Recombinant Marek's virus strain for co-expressing H9 subtype AIV HA gene and IBV S gene as well as construction method and application of recombinant Marek's virus strain

Abstract

The invention discloses a recombinant Marek's virus strain for co-expressing an H9 subtype AIV HA gene and an IBV S gene as well as a construction method and application of the recombinant Marek's virus strain, and belongs to the technical field of medicine or veterinary medicine. The recombinant Marek's virus is obtained by co-transfecting CEF cells with recombinant coplasmids containing HA gene and S gene expression cassettes and recombinant coplasmids containing MDV genome DNA fragments and then carrying out virus rescue. Specifically, by using a recombinant cloning technology, an expression cassette containing an H9 subtype AIV HA gene and an IBV S gene is inserted between 105561-105563 nucleotides of a serum type 1 MDV attenuated live vaccine CVI988 strain, so as to construct and obtain a recombinant clone in which the HA gene and the S gene are inserted in an MDV genome, and the recombinant clone rescues to obtain a recombinant MDV vaccine strain C20301 for co-expressing the AIV HA gene and the IBV S gene. The invention also relates to application of the recombinant MDV vaccine strain in preparation of vaccines for simultaneously preventing H9 subtype avian influenza, infectious bronchitis and Marek's disease.

Description

Technical Field

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

[0002] The H9 subtype avian influenza virus (AIV) was first isolated from a turkey flock in the United States in 1966. It is now distributed worldwide. Its widespread prevalence in poultry poses a continuous threat to the global poultry industry and public health security. Since the H9 subtype AIV was first isolated in Guangdong Province in 1992, the disease has been prevalent in my country for more than 30 years. Broilers are the main host of the H9 subtype AIV. In recent years, the virus has continued to maintain a high level of prevalence in my country and has become one of the most serious pathogens affecting the development of my country's broiler farming industry. In particular, when mixed with pathogens such as infectious bronchitis virus (IBV), broilers will suffer from severe bronchial obstruction symptoms, which seriously affects the health of the flock. AIV belongs to the Orthomyxoviridae family. The HA protein it encodes can recognize specific receptors and is the main host protective antigen of the virus, which can induce the production of neutralizing antibodies. 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 the virus infection, and there is an urgent need to develop a safer and more efficient new vaccine.

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

[0004] Marek's disease (MD) is a highly contagious, lymphoproliferative, immunosuppressive tumor disease of chickens caused by Marek's disease virus (MDV). At present, the attenuated live vaccine CVI988 strain of serotype 1 MDV is widely used for the prevention and control of Marek's disease in chickens at home and abroad. Recombinant live vector vaccine is a live vaccine that uses genetic engineering technology to construct a virus or bacteria into a vector, and then inserts foreign genes into it for efficient expression. MDV has a large genome and many replication-non-essential genes that can be inserted or replaced by foreign genes. It is an ideal viral vector for constructing recombinant live vector vaccines. At the same time, MDV is a strictly cell-bound virus. The virus spreads between cells and is not interfered by maternal antibodies, making it suitable for early immunization. In addition, MDV carries the virus for life after vaccination, and can continuously express foreign protein antigens and induce antibodies, thereby giving the body lasting immunity. Summary of the invention

[0005] The present invention aims to provide a recombinant Marek's virus which co-expresses the HA gene of H9 subtype avian influenza virus and the S gene of infectious bronchitis virus and its application in preparing vaccines for H9 subtype avian influenza, infectious bronchitis and Marek's disease.

[0006] In order to achieve the above object, the present invention adopts the following technical means: The invention discloses a recombinant Marek's virus strain for co-expressing the HA gene of avian influenza virus and the S gene of infectious bronchitis virus, which is obtained by inserting the expression framework CAGW-HA2AS containing the chicken β-actin promoter, the AIV HA gene and the IBV S gene coding sequence HA2AS, the woodchuck hepatitis virus post-transcriptional regulatory sequence, and the rabbit β-globulin polyadenylation sequence into the MDV genome.

[0007] Preferably, the MDV is the serotype 1 MDV attenuated live vaccine CVI988 strain, and the GenBank accession number of its genome sequence is DQ530348.

[0008] Among them, preferably, the recombinant Marek's virus strain is obtained by inserting the expression frame CAGW-HA2AS containing chicken β-actin promoter, AIV HA gene and IBV S gene coding sequence HA2AS, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence into the 105561-105563 nucleotides of the MDV CVI988 strain genome.

[0009] Among them, preferably, the AIV is an H9 subtype AIV strain, and the IBV is a QX type IBV strain.

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

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

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

[0013] Furthermore, the present invention also proposes a method for constructing the recombinant MDV virus strain, comprising co-transfecting CEF cells with recombinant cosmids C1, C2, C3, C5, C6 containing MDV genomic DNA fragments and recombinant cosmid C4-45-HA2AS containing HA gene and S gene expression framework, and then performing a virus rescue step; Among them, the recombinant cosmid C1 contains the nucleotide fragment of positions 1-37644 of the CVI988 genome, C2 contains the nucleotide fragment of positions 29969-68579 of the CVI988 genome, C3 contains the nucleotide fragment of positions 60962-99647 of the CVI988 genome, C5 contains the nucleotide fragment of positions 115670-152340 of the CVI988 genome, and C6 contains the nucleotide fragment of positions 143712-178311 of the CVI988 genome.

[0014] Among them, the recombinant cosmid C4-45-HA2AS is obtained by inserting the expression framework CAGW-HA2AS described in claim 1 into C4 between the nucleotides 105561-105563 corresponding to the genome of the MDV CVI988 strain, and the recombinant cosmid C4 contains the nucleotide fragment of the nucleotides 91415-126182 of the genome of the CVI988 strain.

[0015] Among them, preferably, the expression frame CAGW-HA2AS is obtained by cloning the gene fragment HA2AS containing the HA gene coding region sequence, the self-cleaving peptide P2A sequence and the S gene coding region sequence 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 HA2AS sequence and the rabbit β-globulin polyadenylic acid sequence, wherein the nucleotide sequence of the expression frame CAGW-HA2AS is shown in SEQ ID NO.4.

[0016] Furthermore, the present invention also proposes the use of the recombinant MDV virus strain in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bronchitis and Marek's disease.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes recombinant cloning technology to insert an expression framework containing H9 subtype AIV HA gene and IBV S gene into the nucleotides 105561-105563 of the serum type 1 MDV attenuated live vaccine CVI988 strain, constructs a recombinant clay that inserts the HA gene and S gene expression framework into the MDV genome, and rescues the recombinant MDV vaccine strain C20301 that co-expresses AIV HA gene and IBV S gene. Experiments have shown that the recombinant virus C20301 can co-express HA protein and S protein in infected cells, has good replication ability on CEF, and has good genetic stability. Animal experiments have shown that the recombinant virus C20301 is safe for SPF chickens, and can provide good immune protection against H9 subtype AIV, QX type IBV virulent and MDV virulent after inoculation of SPF chickens. The present invention provides a new technical means for preparing vaccines that simultaneously prevent H9 subtype avian influenza, infectious bronchitis and Marek's disease in chickens. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The PCR identification results of the recombinant cosmid C4-45-HA2AS expressing the HA gene and the S gene; Figure 2 The plaque lesions produced by the recombinant virus C20301 and the parent virus CVI988 on CEF; Figure 3 The results of PCR identification of the genomic DNA of the recombinant virus C20301 and the parental virus CVI988; Figure 4 To detect the expression of HA and S proteins in CEF infected with the recombinant virus C20301; Figure 5 is the replication kinetic curve of the recombinant virus C20301 in CEF cells; Figure 6 This is the PCR test result of the genetic stability of the recombinant virus C20301; Figure 7 This is the test result of the 20th generation recombinant virus C20301 expressing HA and S proteins; Figure 8 The results of the test for the AIV HI antibody titer induced by C20301 in SPF chickens 4 weeks after inoculation; Fig. 9 These are the results of the test on the IBV neutralizing antibody titer induced in SPF chickens 4 weeks after inoculation with C20301. DETAILED DESCRIPTION

[0019] The present invention is further described below. The implementation cases introduced in this description are only exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that without departing from the principles and methods of the present invention, the details and forms of the technical solution of the present invention may be partially modified or replaced, but such modification or replacement is within the scope of protection of the present invention. Example 1: Construction and identification of recombinant MDV expressing H9 subtype AIV HA gene and IBV S gene

[0020] 1.1 Establishment of the reverse genetics operating system for MDV CVI988 strain Extract the genomic DNA of the serotype 1 MDV attenuated live vaccine CVI988 strain, the GenBank accession number of the genomic sequence of the MDV attenuated live vaccine CVI988 strain is DQ530348. According to the instructions of the CopyControl Fosmid Library ProductionKit kit, the CVI988 strain genome is segmented and cloned into the pCC1Fos vector. According to the results of the recombinant cosmid terminal sequencing, select 6 recombinant cosmids C1, C2, C3, C4, C5, and C6 that clone the CVI988 strain genomic DNA fragments and can be spliced ​​to cover the complete CVI988 genome. Among them, C1 contains a nucleotide fragment of 1-37644 of the CVI988 genome, C2 contains a nucleotide fragment of 29969-68579 of the CVI988 genome, C3 contains a nucleotide fragment of 60962-99647 of the CVI988 genome, C4 contains a nucleotide fragment of 91415-126182 of the CVI988 genome, C5 contains a nucleotide fragment of 115670-152340 of the CVI988 genome, and C6 contains a nucleotide fragment of 143712-178311 of the CVI988 genome. The above 6 recombinant cosmids cloned with CVI988 genomic DNA fragments were extracted, and the 6 cosmids were co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. The appearance of cytopathic effect can be observed 4-5 days after transfection, that is, the parental virus strain CVI988 was rescued.

[0021] 1.2 Construction and identification of recombinant cosmids expressing HA and S genes According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in our laboratory, the porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence was added to its 3' end, and the target gene H9HA (shown in SEQ ID NO.1) was synthesized after chicken codon optimization. According to the S protein sequence of the QX type IBV SX strain (GenBank accession number OQ189490), its S1-S2 cleavage site was mutated from RRRR to GSAS, and the amino acids at positions 695, 768, 775, 817, 862 and 863 were mutated to proline, and the amino acids at positions 1097-1165 at its C-terminus were replaced with the T4 fiber protein trimer domain, and the target gene IBVS6P (shown in SEQ ID NO.2) was synthesized after chicken codon optimization. The target gene HA2AS (shown in SEQ ID NO.3) containing HA and S coding sequences was obtained by fusion PCR amplification using primers H9P1F, H9S6P1R, H9S6P2F and S6P2R in Table 1, wherein the HA gene and the S gene coding regions were connected by a self-cleaving peptide P2A. The target gene PCR product was purified and cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector; at the same time, the woodchuck hepatitis virus post-transcriptional regulatory sequence was inserted between the target gene HA2AS and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-HA2AS that co-expressed the H9 subtype AIV HA gene and the IBV S gene. The recombinant plasmid was sequenced using primers CAGF and CAGR in Table 1, and it was found that the cloned target gene expression cassette sequence was correct.

[0022] According to the instructions of the Counter Selection BAC Modification Kit, the expression framework CAGW-HA2AS (shown in SEQ ID NO.4) containing the chicken β-actin promoter, the target gene HA2AS, the woodchuck hepatitis virus post-transcriptional regulatory sequence and the rabbit β-globulin polyadenylation sequence was cloned into the recombinant cosmid C4 between nucleotides 105561-105563 of the CVI988 genome to construct the recombinant cosmid C4-45-HA2AS that co-expresses the HA gene and the S gene. The recombinant cosmid C4-45-HA2AS was identified by PCR using the target gene-specific primer IBVS2046F and the downstream homology arm primer MDV46R in Table 1. The results showed that the amplified PCR fragment of about 2700 bp ( Figure 1 ), which was consistent with expectations; sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The parental cosmid C4 had no target gene insertion sequence, and the PCR result was negative.

[0023]

[0024] 1.3 Rescue and identification of recombinant MDV expressing HA and S genes The recombinant cosmid C4-45-HA2AS and five parental cosmids C1, C2, C3, C5, and C6 cloned with genomic fragments of MDV CVI988 strain were extracted using a plasmid extraction kit. The above recombinant cosmids and parental cosmids were co-transfected into CEF cells using the calcium phosphate transfection method. After 4-5 days of culture and the appearance of plaque lesions, the virus was harvested and continuously passaged and preserved in CEF cells. The recombinant virus with the target gene HA2AS expression framework inserted between nucleotides 105561-105563 of the CVI988 strain genome was rescued and named C20301. The plaque lesions produced by the recombinant virus C20301 on CEF are as follows: Figure 2 shown.

[0025] The genomic DNA of the recombinant virus C20301 was extracted, and the genomic DNA of the parent virus CVI988 was set as a control. The recombinant virus was identified by PCR using the target gene specific primer IBVS2046F and the target gene downstream homology arm primer MDV46R in Table 1, and the PCR product was further sequenced and analyzed. The PCR results showed that the recombinant virus amplified a gene fragment of about 2700 bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the S gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The parent virus CVI988 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene HA2AS was correctly inserted into the genome of the MDV CVI988 strain, and the recombinant virus C20301 was correctly constructed. Example 2: Analysis of in vitro biological characteristics of recombinant MDV expressing H9 subtype AIV HA gene and IBV S gene

[0026] 2.1 Detection of HA and S proteins expressed by recombinant virus C20301 The recombinant virus C20301 and the parental virus CVI988 were inoculated into CEF cells, and after plaque lesions appeared after 3-4 days of culture, the cells were fixed with anhydrous ethanol. The HA protein polyclonal antibody and IBV positive serum were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG and FITC-labeled rabbit anti-chicken IgG were used as secondary antibodies. The expression of HA protein and S protein was detected by indirect immunofluorescence test, and CEF infected with the parental virus CVI988 was used as a negative control. The results showed that the cells infected with the recombinant virus C20301 could react with both the HA protein polyclonal antibody and IBV positive serum, showing green fluorescence signals ( Figure 4). No fluorescence was observed in cells infected with the parental virus MDV CVI988 strain and in control cells that were not infected. The above results indicate that the recombinant virus C20301 can co-express HA protein and S protein in infected cells.

[0027] 2.2 Analysis of in vitro replication characteristics of recombinant virus C20301 The recombinant virus C20301 and the parental virus CVI988 were inoculated on CEF in a 6-well plate at a dose of 100 plaque-forming units (PFU). After infection, the virus-containing cells were collected every 24 hours until 144 hours after infection. The viruses collected at each time point were inoculated into CEF, the number of plaques in the virus solution at each time point was determined, and the in vitro replication kinetic curve of the virus was drawn to analyze the in vitro replication characteristics of the recombinant virus C20301 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus C20301 and the parental virus CVI988 reached the highest peak 120 hours after infection, which were 1.22×10 5 PFU / ml and 1.31×10 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 C20301 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus CVI988 strain.

[0028] 2.3 Genetic stability test of recombinant virus C20301 The recombinant virus C20301 was continuously passaged on CEF to the 20th generation. The 10th and 20th generation cytotoxic cells were selected, and the viral genomic DNA was extracted. The target gene-specific primer IBVS2046F and the downstream homology arm primer MDV46R in Table 1 were used for PCR identification. At the same time, the primers CAGF and CAGR were used to perform PCR and sequencing on the viral genomic DNA to detect the genetic stability of the target gene sequence in the recombinant viral genome. The above-mentioned 20th generation recombinant virus was inoculated into CEF cells, and an indirect immunofluorescence test was performed using HA protein polyclonal antibodies and IBV positive serum to detect the expression stability of the target genes HA and S during the passage of the recombinant virus.

[0029] PCR results showed that the target genes HA and S were stably present in the MDV genome, and the target gene bands of about 2700 bp could be amplified in the 10th and 20th generation recombinant viruses ( Figure 6), which is consistent with expectations. The primers CAGF and CAGR in Table 1 were used to amplify the genomic DNA of the 20th generation recombinant virus and sequence the PCR products, and it was found that the target gene sequence inserted into the MDV genome was correct. The above 20th generation recombinant virus was taken for indirect immunofluorescence test to detect the expression of the target genes HA and S during the passage process. The results showed that after the recombinant virus C20301 was continuously passed on CEF cells to the 20th generation, the target genes HA and S could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus C20301 has good genetic stability. Example 3: Safety and immunogenicity testing of recombinant virus C20301

[0030] 3.1 Safety testing of recombinant virus C20301 The fifth-generation recombinant virus C20301 was inoculated into one-day-old SPF chickens at a dose of 2000 PFU / chicken, and the clinical symptoms of the chickens in each group were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their weights were weighed to evaluate the effect of the recombinant virus C20301 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the organs such as the bursa of Fabricius, thymus, spleen, and liver were collected, weighed, and observed for atrophy or swelling. The results showed that the recombinant virus C20301 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 C20301 inoculation group was not significantly different from that of the normal uninoculated control group. The test chickens were autopsied, and the bursa of Fabricius, thymus, spleen, liver and other organs were collected. The results showed that the above organs were normal and no obvious clinical lesions were found. The above results show that the recombinant virus C20301 is safe for SPF chickens.

[0031] 3.2 Immunoprotective test of recombinant virus C20301 against H9 subtype AIV Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus C20301 at a dose of 2000 PFU / chicken, Group 2 was inoculated with the parental virus CVI988 at the same dose, and Group 3 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the HI antibody titer of chickens in each group was detected by hemagglutination inhibition test. 28 days after immunization, Groups 1 and 2 were nasally challenged with H9 subtype AIV LC18 strain respectively; Group 3 was not challenged and served as a healthy control group. Five days after the challenge, laryngeal and cloacal swabs were collected from each chicken in each group, mixed and stored in PBS containing double antibodies for virus isolation. The collected cotton swabs were repeatedly frozen and thawed 3 times and then centrifuged. The supernatant was taken and inoculated into 10-day-old SPF chicken embryos through the allantoic cavity. The chicken embryos were placed in an incubator at 37°C, and the allantoic fluid was collected after 96 hours of incubation to determine the HA titer. A titer of no less than 1:16 was considered positive for virus isolation. Samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0032] The results showed that 28 days after the recombinant virus C20301 was inoculated into one-day-old SPF chicks, the average titer of HI antibody in the test chickens could reach 1:549, while the HI antibody titer in the parent virus CVI988 inoculation group was negative ( Figure 8 ). The above results show that the recombinant virus C20301 induced a good immune response after inoculation of chicks. After the experimental chickens were challenged with H9 subtype AIV, 9 out of 10 chickens in the parental virus CVI988 strain inoculation group were positive for virus isolation, and the virus isolation of the experimental chickens in the blank control group without infection was negative, and the infection test was established. After the recombinant virus C20301 was inoculated with chickens to challenge H9 subtype AIV, during the observation period, all chickens had normal feeding, drinking, mental state, movement, etc., without obvious adverse clinical symptoms, and virus isolation was negative.

[0033] 3.3 Immunoprotective test of recombinant virus C20301 against virulent IBV Thirty one-day-old SPF chicks were randomly divided into three groups, with 10 in each group. Group 1 was subcutaneously inoculated with the recombinant virus C20301 at a dose of 2000 PFU / chicken, Group 2 was inoculated with the parental virus CVI988 at the same dose, and Group 3 was not immunized as a blank control group. Blood was collected 28 days after immunization, serum was separated, and the titer of IBV neutralizing antibodies was detected. 28 days after immunization, Group 1 and Group 2 were challenged with the QX-type virulent SD strain by nose drops and eye drops, respectively; Group 3 was not challenged and served as a healthy control group. After 14 days of observation, the clinical symptoms and deaths of the experimental chickens in each group were recorded; 14 days after the challenge, all experimental chickens were killed and dissected, and the necropsy lesions were observed.

[0034] The results showed that 28 days after the recombinant virus C20301 was inoculated into one-day-old SPF chicks, the average titer of IBV neutralizing antibodies in the experimental chickens could reach 1:147, while the titer of IBV neutralizing antibodies in the parent virus CVI988 inoculation group was negative ( Fig. 9 ). The above results show that the recombinant virus C20301 induced a good immune response after inoculation of chicks. After the 10 chickens in the parental virus CVI988 inoculation group were challenged with IBV, they all showed clinical symptoms such as depression, runny nose, and messy feathers. Among them, 5 died. The autopsy of the diseased chickens showed lesions such as kidney enlargement, spotted kidney, laryngeal and tracheal bleeding, and tracheal mucus. After the 10 chickens in the recombinant virus C20301 inoculation group were challenged with IBV, all the test chickens survived during the observation period. One chicken showed symptoms of depression, and the other 9 chickens had normal feeding, drinking, mental state, and movement, without obvious adverse clinical symptoms; the autopsy showed that the laryngeal bleeding point lesions were observed in the one chicken that was sick, and no obvious autopsy lesions were found in the other 9 chickens. The test chickens in the blank control group without infection showed no abnormal clinical and autopsy symptoms.

[0035] 3.4 Immunoprotective test of recombinant virus C20301 against virulent MDV 40 one-day-old SPF chicks were randomly divided into two groups, 20 in each group. Group 1 was subcutaneously inoculated with the recombinant virus C20301 at a dose of 2000 PFU / chicken, and Group 2 was not inoculated as a control group. Seven days after inoculation, each group of experimental chickens was intraperitoneally injected with 1000 PFU / chicken with a strong GA strain of MDV. After the challenge, the clinical symptoms of the experimental chickens were observed until 70 days after the challenge, and the incidence and mortality of the chickens in each group were counted. Dead chickens were dissected in time, and at the end of the observation period, all experimental chickens were dissected and the lesions of various organs were recorded.

[0036] The results showed that 15 chickens died after the non-immune control group was challenged with MDV, and no typical clinical symptoms of MD were found in the surviving chickens during the observation period. Autopsy revealed that 18 chickens in this group had MD-characteristic autopsy lesions such as hepatosplenomegaly and organ tumors, and the MD-positive rate was 90%. All 20 chickens in the recombinant virus C20301-inoculated group survived after being challenged with strong MDV, without clinical symptoms of MD or characteristic autopsy lesions, and the MD-positive reduction rate was 100%. The above results show that the recombinant virus C20301 has a good immune protection effect against strong MDV.

Claims

1. A recombinant Marek's disease virus (MDV) strain co-expressing the HA gene of avian influenza virus (AIV) and the S gene of infectious bronchitis virus (IBV), characterized in that: The expression frame CAGW-HA2AS containing chicken beta actin promoter, AIV HA gene and IBV S gene coding sequence HA2AS, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit beta globulin polyadenylation sequence is inserted into the MDV genome to obtain the expression frame CAGW-HA2AS.

2. The recombinant MDV strain according to claim 1, characterized in that The MDV is a serum type 1 MDV attenuated live vaccine CVI988 strain, and the GenBank accession number of its genome sequence is DQ530348.

3. The recombinant MDV strain according to claim 1, characterized in that The expression frame CAGW-HA2AS including chicken beta actin promoter, AIV HA gene and IBV S gene coding sequence HA2AS, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit beta globulin polyadenylation sequence is inserted into the 105561-105563 nucleotides of the MDV CVI988 strain genome.

4. The recombinant MDV virus strain expressing the AIV HA gene and the IBV S gene according to claim 1, characterized in that: The AIV is an H9 subtype AIV strain, and the IBV is a QX type IBV strain.

5. The recombinant MDV virus strain according to claim 4, characterized in that The HA gene is obtained by optimizing the chicken codon of the H9 subtype AIV HA gene coding region, and its nucleotide sequence is shown in SEQ ID NO.

1.

6. The recombinant MDV strain according to claim 4, characterized in that The S gene is obtained by mutating the S1-S2 cleavage site in the QX type IBV S protein sequence from RRRR to GSAS, mutating the amino acids at positions 695, 768, 775, 817, 862 and 863 to proline, and replacing the amino acids at positions 1097-1165 at the C-terminus with the T4 fiber protein trimer domain, and optimizing the chicken codon design. The nucleotide sequence is shown in SEQ ID NO.

2.

7. The recombinant MDV strain according to claim 1, characterized in that The AIV HA gene and IBV S gene coding sequence HA2AS is obtained by connecting the AIV HA gene coding sequence and the IBV S gene coding sequence with the porcine Teschovirus 2A (P2A) self-cleavage peptide coding sequence, and its nucleotide sequence is shown in SEQ ID NO.

3.

8. A method for constructing the recombinant MDV strain according to any one of claims 1 to 7, characterized in that: The method comprises the steps of co-transfecting CEF cells with recombinant cosmids C1, C2, C3, C5, C6 containing MDV genomic DNA fragments and recombinant cosmid C4-45-HA2AS containing HA gene and S gene expression cassettes, and then performing virus rescue; The recombinant cosmid C1 comprises a nucleotide fragment of positions 1-37644 of the genome of the CVI988 strain, C2 comprises a nucleotide fragment of positions 29969-68579 of the genome of the CVI988 strain, C3 comprises a nucleotide fragment of positions 60962-99647 of the genome of the CVI988 strain, C5 comprises a nucleotide fragment of positions 115670-152340 of the genome of the CVI988 strain, and C6 comprises a nucleotide fragment of positions 143712-178311 of the genome of the CVI988 strain. Among them, the recombinant cosmid C4-45-HA2AS is obtained by inserting the expression framework CAGW-HA2AS described in claim 1 into the nucleotides corresponding to positions 105561-105563 of the CVI988 strain genome on the basis of C4, and the recombinant cosmid C4 contains a nucleotide fragment of positions 91415-126182 of the CVI988 strain genome.

9. The method according to claim 8, characterized in that The expression frame CAGW-HA2AS is obtained by cloning the gene fragment HA2AS containing the HA gene coding region sequence, the self-cleaving peptide P2A sequence and the S gene coding region sequence 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 HA2AS sequence and the rabbit β-globulin polyadenylic acid sequence, wherein the nucleotide sequence of the expression frame CAGW-HA2AS is shown in SEQ ID NO.

4.

10. Use of the recombinant MDV strain according to any one of claims 1 to 7 in the preparation of a vaccine for simultaneously preventing H9 subtype avian influenza, infectious bronchitis and Marek's disease.

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