Construction method and application of recombinant herpesvirus turkey for co-expressing NDV F gene and H9 subtype AIV HA gene

Abstract

The invention provides a construction method and application of a recombinant herpesvirus turkey for co-expressing an NDV (Newcastle Disease Virus) F gene and an H9 subtype AIV (Avian Influenza Virus) HA gene, and belongs to the field of genetic engineering vaccines. The recombinant clone technology is utilized, an expression framework containing a Newcastle disease virus gene VII type strain F gene and an H9 subtype avian influenza virus HA gene is inserted between 95322-95323 nucleotides or between 112071-112088 nucleotides of a turkey herpesvirus FC126 strain, and the recombinant clone with the F gene and HA gene expression framework inserted into a turkey herpesvirus genome is constructed and obtained. Recombinant turkey herpesvirus vaccine strains H20901 and H20902 for co-expressing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene are rescued and obtained by the recombinant turkey herpesvirus vaccine strain H20901 and H20902. The vaccine prepared from the recombinant turkey herpesvirus provided by the invention can provide dual protection for the Newcastle disease virus and the 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 construction method and application of a recombinant turkey herpes virus that co-expresses NDV F gene and H9 subtype AIV HA gene. Background Art

[0002] Newcastle disease (ND) is a virulent, highly contagious respiratory 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. Currently, ND is mainly controlled by inactivated vaccines and attenuated live vaccines. Since the immune effect of attenuated live vaccines is easily interfered by maternal antibodies and the immunity duration of inactivated vaccines is not long, the current ND vaccines must be immunized multiple times to achieve the immune effect, which increases the immune pressure and breeding burden of the flock. Therefore, the market currently urgently needs a new ND vaccine that is not interfered by maternal antibodies, suitable for early vaccination of chicks, and has a long immunity duration.

[0003] 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. 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.

[0004] 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.

[0005] 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

[0006] The object of the present invention is to provide a recombinant turkey herpes virus which co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene.

[0007] 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 and the H9 subtype avian influenza virus HA gene.

[0008] The present invention also aims to provide an application of a recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene.

[0009] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0010] A recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene, wherein the recombinant turkey herpes virus is obtained by inserting an expression framework CAGW-F2AHA comprising a chicken β-actin promoter, the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene coding sequence F2AHA, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence into the turkey herpes virus genome;

[0011] 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;

[0012] The expression frame CAGW-F2AHA is obtained by cloning F2AHA 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.

[0013] The method for co-expressing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene of a recombinant turkey herpes virus is characterized by comprising the following steps:

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

[0015] The genomic DNA of strain FC126 was extracted, and the genomic DNA of strain FC126 was cloned into the pCC1Fos vector in segments, and 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that cloned the genomic DNA fragments of strain FC126 and could be spliced ​​to cover the complete HVT genome were selected; among them, HVT01 contained the nucleotide fragment of 1-30189 of the FC126 genome, HVT02 contained the nucleotide fragment of 21572-61476 of the FC126 genome, HVT03 contained the nucleotide fragment of 50871-90478 of the FC126 genome, HVT04 contained the nucleotide fragment of 73762-108139 of the FC126 genome, HVT05 contained the nucleotide fragment of 96776-135815 of the FC126 genome, and HVT06 contained the nucleotide fragment of 129602-159160 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 is constructed, wherein the F gene and HA gene coding regions are connected by a porcine Teschovirus 2A self-cleavage peptide coding sequence; the expression frame CAGW-F2AHA is cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 by the Red / ET recombination method, and a recombinant cosmid that co-expresses the F gene and the HA gene is constructed and named HVT04-53-F2AHA; the expression frame CAGW-F2AHA is cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by the Red / ET recombination method, and a recombinant cosmid that co-expresses the F gene and the HA gene is constructed and named HVT05-65-F2AHA;

[0018] (3) Rescue of recombinant HVT expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0019] Extract recombinant cosmids HVT04-53-F2AHA, HVT05-65-F2AHA and cloned HVT The parental cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 of the genomic fragment of FC126 strain; using the calcium phosphate transfection method, the recombinant cosmid HVT04-53-F2AHA was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT05, and HVT06 into CEF cells, and the recombinant virus H20901 with the expression frame F2AHA inserted between nucleotides 95322-95323 of the HVT genome was rescued; using the calcium phosphate transfection method, the recombinant cosmid HVT05-65-F2AHA was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT06 into CEF cells, and the recombinant virus H20902 with the expression frame F2AHA inserted between nucleotides 112071-112088 of the HVT genome was rescued.

[0020] The invention discloses an application of the recombinant turkey herpes virus which co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene in the preparation of Newcastle disease and avian influenza virus vaccines.

[0021] The invention utilizes the recombinant cloning technology to insert the expression frame containing the Newcastle disease virus gene type VII strain F gene and the H9 subtype avian influenza virus HA gene into the 95322-95323 nucleotides or the 112071-112088 nucleotides of the turkey herpes virus FC126 strain genome, and constructs the recombinant turkey herpes virus vaccine strains H20901 and H20902 which co-express the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene. After the recombinant virus vaccine strain is used to immunize specific pathogen-free (SPF) chickens, the SPF chickens can obtain immune protection against the Newcastle disease virus gene type VII virulent virus and avian influenza, and can be used for the preparation of Newcastle disease and avian influenza virus vaccines.

[0022] The vaccine prepared from the recombinant turkey herpes virus provided by the present invention can simultaneously provide multiple protections against Newcastle disease virus and avian influenza virus infection, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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:

[0024] Figure 1 The figure shows the PCR identification results of the recombinant cosmids HVT04-53-F2AHA and HVT05-65-F2AHA, where M is DL5000 Marker, 1 is HVT04-53-F2AHA, 2 is HVT05-65-F2AHA, and 3 is ddH2O;

[0025] Figure 2 The images are of plaque lesions produced by recombinant viruses H20901 and H20902 on CEF;

[0026] Figure 3 The figure is the PCR identification result of the genomic DNA of the recombinant viruses H20901 and H20902, wherein M is DL5000Marker, 1 is the F2AHA gene identification result of H20901, 2 is the F2AHA gene identification result of FC126, 3 is the F2AHA gene identification result of H20902, and 4 is the F2AHA gene identification result of FC126;

[0027] Figure 4 The indirect immunofluorescence assay was used to detect the expression of F and HA proteins in CEF infected with H20901 and H20902;

[0028] Figure 5 The replication kinetics curves of the recombinant viruses H20901, H20902 and the parental virus FC126 on CEF cells;

[0029] Figure 6 The PCR test results of the genetic stability of the recombinant viruses H20901 and H20902 are shown in Figure 1, where M is DL5000Marker, 1 is the F2AHA gene identification result of H20901-F20, 2 is the F2AHA gene identification result of H20902-F20, and 3 is the negative control ddH2O;

[0030] Figure 7 This is a graph showing the detection results of the 20th generation recombinant viruses H20901 and H20902 expressing F and HA proteins; DETAILED DESCRIPTION

[0031] 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.

[0032] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0033] 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 principles of the present invention should be included in the protection scope of the present invention.

[0034] Example 1: Construction and identification of recombinant HVT expressing NDV F and H9 subtype AIVHA genes

[0035] 1.1 Establishment of reverse genetics system for turkey herpesvirus FC126 strain

[0036] 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 with primers pCC1F and pCC1R (see Table 1 for details). According to the sequencing results, 6 recombinant cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 that cloned the genomic DNA fragments of FC126 strain 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 genomic DNA sequence of the HVTFC126 strain 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.

[0037] 1.2 Construction and identification of recombinant plasmids expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0038] 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.

[0039] 1.3 Construction and identification of the recombinant cosmid HVT04-53-F2AHA expressing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene

[0040] 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 HVT04 using the Red / ET recombination method to construct the recombinant cosmid HVT04-53-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 HVT04-53-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 the expectation. 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. The above results showed that the recombinant cosmid HVT04-53-F2AHA was constructed correctly.

[0041] 1.4 Construction and identification of the recombinant cosmid HVT05-65-F2AHA expressing the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene

[0042] 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 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-F2AHA that co-expresses the NDV F gene and the H9 subtype AIV HA gene. The target gene primer NDH9P2F and the target gene downstream homology arm primer HVT65F were used to identify whether the recombinant cosmid HVT05-65-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 the expectation. 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. The above results showed that the recombinant cosmid HVT05-65-F2AHA was constructed correctly.

[0043] Table 1 PCR primers used to construct and identify F gene and HA gene recombinant cosmids

[0044] Primer name Primer nucleotide sequence number pCC1F SEQ ID NO.3 pCC1R SEQ ID NO.4 NDP1F SEQ ID NO.5 NDH9P1R SEQ ID NO.6 NDH9P2F SEQ ID NO.7 H9P2R SEQ ID NO.8 CAGF SEQ ID NO.9 CAGR SEQ ID NO.10 HVT54R SEQ ID NO.11 HVT65F SEQ ID NO.12

[0045] 1.5 Rescue and identification of recombinant HVT expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0046] The recombinant cosmids HVT04-53-F2AHA, HVT05-65-F2AHA and other parental cosmids cloned with HVT FC126 strain genome fragments were extracted using a plasmid extraction kit. The recombinant cosmid HVT04-53-F2AHA and the parental cosmids HVT01, HVT02, HVT03, HVT05, and HVT06 were co-transfected into CEF cells using the calcium phosphate transfection method. The virus was harvested after 4-5 days of culture until plaque lesions appeared, and was continuously passaged and preserved in CEF cells. The recombinant virus with the F2AHA expression frame inserted between nucleotides 95322-95323 of the HVT genome was rescued and named H20901. The recombinant cosmid HVT05-65-F2AHA was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT06 into CEF cells using the calcium phosphate transfection method. The virus was harvested after plaque lesions appeared after 4-5 days of culture, and was continuously passaged and stored in CEF cells. The recombinant virus with the F2AHA expression framework inserted between nucleotides 112071-112088 of the HVT genome was rescued and named H20902. The plaque lesions produced by the recombinant viruses H20901 and H20902 on CEF are shown in Figure 2. Figure 2 shown.

[0047] The genomic DNA of the recombinant viruses H20901 and H20902 was extracted, and the genomic DNA of the parental virus strain FC126 was used as a control. The recombinant viruses H20901 and H20902 were identified by PCR using the target gene-specific primer NDH9P2F and the target gene downstream homology arm primers HVT54R and HVT65F, respectively, and the PCR products were further sequenced and analyzed. The PCR results showed that the above recombinant viruses could amplify a PCR fragment of about 3065 bp in length, which was consistent with the expected size, such as 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 inserted, and the PCR result was negative. The above results showed that the target gene F2AHA was correctly inserted into the genome of the HVTFC126 strain, and the recombinant viruses H20901 and H20902 were correctly constructed.

[0048] Example 2: In vitro biological characteristics analysis of recombinant HVT expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene

[0049] 2.1 Detection of F and HA proteins expressed by recombinant viruses H20901 and H20902

[0050] The recombinant viruses H20901 and H20902 and the parental virus FC126 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 F protein and HA protein was detected by indirect immunofluorescence test using NDV positive serum and HA protein polyclonal antibody as primary antibodies, FITC-labeled rabbit anti-chicken IgG and FITC-labeled goat anti-rabbit IgG as secondary antibodies, respectively. CEF infected with the parental virus FC126 was used as a negative control. The results showed that cells infected with the recombinant viruses H20901 and H20902 were able to react with NDV positive serum and HA protein polyclonal 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 viruses H20901 and H20902 can co-express F protein and HA protein in infected cells.

[0051] 2.2 Analysis of in vitro replication characteristics of recombinant viruses H20901 and H20902

[0052] The recombinant viruses H20901, H20902 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 of the virus was drawn to analyze the in vitro replication characteristics of the recombinant viruses H20901, H20902 and the parental virus in CEF. The results showed that the replication titers of the recombinant viruses H20901, H20902 and the parental virus FC126 reached the highest peak at 120 hours after infection, which were 1.85×10 5 PFU / ml, 1.96×10 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 replication characteristics of the recombinant viruses H20901 and H20902 in CEF are consistent with those of the parental virus FC126 strain.

[0053] 2.3 Genetic stability testing of recombinant viruses H20901 and H20902

[0054] The 5th generation recombinant viruses H20901 and H20902 were continuously passaged on CEF to the 20th generation. The 20th generation recombinant virus was selected, the viral genomic DNA was extracted, PCR identification and sequencing were performed, and the genetic stability of the target gene sequence in the recombinant viral genome was detected. At the same time, the above-mentioned 20th generation recombinant virus was inoculated into CEF cells, and an indirect immunofluorescence test was performed with NDV positive serum and HA polyclonal antibody to detect the expression stability of the target genes F and HA. The PCR identification results showed that the target genes F and HA can both stably exist in the genomes of the recombinant viruses H20901 and H20902, and the above-mentioned 20th generation recombinant viruses can be amplified to obtain a target gene band of approximately 3065bp ( Figure 6 ), which is consistent with expectations. CAGF and CAGR were used to amplify and sequence the recombinant viral genomic DNA, and it was found that the target gene sequence was correct and no mutation occurred during the passage. The above-mentioned 20th generation recombinant virus was taken for indirect immunofluorescence test to detect the expression of the target genes F and HA during the passage. The results showed that after the recombinant viruses H20901 and H20902 were continuously passed on CEF cells to the 20th generation, the target genes F and HA could still be stably expressed ( Figure 7 ). The above results show that the recombinant viruses H20901 and H20902 have good genetic stability.

[0055] Example 3: Safety and immunogenicity testing of recombinant viruses H20901 and H20902

[0056] 3.1 Safety testing of recombinant viruses H20901 and H20902

[0057] The fifth-generation recombinant viruses H20901 and H20902 were 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 effects of the recombinant virus 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 viruses H20901 and H20902 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 inoculation groups of the recombinant viruses H20901 and H20902 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 observed. The above results indicate that the recombinant viruses H20901 and H20902 are safe for SPF chickens.

[0058] 3.2 Immunogenicity testing of recombinant viruses H20901 and H20902

[0059] 80 one-day-old SPF chicks were randomly divided into 4 groups, 20 in each group; Group 1 was subcutaneously inoculated with recombinant virus H20901 at a dose of 4000 PFU / chicken, Group 2 was subcutaneously inoculated with recombinant virus H20902 at a dose of 4000 PFU / chicken, Group 3 was inoculated with parental virus FC126 at the same dose, and Group 4 was not immunized as a blank control group. 28 days after immunization, 10 chickens in Groups 1-3 were challenged with the virulent GM strain of NDV of type VII, observed for 14 days after the challenge, and the clinical symptoms of the test chickens were recorded, and the survival of the chickens in each group was counted. The remaining 10 chickens in Groups 1-3 were challenged with the H9 subtype AIV LC18 strain, observed for 14 days after the challenge, and the clinical symptoms of the test chickens were recorded; 5 days after the challenge, the laryngeal and cloacal swabs of each chicken in each group were collected, 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 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. 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. Group 4 was not challenged with virus and served as the healthy control group.

[0060] The results showed that all 10 chickens in the parental virus FC126 inoculation group died after NDV infection, while all the experimental chickens in the recombinant virus H20901 and H20902 inoculation groups survived after NDV infection without obvious clinical symptoms. After the experimental chickens were infected with H9 subtype AIV, the virus isolation of 10 chickens in the parental virus FC126 inoculation group was positive, and the virus isolation of the experimental chickens in the blank control group without infection was negative. After the recombinant virus H20901 inoculated chickens with H9 subtype AIV, the feeding, drinking, mental state, and movement of all chickens were normal during the observation period, without obvious adverse clinical symptoms, of which 1 chicken was positive for virus isolation, and the virus isolation of the remaining 9 chickens was negative. After the recombinant virus H20902 inoculated chickens with H9 subtype AIV, the feeding, drinking, mental state, and movement of all chickens were normal during the observation period, without obvious adverse clinical symptoms, and the virus isolation was negative. The above results show that the recombinant viruses H20901 and H20902 have good immune protection effects against NDV genotype VII virulent and H9 subtype AIV.

Claims

1. A recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene, characterized in that: The recombinant turkey herpes virus is obtained by inserting the expression framework CAGW-F2AHA containing chicken β-actin promoter, Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene coding sequence F2AHA, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence into the turkey herpes virus genome; 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; The expression frame CAGW-F2AHA is obtained by cloning F2AHA 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.

2. A method for constructing a recombinant turkey herpes virus that co-expresses the Newcastle disease virus F gene and the H9 subtype avian influenza virus HA gene as described in claim 1, characterized in that: The following steps are involved: (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 HVT01, HVT02, HVT03, HVT04, HVT05 and HVT06 were selected, which contained genomic DNA fragments of FC126 strain and could be assembled to cover the complete HVT genome. Among them, HVT01 contained nucleotide fragments of 1-30189 of FC126 genome, HVT02 contained F The nucleotide fragment of C126 genome is 21572-61476, HVT03 contains the nucleotide fragment of FC126 genome at 50871-90478, HVT04 contains the nucleotide fragment of FC126 genome at 73762-108139, HVT05 contains the nucleotide fragment of FC126 genome at 96776-135815, and HVT06 contains the nucleotide fragment of FC126 genome at 129602-159160; (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 containing a chicken β-actin promoter, a Newcastle disease virus F gene and an H9 subtype avian influenza virus HA gene coding sequence, 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 a porcine Teschovirus 2A self-cleavage peptide coding sequence; the expression framework CAGW-F2AHA was recombined using the Red / ET recombination method. The expression frame CAGW-F2AHA was cloned into the 95322-95323 nucleotides of the FC126 genome in the recombinant cosmid HVT04 to construct a recombinant cosmid that co-expresses the F gene and the HA gene, named HVT04-53-F2AHA; the expression frame CAGW-F2AHA was cloned into the 112071-112088 nucleotides of the FC126 genome in the recombinant cosmid HVT05 by the Red / ET recombination method to construct a recombinant cosmid that co-expresses the F gene and the HA gene, named HVT05-65-F2AHA; (3) Rescue of recombinant HVT expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene. Extraction of recombinant cosmids HVT04-53-F2AHA, HVT05-65-F2AHA and cloned HVT The parental cosmids HVT01, HVT02, HVT03, HVT04, HVT05, and HVT06 of the genomic fragment of FC126 strain; using the calcium phosphate transfection method, the recombinant cosmid HVT04-53-F2AHA was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT05, and HVT06 into CEF cells, and the recombinant virus H20901 with the expression frame F2AHA inserted between nucleotides 95322-95323 of the HVT genome was rescued; using the calcium phosphate transfection method, the recombinant cosmid HVT05-65-F2AHA was co-transfected with the parental cosmids HVT01, HVT02, HVT03, HVT04, and HVT06 into CEF cells, and the recombinant virus H20902 with the expression frame F2AHA inserted between nucleotides 112071-112088 of the HVT genome was rescued.

3. Use of the recombinant turkey herpes virus co-expressing Newcastle disease virus F gene and H9 subtype avian influenza virus HA gene as claimed in claim 1 in the preparation of Newcastle disease and avian influenza virus vaccines.

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