Recombinant Marek's disease virus co-expressing H9 subtype AIV HA gene and IBDV novel variant VP2 gene and application thereof

Abstract

The invention provides a recombinant chicken Marek's disease virus for co-expressing an H9 subtype avian influenza virus HA gene and a novel variant VP2 gene of an infectious bursal disease virus as well as a construction method and application of the recombinant chicken Marek's disease virus. According to the invention, by using a recombinant cloning technology, an expression framework containing an H9 subtype avian influenza virus HA gene and a novel variant VP2 gene of an infectious bursal disease virus is inserted between 105561-105563 nucleotides of a serum type 1 chicken Marek's disease virus CVI988 strain genome; according to the method, a recombinant cosmid is constructed and obtained, wherein an H9 subtype avian influenza virus HA gene and an infectious bursal disease virus novel variant VP2 gene expression framework are inserted into a chicken Marek's disease virus genome, and a recombinant virus C20403 strain co-expressing the H9 subtype avian influenza virus HA gene and the infectious bursal disease virus novel variant VP2 gene is rescued and obtained by the recombinant cosmid. The vaccine prepared from the recombinant chicken Marek's disease virus provided by the invention can prevent avian influenza, chicken infectious bursal disease and chicken Marek's disease at the same time, 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 Marek's disease virus that co-expresses the HA gene of the H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus and an application thereof. Background Art

[0002] Infectious bursal disease (IBD) in chickens is an acute, highly contagious, immunosuppressive infectious disease caused by infectious bursal virus (IBDV) that mainly harms chicks. Since 2017, atypical IBD outbreaks have occurred in many provinces in my country. The production performance and immune function of sick chickens are low, and autopsy shows severe atrophy of the bursa of Fabricius. Studies have found that the strain causing the outbreak is a new variant of IBDV (varIBDV). However, the commercial IBD vaccines currently in use can no longer provide effective immune protection against the newly emerged varIBDV, so there is an urgent need for an IBD vaccine that can prevent new variants. The H9 subtype avian influenza virus (AIV) is another pathogen that seriously harms China's poultry industry. The isolation rate of this virus in my country has increased year by year in recent years, indicating that traditional inactivated vaccine immunization can no longer effectively prevent and control H9 subtype AIV infection, and there is an urgent need to develop a safer and more efficient new vaccine.

[0003] Marek's disease (MD) is a highly contagious, lymphoproliferative, immunosuppressive tumor disease of chickens caused by Marek's disease virus (MDV). At present, the serotype 1 MDV attenuated live vaccine CVI988 strain 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. This type of vaccine induces a wide range of immunity produced by the body, can express multiple antigens at the same time, and is made into a multivalent or multi-unit vaccine, which has the effect of preventing multiple diseases with one shot. It is one of the main directions of current and future vaccine research and development. 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, which is 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

[0004] The object of the present invention is to provide a recombinant Marek's disease virus which co-expresses the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus.

[0005] The present invention also aims to provide a method for constructing a recombinant Marek's disease virus that co-expresses the HA gene of the H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus.

[0006] The present invention also aims to provide an application of a recombinant Marek's disease virus that co-expresses the HA gene of the H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus.

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

[0008] A recombinant Marek's disease virus that co-expresses the HA gene of an H9 subtype avian influenza virus and the VP2 gene of a new variant of infectious bursal disease virus. The recombinant Marek's disease virus is obtained by inserting an expression framework CAGW-HAA2 comprising a chicken β-actin promoter, the HA gene of an H9 subtype avian influenza virus and the VP2 gene coding sequence HAA2 of a new variant of infectious bursal disease virus, a post-transcriptional regulatory sequence of woodchuck hepatitis virus, and a rabbit β-globulin polyadenylic acid sequence into the Marek's disease virus genome.

[0009] The coding sequence HAA2 is obtained by connecting the coding sequence of the HA gene of the H9 subtype avian influenza virus and the coding sequence of the VP2 gene of the new variant of the infectious bursal disease virus with the coding sequence of the porcine teschovirus 2A self-cleavage peptide. The nucleotide sequence of the coding sequence HAA2 is shown in SEQ ID NO.1.

[0010] The expression frame CAGW-HAA2 is obtained by cloning HAA2 into the chicken β-actin promoter and the rabbit β-globulin polyadenylic acid sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between the HAA2 sequence and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-HAA2 is shown in SEQ ID NO.2.

[0011] The recombinant Marek's disease virus is obtained by co-transfecting CEF cells with recombinant cosmids C1, C2, C3, C5, and C6 containing genomic DNA fragments of serotype 1 Marek's disease virus CVI988 strain and recombinant cosmid C4-45-HAA2 containing the HA gene of H9 subtype avian influenza virus and the VP2 gene expression framework of the new variant of infectious bursal disease virus, and then rescuing the virus; the recombinant cosmids C1, C2, C3, C5, and C6 contain nucleotide fragments at positions 1-37644, 29969-68579, 60962-99647, 115670-152340, and 143712-178311 of the genome of Marek's disease virus CVI988 strain, respectively;

[0012] The recombinant cosmid C4-45-HAA2 is obtained by inserting the expression framework CAGW-HAA2 between the 105561-105563 nucleotides of the CVI988 genome on the basis of C4. The recombinant cosmid C4 contains the 91415-126182 nucleotide fragment of the CVI988 genome.

[0013] A use of the recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus as claimed in claim 1 in the preparation of vaccines for H9 subtype avian influenza, infectious bursal disease and Marek's disease in chickens.

[0014] The invention utilizes the recombinant cloning technology to insert the expression frame including the HA gene of the H9 subtype avian influenza virus and the VP2 gene of the new variant of the infectious bursal disease virus into the nucleotides 105561-105563 of the genome of the Marek's virus CVI988 strain, and constructs the recombinant Marek's disease virus vaccine strain C20403 strain which co-expresses the HA gene of the H9 subtype avian influenza virus and the VP2 gene of the new variant of the infectious bursal disease virus. After the recombinant virus vaccine strain is used to immunize specific pathogen-free (SPF) chickens, the SPF chickens can obtain immune protection against the H9 subtype avian influenza virus, the new variant of the infectious bursal disease virus and the strong strain of the chicken Marek's disease virus, and can be used for the preparation of vaccines for avian influenza, chicken infectious bursal disease and chicken Marek's disease.

[0015] The vaccine prepared by the recombinant Marek's disease virus provided by the present invention can simultaneously provide multiple protections against avian influenza virus, new variants of infectious bursal disease virus and strong Marek's disease virus infection in chickens, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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:

[0017] Figure 1 The figure is the PCR identification result of the recombinant cosmid C4-45-HAA2 expressing the HA gene and the VP2 gene, wherein M is the DL5000 Marker, 1 is the amplification result of the recombinant cosmid C4-45-HAA2, and 2 is the amplification result of the parental cosmid C4;

[0018] Figure 2 The images are of plaque lesions produced by the recombinant virus C20403 and the parental virus CVI988 on CEF;

[0019] Figure 3The figure is the result of PCR identification of the genomic DNA of the recombinant virus C20403 and the parental virus CVI988, wherein M is DL5000 Marker, 1 is the recombinant virus C20403, and 2 is the parental virus CVI988;

[0020] Figure 4 The indirect immunofluorescence assay was used to detect the expression of HA and VP2 proteins in CEF infected with the recombinant virus C20403;

[0021] Figure 5 This is a replication kinetic curve of the recombinant virus C20403 on CEF cells;

[0022] Figure 6 The figure is the result of PCR test for genetic stability of recombinant virus C20403, where M is DL5000 Marker, 1 is the 10th generation C20403, 2 is the 15th generation C20403, 3 is the 20th generation C20403, and 4 is the parent virus CVI988;

[0023] Figure 7 This is the result of indirect immunofluorescence test of HA and VP2 proteins expressed by the 20th generation recombinant virus C20403;

[0024] Figure 8 This is the result of the test of the titer of H9 subtype AIV HI antibody induced in chickens 4 weeks after inoculation of the recombinant virus C20403;

[0025] Fig. 9 This is the statistical result of bursal index of chickens challenged with new variant strain of IBDV after inoculation of recombinant virus C20403. DETAILED DESCRIPTION

[0026] 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.

[0027] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0028] 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.

[0029] Example 1: Construction and identification of recombinant MDV expressing H9 subtype AIV HA gene and varIBDVVP2 gene

[0030] 1.1 Establishment of the reverse genetics operating system of MDV CVI988 strain

[0031] The genomic DNA of serotype 1 MDV attenuated live vaccine CVI988 strain was extracted, and the CVI988 strain genome was cloned into the pCC1Fos vector according to the instructions of the CopyControlFosmidLibraryProduction Kit. According to the results of terminal sequencing of the recombinant cosmid, 6 recombinant cosmids C1, C2, C3, C4, C5, and C6 that cloned the genomic DNA fragments of the CVI988 strain and could be spliced ​​to cover the complete CVI988 genome were selected. Wherein, C1 comprises the nucleotide fragment of CVI988 genome 1-37644, C2 comprises the nucleotide fragment of CVI988 genome 29969-68579, C3 comprises the nucleotide fragment of CVI988 genome 60962-99647, C4 comprises the nucleotide fragment of CVI988 genome 91415-126182, C5 comprises the nucleotide fragment of CVI988 genome 115670-152340, and C6 comprises the nucleotide fragment of CVI988 genome 143712-178311. The GenBank accession number of the described MDV attenuated live vaccine CVI988 strain genome sequence is DQ530348. The above-mentioned 6 recombinant clays cloned with CVI988 genomic DNA fragments are extracted, and the 6 clays are co-transfected into chicken embryo fibroblasts (CEF) by calcium phosphate transfection method. The appearance of cytopathic effects can be observed 4-5 days after transfection, indicating that the parental virus strain CVI988 was rescued.

[0032] 1.2 Construction and identification of recombinant cosmids expressing H9 subtype AIVHA gene and varIBDVVP2 gene

[0033] According to the HA gene coding region sequence of the H9 subtype AIV TJ strain isolated and identified in this laboratory, the porcine Teschen virus 2A (P2A) self-cleavage peptide sequence was added to its 3' end, and the target gene H9HA was synthesized after chicken codon optimization. According to the VP2 gene sequence of the varIBDV FJ-18 strain (GenBank accession number OK167034), the target gene IBDA2 was synthesized after chicken codon optimization design. The target gene HAA2 containing the HA gene and VP2 gene coding sequences was obtained by fusion PCR amplification using primers H9P1F, H9A2P1R, H9A2P2F and A2P2R. The sequence is shown in SEQ ID NO.1, in which the HA gene and VP2 gene coding regions are connected by the self-cleavage peptide P2A coding sequence. The purified PCR product was cloned into the downstream of the chicken β-actin promoter of the pCAGGS vector through the EcoRI and ClaI restriction sites; at the same time, in order to enhance the transcription and translation efficiency of the target gene, the woodchuck hepatitis virus post-transcriptional regulatory sequence was inserted between the target gene HAA2 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-HAA2 that co-expresses the AIVHA gene and varIBDV VP2 gene. The recombinant plasmid was sequenced using primers CAGF and CAGR, and it was found that the cloned target gene expression cassette sequence was correct.

[0034] According to the instructions of the Counter VP2election BAC Modification Kit, the gene expression framework CAGW-HAA2 (SEQ ID NO.2) was cloned into the nucleotides 105561-105563 of the CVI988 genome in the recombinant cosmid C4 using the Red / ET recombination method to construct the recombinant cosmid C4-45-HAA2 that co-expresses the HA gene and VP2 gene. The target gene primer H9A2P2F and the target gene downstream homology arm primer MDV46R were used to identify whether the recombinant cosmid C4-45-HAA2 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 2768 bp was obtained ( Figure 1 ), the size was consistent with the expectation. The sequencing results showed that the PCR product contained the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. The above results indicate that the recombinant cosmid C4-45-HAA2 was constructed correctly.

[0035] Table 1 PCR primers used to construct and identify recombinant cosmids of HA gene and VP2 gene

[0036] Primer name Primer sequence code H9P1F SEQ ID NO.3 H9A2P1R SEQ ID NO.4 H9A2P2F SEQ ID NO.5 A2P2R SEQ ID NO.6 CAGF SEQ ID NO.7 CAGR SEQ ID NO.8 MDV46R SEQ ID NO.9

[0037] 1.3 Rescue and identification of recombinant MDV expressing HA and VP2 genes

[0038] The recombinant cosmid C4-45-HAA2 and five parental cosmids C1, C2, C3, C5, and C6 cloned with genomic fragments of the 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. The virus was harvested after plaque lesions appeared after 4-5 days of culture, and was continuously passaged and preserved in CEF cells. The recombinant virus with the target gene HAA2 expression framework inserted between nucleotides 105561-105563 of the MDV CVI988 strain genome was rescued and named C20403. The plaque lesions produced by the recombinant virus C20403 on CEF are as follows: Figure 2 shown.

[0039] The genomic DNA of the recombinant virus C20403 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 H9A2P2F and the target gene downstream homology arm primer MDV46R, and the PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by amplification of the recombinant virus was about 2768bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parent virus CVI988 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene HAA2 was correctly inserted into the genome of the MDV CVI988 strain, and the recombinant virus C20403 was correctly constructed.

[0040] Example 2: Analysis of in vitro biological characteristics of recombinant MDV expressing H9 subtype AIV HA gene and varIBDVVP2 gene

[0041] 2.1 Detection of HA and VP2 proteins expressed by recombinant virus C20403

[0042] The recombinant virus C20403 and the parent 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 the VP2 protein monoclonal antibody were used as primary antibodies, and FITC-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies. The expression of HA protein and VP2 protein was detected by indirect immunofluorescence test, and CEF infected with the parent virus CVI988 was used as a negative control. The results showed that the cells infected with the recombinant virus C20403 were able to react with the HA protein polyclonal antibody and the VP2 protein monoclonal antibody, showing green fluorescence signals ( Figure 4). No fluorescence was observed in cells infected with the parental virus CVI988 strain and in control cells that were not infected. The above results indicate that the recombinant virus C20403 can co-express HA protein and VP2 protein in infected cells.

[0043] 2.2 Analysis of in vitro replication characteristics of recombinant virus C20403

[0044] The recombinant virus C20403 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 C20403 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus C20403 and the parental virus CVI988 reached the highest peak at 120 hours after infection, which were 9.26×10 4 PFU / ml and 9.68×10 4 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 C20403 has good replication ability on CEF, and its in vitro replication characteristics are consistent with those of the parent virus CVI988 strain.

[0045] 2.3 Genetic stability test of recombinant virus C20403

[0046] The recombinant virus C20403 was continuously passaged on CEF to the 20th generation. The 10th, 15th, and 20th generation cytotoxic cells were selected, and the viral genomic DNA was extracted. The target gene-specific primer H9A2P2F and the downstream homology arm primer MDV46R 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 VP2 protein monoclonal antibodies to detect the expression stability of the target genes HA and VP2.

[0047] PCR identification results showed that the target genes HA and VP2 existed stably in the MDV genome, and the target gene bands of about 2768 bp could be amplified in the 10th, 15th, and 20th generation recombinant viruses ( Figure 6), which is consistent with expectations. The 20th generation recombinant virus genomic DNA was amplified using primers CAGF and CAGR and the PCR products were sequenced, and it was found that the 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 target genes HA and VP2 during the passage process. The results showed that after the recombinant virus C20403 was continuously passed on CEF cells to the 20th generation, the target genes HA and VP2 could still be stably expressed ( Figure 7 ). The above results show that the recombinant virus C20403 has good genetic stability.

[0048] Example 3: Safety and immunogenicity testing of recombinant virus C20403

[0049] 3.1 Safety testing of recombinant virus C20403

[0050] The recombinant virus C20403 was inoculated into 1-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 C20403 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 C20403 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 C20403 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 C20403 is safe for SPF chickens.

[0051] 3.2 Immunoprotective test of recombinant virus C20403 against H9 subtype AIV

[0052] 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 C20403 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 for incubation, and the embryos were illuminated once every 24 hours, and dead chicken embryos were removed in time. After 96 hours of incubation, the allantoic fluid was collected and the HA titer was determined. The titer was not less than 1:16, which was considered positive for virus isolation. The samples with negative virus isolation were blindly propagated for one generation and then tested again.

[0053] The results showed that 28 days after the recombinant virus C20403 was inoculated into one-day-old SPF chicks, the average titer of HI antibody in the test chickens could reach 1:338, while the HI antibody titer in the parent virus CVI988 inoculation group was negative ( Figure 8 ). The above results show that the recombinant virus C20403 induced a good immune response after inoculation of chicks. After the experimental chickens were challenged with H9 subtype AIV, the virus isolation of 10 chickens in the parental virus CVI988 strain inoculation group was positive, 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 C20403 was inoculated with chickens to challenge H9 subtype AIV, during the observation period, all chickens' eating, drinking, mental state, and movement were normal, with no obvious adverse clinical symptoms, and the virus isolation was negative, with an infection protection rate of 100%.

[0054] 3.3 Immunoprotection test of recombinant virus C20403 against varIBDV

[0055] 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 C20403 at a dose of 2000 PFU / chicken, Group 2 was inoculated with the parent virus CVI988 at the same dose, and Group 3 was not immunized as a blank control group. 28 days after immunization, Groups 1 and 2 were challenged with the new variant strain FJ of IBDV, respectively. After 7 days of challenge, the clinical symptoms of the experimental chickens were recorded; 7 days after challenge, the lesions of the bursa of Fabricius were observed and the bursa weight ratio (F / B) and bursa index (BBIX) were calculated. F / B = (bursa weight / body weight) × 1000, BBIX = bursa weight ratio of chickens in the experimental group / cyst ratio of chickens in the blank control group; when BBIX < 0.7, it was judged as atrophy of the bursa of Fabricius; when BBIX ≥ 0.7, it was judged as normal bursa of Fabricius. Group 3 was not challenged and served as a healthy control group.

[0056] The results showed that after the parent virus CVI988 inoculation group was challenged with varIBDV, the bursa of Fabricius showed atrophic lesions, and BBIX was less than 0.7 ( Fig. 9 ); The blank control group without challenge showed no obvious lesions in the bursa of Fabricius. After the recombinant virus C20403 inoculated group was challenged with varIBDV, all survived during the observation period without obvious clinical symptoms. The autopsy showed that there were no obvious lesions in the bursa of Fabricius of 10 chickens, and BBIX was greater than 0.7. The protection rate of C20403 against varIBDV was 100%.

[0057] 3.4 Immunoprotective test of recombinant virus C20403 against virulent MDV

[0058] 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 C20403 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.

[0059] 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. The autopsy showed that 17 chickens in this group had MD characteristic autopsy lesions such as hepatosplenomegaly and organ tumors, and the MD positive rate was 85%. All 20 chickens in the recombinant virus C20403 vaccination group survived after the MDV strong virus was challenged. The autopsy showed that one chicken in this group had splenomegaly lesions, and the remaining 19 chickens did not have MD characteristic lesions. The MD positive reduction rate of the recombinant virus C20403 immunization group was 94%. The above results show that the recombinant virus C20403 has a good immune protection effect against MDV strong virus.

Claims

1. A recombinant Marek's disease virus that co-expresses the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus, characterized in that: The recombinant Marek's disease virus is obtained by inserting the expression framework CAGW-HAA2 containing chicken beta-actin promoter, H9 subtype avian influenza virus HA gene and infectious bursal disease virus novel variant VP2 gene coding sequence HAA2, woodchuck hepatitis virus post-transcriptional regulatory sequence and rabbit beta-globulin polyadenylic acid sequence into the chicken Marek's disease virus genome.

2. The recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus according to claim 1, characterized in that: The coding sequence HAA2 is obtained by connecting the coding sequence of the HA gene of the H9 subtype avian influenza virus and the coding sequence of the VP2 gene of the new variant of the infectious bursal disease virus with the coding sequence of the porcine teschovirus 2A self-cleavage peptide. The nucleotide sequence of the coding sequence HAA2 is shown in SEQ ID NO.

1.

3. The recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus according to claim 1, characterized in that: The expression frame CAGW-HAA2 is obtained by cloning HAA2 into the chicken β-actin promoter and the rabbit β-globulin polyadenylic acid sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between HAA2 and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-HAA2 is shown in SEQID NO.

2.

4. The recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus according to claim 1, characterized in that: The recombinant Marek's disease virus is obtained by co-transfecting CEF cells with recombinant cosmids C1, C2, C3, C5, and C6 containing genomic DNA fragments of serotype 1 chicken Marek's disease virus CVI988 strain and recombinant cosmid C4-45-HAA2 containing the HA gene of H9 subtype avian influenza virus and the VP2 gene expression framework of the new variant of infectious bursal disease virus, and then rescuing the virus through virus rescue; the recombinant cosmids C1, C2, C3, C5, and C6 contain nucleotide fragments at positions 1-37644, 29969-68579, 60962-99647, 115670-152340, and 143712-178311 of the Marek's disease virus CVI988 strain genome, respectively.

5. The recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus according to claim 4, characterized in that: The recombinant cosmid C4-45-HAA2 is obtained by inserting the expression frame CAGW-HAA2 between the 105561-105563 nucleotides of the genome of the Marek's disease virus CVI988 strain on the basis of C4. The recombinant cosmid C4 contains the nucleotide fragment of the 91415-126182 nucleotides of the genome of the CVI988 strain.

6. Use of the recombinant Marek's disease virus co-expressing the HA gene of H9 subtype avian influenza virus and the VP2 gene of a novel variant of infectious bursal disease virus as claimed in claim 1 in the preparation of vaccines for avian influenza, infectious bursal disease and Marek's disease in chickens.

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