401 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. 28 days after immunization, 10 chickens in Group 1 and Group 2 were respectively challenged with the IBDV super-virulent strain HN strain, and observed for 7 days after the challenge, and the clinical symptoms of the test chickens were recorded, and the survival was counted; 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 respectively challenged with the new variant strain FJ strain of IBDV, and observed for 7 days after the challenge, and the clinical symptoms of the test chickens were recorded; 7 days after the challenge, the bursa of Fabricius was observed for lesions and the bursa weight ratio (F / B) and bursa index (BBIX) were counted. F / B = (bursa weight / body weight) × 1000, BBIX = bursa weight ratio of chickens in the experimental group / cyst ratio of chickens in the blank control group; when BBIX < 0.7, it is judged as bursa atrophy; when BBIX ≥ 0.7, it is judged as normal bursa. Group 3 was not challenged with virus and served as the healthy control group.

[0064] The results showed that 9 out of 10 chickens died after the parental virus FC126 inoculation group was challenged with super-strong IBDV ( Fig. 9 ), autopsy showed that the bursa of 10 chickens showed atrophy, strip-like hemorrhage or yellow jelly-like infiltration lesions; all 10 chickens inoculated with recombinant virus H20401 survived the observation period after the IBDV super-strong virus was inoculated, and there was no obvious lesion in the bursa of Fabricius. After the parent virus FC126 inoculation group was inoculated with the new variant of IBDV, the bursa of Fabricius showed atrophy lesions, and BBIX was less than 0.7 ( Fig.10 ); After the H20401 inoculation group was challenged with the new variant of IBDV, all survived during the observation period without obvious clinical symptoms. The autopsy showed that one of the 10 chickens had atrophic lesions in the bursa of Fabricius, and the other 9 chickens had no obvious lesions in the bursa of Fabricius, and the BBIX was greater than 0.7. The above results show that the protection rates of the recombinant virus H20401 against the super-virulent strain of IBDV and the new variant were 100% and 90%, respectively.

Claims

1. A recombinant turkey herpes virus that co-expresses the VP2 gene of a very virulent strain and a novel variant of infectious bursal disease virus of chickens and the HA gene of H9 subtype avian influenza virus, characterized in that: The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-HAA2 comprising a chicken beta-actin promoter, an H9 subtype avian influenza virus HA gene, a VP2 gene coding sequence HAA2 of a new variant of infectious bursal disease virus, 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-IBDV2 comprising a mouse cytomegalovirus promoter, a VP2 gene coding sequence of a very virulent strain of infectious bursal disease virus, 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 super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 1, characterized in that: The coding sequence HAA2 is obtained by connecting the coding sequence of the HA gene of the H9 subtype avian influenza virus and the coding sequence of the VP2 gene of the new variant of the infectious bursal disease virus with the coding sequence of the porcine teschovirus 2A self-cleavage peptide. The nucleotide sequence of the coding sequence HAA2 is shown in SEQ ID NO.

1.

3. The recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 1, characterized in that: The expression frame CAGW-HAA2 is obtained by cloning the target gene coding sequence HAA2 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 HAA2 and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-HAA2 is shown in SEQ ID NO.

2.

4. The recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 1, characterized in that: The expression framework CMV-IBDVC2 is obtained by cloning the VP2 gene of the super-virulent strain of chicken infectious bursal virus into the mouse cytomegalovirus promoter and the SV40 polyadenylic acid sequence of the pCMV vector.

5. The recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 1, characterized in that: The recombinant turkey herpes virus is obtained by co-transfecting recombinant cosmids HVT01, HVT02, HVT03, and HVT06 containing genomic DNA fragments of turkey herpes virus FC126 strain, recombinant cosmid HVT04-53-HAA2 containing the HA gene of H9 subtype avian influenza virus and the VP2 gene expression framework of a new variant of infectious bursal disease virus of chicken, and recombinant cosmid HVT05-65-IBDV2 containing the VP2 gene expression framework of a very virulent strain of infectious bursal disease virus of chicken into chicken embryo fibroblasts, and then rescued by virus; The recombinant cosmids HVT01, HVT02, HVT03 and HVT06 respectively contain nucleotide fragments at positions 1-30189, 21572-61476, 50871-90478 and 129602-159160 of the genome of the turkey herpesvirus FC126 strain.

6. The recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 5, characterized in that: The recombinant cosmid HVT04-53-HAA2 is obtained by inserting the expression framework CAGW-HAA2 between the nucleotides at positions 95322-95323 of the HVT genome on the basis of HVT04; the recombinant cosmid HVT04 contains a nucleotide fragment at positions 73762-108139 of the FC126 strain genome.

7. The recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the novel variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus according to claim 5, characterized in that: The recombinant cosmid HVT05-65-IBDV2 is obtained by inserting the expression framework CMV-IBDV2 between nucleotides 112071-112088 of the HVT genome on the basis of HVT05; the recombinant cosmid HVT05 contains a nucleotide fragment of nucleotides 96776-135815 of the FC126 strain genome.

8. Use of the recombinant turkey herpes virus co-expressing the VP2 gene of the super virulent strain and the new variant of infectious bursal disease virus of chicken and the HA gene of H9 subtype avian influenza virus as claimed in claim 1 in the preparation of avian influenza and infectious bursal disease vaccines.

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