Recombinant herpesvirus turkey for expressing NDV F gene, H9 subtype AIV HA gene and IBDV VP2 gene and application of recombinant herpesvirus turkey

Abstract

The invention provides a recombinant herpesvirus turkey for expressing an NDV F gene, an H9 subtype AIV HA gene and an IBDV VP2 gene and application of the recombinant herpesvirus turkey, and belongs to the field of genetic engineering vaccines. According to the invention, by using a recombinant cloning technology, an expression framework containing a Newcastle disease virus F gene and an H9 subtype avian influenza virus HA gene is inserted between 95322-95323 nucleotides of a turkey herpesvirus FC126 strain, and an expression framework containing an infectious bursal disease virus VP2 gene is inserted between 112071-112088 nucleotides of a genome of the turkey herpesvirus FC126 strain; and rescuing to obtain the recombinant turkey herpesvirus vaccine strain which co-expresses the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene. The vaccine prepared by the construction method can provide multiple protection against Newcastle disease, avian influenza and infectious bursal 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 expressing NDV F gene, H9 subtype AIV HA gene and IBDV VP2 gene and an application thereof. Background Art

[0002] Newcastle disease (ND) is a virulent, highly contagious, respiratory infectious disease of poultry caused by Newcastle disease virus (NDV). Currently, Newcastle disease is distributed globally, causing huge losses to the breeding industry and import and export trade of related products around the world. At present, the market urgently needs a new ND vaccine that is homologous to the currently popular genotype VII strain, is not interfered by maternal antibodies, is suitable for early vaccination of chicks, and has a long duration of immunity. 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. The H9 subtype avian influenza is currently mainly controlled by inactivated vaccines. However, in recent years, the isolation rate of H9 subtype AIV in my country has increased year by year, indicating that traditional inactivated vaccine immunization can no longer effectively prevent and control H9 subtype AIV infection, and there is an urgent need to develop a safer and more efficient new vaccine. Infectious bursal disease (IBD) in chickens is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal virus (IBDV) that mainly harms chicks. The virus of this disease mainly attacks the chicken's immune organ, the bursa of Fabricius, causing immune dysfunction in infected chickens and reducing the immune effect of vaccines. The clinical manifestations are depression, anorexia, diarrhea and severe weakness in large groups of chickens. The autopsy is characterized by atrophy, hemorrhage and yellow jelly-like infiltration of the bursa of Fabricius.

[0003] 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.

[0004] 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. Summary of the invention

[0005] The object of the present invention is to provide a recombinant turkey herpes virus which co-expresses the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene.

[0006] The present invention also aims to provide a method for constructing a recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene.

[0007] The present invention also aims to provide an application of a recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene.

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

[0009] A recombinant turkey herpes virus expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene, characterized in that the recombinant turkey herpes virus is obtained by inserting the expression frame CAGW-F2AHA containing the chicken β-actin promoter, the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene coding sequence F2AHA, the woodchuck hepatitis virus post-transcriptional regulatory sequence, and the rabbit β-globulin polyadenylic acid sequence into the 95322-95323 nucleotides of the HVT genome, and inserting the expression frame CMV-IBDVC2 containing the mouse cytomegalovirus promoter, the infectious bursal disease virus VP2 gene coding sequence and the SV40 polyadenylic acid sequence into the 112071-112088 nucleotides of the HVT genome;

[0010] The coding sequence F2AHA is obtained by connecting the Newcastle disease virus F gene coding sequence and the H9 subtype avian influenza virus HA gene coding sequence with the porcine Teschovirus 2A self-cleavage peptide coding sequence, and the nucleotide sequence of the coding sequence F2AHA is shown in SEQ ID NO.1;

[0011] The expression frame CAGW-F2AHA is obtained by cloning the F2AHA gene 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 the F2AHA sequence and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-F2AHA is shown in SEQ ID NO.2.

[0012] The expression frame CMV-IBDVC2 is obtained by cloning the infectious bursal virus VP2 gene coding sequence into the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence. The nucleotide sequence of the expression frame CMV-IBDVC2 is shown in SEQ ID NO.3.

[0013] The method for constructing a recombinant turkey herpes virus expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene comprises the following steps:

[0014] (1) Establishment of a multi-segment cosmid rescue system for turkey herpesvirus FC126 strain

[0015] The genomic DNA of FC126 strain of turkey herpesvirus was extracted and cloned into pCC1Fos vector. Six recombinant cosmids H434, H481, H159, H483, H361 and H220 were selected, which contained genomic DNA fragments of FC126 strain and could be assembled to cover the complete HVT genome. Among them, H434 contained the nucleotide fragment of 1-40394 of FC126 genome, H481 contained The nucleotide fragment of FC126 genome includes nucleotides at positions 31586-69191, H159 includes nucleotides at positions 58293-92645 of the FC126 genome, H483 includes nucleotides at positions 76940-110712 of the FC126 genome, H361 includes nucleotides at positions 103006-134393 of the FC126 genome, and H220 includes nucleotides at positions 123621-159160 of the FC126 genome;

[0016] (2) Construction of recombinant cosmids expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0017] A recombinant expression plasmid comprising a chicken β-actin promoter, NDV F gene and AIV HA gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence and a rabbit β-globulin polyadenylation sequence expression frame CAGW-F2AHA was constructed, wherein the F gene and HA gene coding regions were connected by a porcine Teschovirus 2A self-cleavage peptide coding sequence; the expression frame CAGW-F2AHA was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid H483 by using the Red / ET recombination method, and a recombinant cosmid that co-expressed the F gene and the HA gene was constructed and named H483-53-F2AHA;

[0018] (3) Construction of recombinant cosmid expressing infectious bursal disease virus VP2 gene

[0019] A recombinant expression plasmid comprising a mouse cytomegalovirus promoter, an IBDVVP2 gene coding sequence and an SV40 polyadenylation sequence expression frame CMV-IBDVC2 was constructed; the expression frame CMV-IBDVC2 was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 by using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDV VP2 gene, which was named H361-65-IBDVC2;

[0020] (4) Rescue of recombinant turkey herpesvirus co-expressing Newcastle disease virus F gene, H9 subtype avian influenza virus HA gene and infectious bursal disease virus VP2 gene

[0021] The recombinant cosmids H483-53-F2AHA, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the HVTFC126 strain genome fragment were extracted; the above-mentioned recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, the viruses were harvested after the appearance of plaque lesions in culture, and continuously passaged and preserved in CEF to rescue the recombinant virus in which the expression frame CAGW-F2AHA was inserted between nucleotides 95322-95323 of the HVT FC126 strain genome, and the expression frame CMV-IBDVC2 was inserted between nucleotides 112071-112088 of the HVTFC126 strain genome, and the recombinant virus was named H30501.

[0022] The recombinant turkey herpes virus expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene is used in the preparation of Newcastle disease, avian influenza and infectious bursal disease vaccines.

[0023] The invention utilizes the recombinant cloning technology to insert an expression frame containing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene into the 95322-95323 nucleotides of the turkey herpes virus FC126 strain, and simultaneously inserts an expression frame containing the infectious bursal disease virus VP2 gene into the 112071-112088 nucleotides of the turkey herpes virus FC126 strain genome, thereby rescuing and obtaining the recombinant turkey herpes virus vaccine strain H30501 which co-expresses the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene. After immunizing specific pathogen-free (SPF) chickens with the recombinant virus vaccine strain, the SPF chickens can obtain immune protection against the Newcastle disease virus gene type VII strong virus, the H9 subtype avian influenza virus and the infectious bursal disease super strong virus, and can be used for the preparation of Newcastle disease, avian influenza and infectious bursal disease vaccines.

[0024] The vaccine prepared from the recombinant turkey herpes virus provided by the present invention can simultaneously provide multiple protections against Newcastle disease virus, avian influenza virus and infectious bursal disease virus infection, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute 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 improper limitations on the present invention. In the accompanying drawings:

[0026] Figure 1 Figure 1 is the PCR identification result of the recombinant cosmids H483-53-F2AHA and H361-65-IBDVC2, wherein M is DL5000 Marker, 1 is the identification result of the F2AHA gene of the H483-53-F2AHA cosmid, 2 is the identification result of the IBDVC2 gene of the H361-65-IBDVC2 cosmid, 3 is the identification result of the F2AHA gene of the H483 cosmid, and 4 is the identification result of the IBDVC2 gene of the H361 cosmid;

[0027] Figure 2 The images are of plaque lesions produced by the recombinant virus H30501 and the parental virus FC126 on CEF;

[0028] Figure 3 Figure 1 is the PCR identification result of the recombinant virus H30501 and the parent virus FC126 genomic DNA, where M is the DL5000 Marker, 1 is the F2AHA gene identification result of H30501, 2 is the IBDVC2 gene identification result of H30501, and 3 is the negative control ddH 2 O;

[0029] Figure 4The indirect immunofluorescence assay was used to detect the expression of F, HA and VP2 proteins in CEF infected with H30501;

[0030] Figure 5 This is a replication kinetic curve of the recombinant virus H30501 on CEF cells;

[0031] Figure 6 The figure is the result of PCR test for genetic stability of recombinant virus H30501, wherein M is DL5000 Marker, 1 is the result of F2AHA gene identification of H30501-F10, 2 is the result of IBDVC2 gene identification of H30501-F10, 3 is the result of F2AHA gene identification of H30501-F20, and 4 is the result of IBDVC2 gene identification of H30501-F20;

[0032] Figure 7 This is the result of indirect immunofluorescence test on the expression of F, HA and VP2 proteins by the 20th generation recombinant virus H30501. 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 HVT co-expressing NDV F gene, H9 subtype AIV HA gene and IBDV VP2 gene

[0037] 1.1 Establishment of reverse genetics system for turkey herpesvirus FC126 strain

[0038] The genomic DNA of FC126 strain of turkey herpesvirus was extracted, and the genomic DNA of FC126 strain was cloned into the pCC1Fos vector according to the instructions of the CopyControl Fosmid LibraryProduction Kit. The constructed recombinant cosmid was terminally sequenced using primers pCC1F and pCC1R (see Table 1 for details). According to the sequencing results, 6 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. Wherein, H434 comprises the nucleotide fragment of 1-40394 position of FC126 genome, H481 comprises the nucleotide fragment of 31586-69191 position of FC126 genome, H159 comprises the nucleotide fragment of 58293-92645 position of FC126 genome, H483 comprises the nucleotide fragment of 76940-110712 position of FC126 genome, H361 comprises the nucleotide fragment of 103006-134393 position of FC126 genome, and H220 comprises the nucleotide fragment of 123621-159160 position of FC126 genome. The GenBank accession number of described HVT FC126 strain genomic DNA is AF291866. The above-mentioned 6 clones with the recombinant clay of FC126 genomic DNA fragment are extracted, and 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.

[0039] 1.2 Construction and identification of recombinant cosmids expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0040] According to the F gene coding region sequence of genotype VII NDVGM strain (GenBank accession number DQ486859), the porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its C-terminus, and the target gene NDF was designed and synthesized after chicken codon optimization. According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in this laboratory, the target gene H9HA was synthesized after chicken codon optimization. The target gene F2AHA (SEQ ID NO.1) containing the F gene and HA gene coding sequences was obtained by fusion PCR amplification using primers NDP1F, NDH9P1R, NDH9P2F and H9P2R, wherein the F gene and HA 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 F2AHA and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-F2AHA that co-expresses the NDV F gene and the H9 subtype AIVHA gene. The recombinant plasmid was sequenced using primers CAGF and CAGR, and it was found that the cloned target gene sequence was correct.

[0041] According to the instructions of the Counter Selection BAC Modification Kit, the F2AHA gene expression framework CAGW-F2AHA (SEQ ID NO.2) was inserted 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-F2AHA that co-expresses the NDV F gene and the H9 subtype AIVHA gene. The target gene primer NDH9P2F and the target gene downstream homology arm primer HVT54R were used to identify whether the recombinant cosmid H483-53-F2AHA was inserted into the target gene expression framework by PCR, and the PCR product was sequenced and identified. The results showed that a PCR fragment of approximately 3065 bp was obtained ( Figure 1 ), the size was consistent with expectations. Sequencing results showed that the PCR product contained the HA gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parent cosmid H483 had no target gene insertion sequence, and the PCR result was negative. The above results indicate that the recombinant cosmid H483-53-F2AHA was constructed correctly.

[0042] 1.3 Construction and identification of recombinant cosmids expressing IBDV VP2 gene

[0043] The target gene IBDVC2 was synthesized according to the VP2 gene sequence of IBDV Faragher 52 / 70 strain (GenBank accession number HG974565). The recombinant plasmid pUC57-IBDVC2 cloned with the IBDVC2 gene was used as a template to obtain the IBDVC2 gene by PCR amplification using primers IBDVC2F and IBDVC2R. 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.

[0044] According to the instructions of the Counter Selection BAC Modification Kit, the IBDVC2 gene expression framework CMV-IBDVC2 (SEQ ID NO.3) 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 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 the expectation. The 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 showed that the recombinant cosmid H361-65-IBDVC2 was constructed correctly.

[0045] Table 1 PCR primers used to construct and identify recombinant cosmids

[0046]

[0047]

[0048] 1.4 Rescue and identification of recombinant HVT co-expressing Newcastle disease virus F gene, H9 subtype avian influenza virus HA gene and IBDV VP2 gene

[0049] The recombinant cosmids H483-53-F2AHA, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the genome fragment of HVT FC126 strain were extracted using a plasmid extraction kit. The above six recombinant cosmids and the parental cosmids were linearized by SbfI and co-transfected into CEF cells using the calcium phosphate transfection method. After 4-5 days of culture, the virus was harvested after the appearance of plaque lesions, and was continuously passaged and preserved in CEF cells. The recombinant virus with the target gene F2AHA expression framework inserted between nucleotides 95322-95323 of the HVT FC126 genome and the IBDV VP2 gene expression framework inserted between nucleotides 112071-112088 was rescued and named H30501. The plaque lesions produced by the recombinant virus on CEF are as follows: Figure 2 shown.

[0050] The recombinant virus genomic DNA was extracted, and the parent virus FC126 strain genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene specific primer NDH9P2F 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 3065bp in length, which was consistent with the expected size. Figure 3 As shown. The sequencing results showed that the PCR product contained the HA 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 insertion and the PCR result was negative. The above results showed that the target gene expression framework CAGW-F2AHA was correctly inserted into the genome of the HVT FC126 strain.

[0051] At the same time, the extracted recombinant virus genomic DNA was used to perform PCR identification of the recombinant virus 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 amplifying the recombinant virus was about 1800bp in length, which was consistent with the expected size ( Figure 3 ). The 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. However, the parent virus FC126 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene expression framework CMV-IBDVC2 was correctly inserted into the genome of the HVT FC126 strain.

[0052] Example 2: Analysis of in vitro biological characteristics of recombinant HVT co-expressing Newcastle disease virus F gene, H9 subtype avian influenza virus HA gene and infectious bursal disease virus VP2 gene

[0053] 2.1 Detection of F, HA and VP2 proteins expressed by recombinant virus H30501

[0054] The recombinant virus H30501 and the parental virus 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. NDV-positive serum, HA protein polyclonal antibody and VP2 protein monoclonal antibody were used as primary antibodies, FITC-labeled rabbit anti-chicken IgG, FITC-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies, and indirect immunofluorescence test was used to detect the expression of F protein, HA protein and VP2 protein. CEF inoculated with the parental virus FC126 was used as a negative control. The results showed that cells infected with the recombinant virus H30501 could react with NDV-positive serum, HA protein polyclonal antibody and VP2 protein monoclonal antibody, 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 H30501 can co-express NDV F protein, AIV HA protein and IBDV VP2 protein in infected cells.

[0055] 2.2 Analysis of in vitro replication characteristics of recombinant virus H30501

[0056] The recombinant virus H30501 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 H30501 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus H30501 and the parental virus FC126 reached the highest peak at 120 hours after infection, which were 1.91×10 5 PFU / ml and 2.16×10 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 H30501 has good replication ability in CEF and its in vitro replication characteristics are consistent with those of the parent virus FC126 strain.

[0057] 2.3 Genetic stability test of recombinant virus H30501

[0058] The 5th generation recombinant virus H30501 was continuously passaged on CEF to the 20th generation. The 10th and 20th generation cytotoxic cells were selected to extract viral genomic DNA, and PCR identification and sequencing were performed to detect the genetic stability of the target gene sequence in the recombinant virus genome. The results showed that the 10th and 20th generation recombinant viruses were identified by PCR using the target gene-specific primer NDH9P2F and the target gene downstream homology arm primer HVT54R, and a fragment of about 3065bp could be amplified, which was consistent with the expected size ( Figure 6 ). The 20th generation recombinant virus genomic DNA was amplified and sequenced using primers CAGF and CAGR, and the inserted target gene F2AHA sequence was found to be correct. The 10th and 20th generation recombinant viruses were identified by PCR using the target gene primer IBDVC2F and the target gene downstream homology arm primer HVT65F, and a fragment of about 1800 bp was amplified, which was consistent with the expected size ( Figure 6 ). The 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 show that the target genes F, HA, and VP2 inserted into the HVT genome can exist stably during the passage process.

[0059] At the same time, the 20th generation recombinant virus was inoculated into CEF cells, and indirect immunofluorescence tests were performed using NDV positive serum, HA protein polyclonal anti-epidemic and VP2 protein monoclonal antibodies to detect the stability of target gene expression. The results showed that after the recombinant virus H30501 was continuously propagated on CEF cells to the 20th generation, the exogenous genes F, HA and VP2 inserted into the HVT genome could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus H30501 has good genetic stability.

[0060] Example 3: Safety and immunogenicity testing of recombinant virus H30501

[0061] 3.1 Safety testing of recombinant virus H30501

[0062] The fifth-generation recombinant virus H30501 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 H30501 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 H30501 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 H30501 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 H30501 is safe for SPF chickens.

[0063] 3.2 Immunoprotective test of recombinant virus H30501 against NDV, H9 subtype AIV and IBDV

[0064] 90 one-day-old SPF chicks were randomly divided into 3 groups, 30 in each group. Group 1 was subcutaneously inoculated with the recombinant virus H30501 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, Group 1 and Group 2 were subjected to NDV, H9 subtype AIV and IBDV virulent challenge tests, respectively.

[0065] Among them, 10 chickens were taken from Group 1 and Group 2 to attack the virulent GM strain of NDV of gene type VII, and observed for 14 days after the attack, and the clinical symptoms and survival of the test chickens were recorded. At the same time, 10 chickens were taken from Group 1 and Group 2 to attack the H9 subtype AIV LC18 strain, and observed for 14 days after the attack, and the clinical symptoms of the test chickens were recorded; 5 days after the attack, the laryngeal and cloacal swabs of each chicken in each group were collected, mixed and added to PBS containing double antibodies for preservation, and virus isolation was performed. The collected cotton swabs were repeatedly frozen and thawed 3 times and then centrifuged, and 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, and the allantoic fluid was collected after 96 hours of incubation, 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. In addition, the remaining 10 chickens in Group 1 and Group 2 were challenged with the highly virulent HN strain of IBDV, and observed for 7 days after the challenge, and the clinical symptoms and survival of the test chickens were recorded. The bursa of Fabricius was necropsied and observed for atrophy, hemorrhage, or yellow jelly-like exudation 7 days after the challenge. Group 3 was not challenged and served as a healthy control group.

[0066] The results showed that all 10 chickens in the parental virus FC126 inoculation group died after being challenged with strong NDV, while all 10 chickens in the recombinant virus H30501 inoculation group survived after being challenged with NDV, with no obvious clinical symptoms. After the 10 chickens in the parental virus FC126 inoculation group were challenged with H9 subtype AIV, virus isolation was positive, while virus isolation was negative in the blank control group; after the 10 chickens in the recombinant virus H30501 inoculation group were challenged with H9 subtype AIV, during the observation period, all chickens had normal feeding, drinking, mental state, and movement, with no obvious adverse clinical symptoms, of which one chicken had positive virus isolation, and the remaining 9 chickens had negative virus isolation. After the parental virus FC126 strain inoculation group was challenged with IBDV super-virulent virus, all 10 chickens became ill, 8 of which died. Autopsy showed that the bursa of Fabricius of all 10 chickens showed atrophy, strip-shaped bleeding or yellow jelly-like infiltration lesions; all 10 chickens in the recombinant virus H30501 inoculation group survived after being challenged with IBDV super-virulent virus, and no obvious lesions in the bursa of Fabricius were found in the autopsy. The experimental chickens in the non-challenged control group survived healthily during the observation period, and no adverse clinical symptoms or autopsy lesions were observed. The above results show that the recombinant virus H30501 has a good immune protection effect against NDV genotype VII virulent virus, H9 subtype AIV and IBDV super-virulent virus.

Claims

1. A recombinant turkey herpes virus expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene, characterized in that: The recombinant turkey herpes virus is obtained by inserting an expression frame CAGW-F2AHA comprising a chicken β-actin promoter, a Newcastle disease virus F gene and an H9 subtype avian influenza virus HA gene coding sequence F2AHA, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylic acid sequence into the 95322-95323 nucleotides of the HVT genome, and inserting an expression frame CMV-IBDVC2 comprising a mouse cytomegalovirus promoter, an infectious bursal disease virus VP2 gene coding sequence, and an SV40 polyadenylic acid sequence into the 112071-112088 nucleotides of the HVT genome; the coding sequence F2AHA is obtained by connecting a Newcastle disease virus F gene coding sequence and an H9 subtype avian influenza virus HA gene coding sequence with a porcine Teschovirus 2A self-cleaving peptide coding sequence, and the nucleotide sequence of the coding sequence F2AHA is shown in SEQ ID NO.1; The expression frame CAGW-F2AHA is obtained by cloning the F2AHA gene 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 the F2AHA sequence and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-F2AHA is shown in SEQ ID NO.

2. The expression frame CMV-IBDVC2 is obtained by cloning the infectious bursal virus VP2 gene coding sequence into the mouse cytomegalovirus promoter and the SV40 polyadenylation sequence. The nucleotide sequence of the expression frame CMV-IBDVC2 is shown in SEQ ID NO.

3.

2. A method for constructing a recombinant turkey herpes virus expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene as claimed in claim 1, characterized in that: The construction method comprises the following steps: (1) Establishment of a multi-segment cosmid rescue system for turkey herpesvirus FC126 strain The genomic DNA of FC126 strain of turkey herpesvirus was extracted and cloned into pCC1Fos vector. Six recombinant cosmids H434, H481, H159, H483, H361 and H220 were selected, which contained genomic DNA fragments of FC126 strain and could be assembled to cover the complete HVT genome. Among them, H434 contained nucleotide fragments of 1-40394 of FC126 genome, H481 contained nucleotide fragments of FC1 26 genome nucleotide fragment 31586-69191, H159 contains the nucleotide fragment 58293-92645 of the FC126 genome, H483 contains the nucleotide fragment 76940-110712 of the FC126 genome, H361 contains the nucleotide fragment 103006-134393 of the FC126 genome, and H220 contains the nucleotide fragment 123621-159160 of the FC126 genome; (2) Construction of a recombinant cosmid expressing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene. A recombinant expression plasmid comprising a chicken β-actin promoter, the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene coding sequences, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence expression framework CAGW-F2AHA was constructed, wherein the F gene and HA gene coding regions were connected by the porcine Teschovirus 2A self-cleaving peptide coding sequence; the expression framework CAGW-F2AHA was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid H483 by the Red / ET recombination method, and a recombinant cosmid expressing both the F gene and the HA gene was constructed and named H483-53-F2AHA; (3) Construction of recombinant cosmid expressing IBDV VP2 gene A recombinant expression plasmid comprising a mouse cytomegalovirus promoter, an IBDVVP2 gene coding sequence and an SV40 polyadenylation sequence expression frame CMV-IBDVC2 was constructed; the expression frame CMV-IBDVC2 was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid H361 by using the Red / ET recombination method to construct a recombinant cosmid expressing the IBDV VP2 gene, which was named H361-65-IBDVC2; (4) Rescue of recombinant HVT co-expressing Newcastle disease virus F gene, H9 subtype avian influenza virus HA gene and IBDV VP2 gene The recombinant cosmids H483-53-F2AHA, H361-65-IBDVC2 and the other four parental cosmids H434, H481, H159 and H220 cloned with the HVTFC126 strain genome fragment were extracted; the above-mentioned recombinant cosmids and the parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method, the viruses were harvested after the appearance of plaque lesions in culture, and were serially passaged and preserved in CEF cells to rescue the recombinant virus in which the expression framework CAGW-F2AHA was inserted between nucleotides 95322-95323 of the HVT FC126 strain genome, and the expression framework CMV-IBDVC2 was inserted between nucleotides 112071-112088 of the HVTFC126 strain genome, and the recombinant virus was named H30501.

3. A method of preparing a vaccine for Newcastle disease, avian influenza and infectious bursal disease of chickens comprising the steps of: expressing the Newcastle disease virus F gene, the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus VP2 gene of a recombinant turkey herpes virus as claimed in claim 1.

Images