CN118325855A - Recombinant Marek's disease virus strain expressing H9N2 subtype AIV HA and IBDV VP2 genes and its construction method and application - Google Patents

Abstract

The invention discloses a recombinant Marek's disease virus strain expressing H9N2 subtype AIV HA and IBDV VP2 genes, and a construction method and application thereof. The invention utilizes recombinant cloning technology to insert a gene fragment CMV-VP2 comprising a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence into the interior of an MDV gene deletion vaccine strain (rMS delta Meq strain) US2 gene, and simultaneously insert a gene fragment SV40-HA comprising an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence into the interior of an rMS delta Meq strain UL41 gene to obtain a recombinant Marek's disease virus strain expressing the H9N2 subtype AIV HA gene and the IBDV VP2 gene, which is named rMDV-VP2-HA. The rMDV-VP2-HA HAs the same in vitro replication capacity and good genetic stability as the parent virus rMS delta Meq, and can provide good protection effect on IBDV super-virulent strain and H9N2 subtype AIV after immunization of chickens. The invention provides a technical means for developing recombinant MDV live vector vaccine for simultaneously preventing IBDV and H9N2 subtype AIV infection.

Description

Recombinant Marek's disease virus strain expressing H9N2 subtype AIV HA and IBDV VP2 genes, construction method and application thereof

Technical Field

The present invention relates to a recombinant Marek's disease virus strain and a construction method and application thereof, and in particular to a recombinant Marek's disease virus strain rMDV-VP2-HA expressing IBDV VP2 gene and H9N2 subtype AIV HA gene and a construction method and application thereof. The present invention belongs to the field of biotechnology.

Background technique

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive, and fatal infectious disease that mainly harms chicks and is caused by the infectious bursal disease virus (IBDV). It is listed as an "important disease affecting the socio-economic situation" by the International Office of Epizootics (OIE). The disease first broke out in the United States in 1957 and is now widely prevalent in Europe, Southeast Asia, Africa, and South America. The prevalence of IBDV in my country is also relatively serious, and it has always threatened the healthy development of the poultry industry. In recent years, the emergence of IBDV super-virulent strains (vvIBDV) and antigenic variants has further aggravated the harm of the disease. The mortality rate of vvIBDV is as high as more than 60%, and the resistant chickens will show immune failure to vaccines such as avian influenza vaccine and Newcastle disease vaccine. Vaccine immunization is the main means of preventing and controlling vvIBDV, but vvIBDV is highly pathogenic and not suitable for cell culture. Vaccine strains can only be obtained by attenuating wild vvIBDV by propagation in chicken embryos or CEF cells. This traditional domestication method is time-consuming and labor-intensive, and the results are random. For the prevention and control of highly pathogenic and easily mutated vvIBDV, especially for the prevention and control of sudden outbreaks, it is urgent to conduct research on the rapid construction of new vaccines. At the same time, the widespread use of existing IBD attenuated vaccines and moderately toxic vaccines not only causes immunosuppression in chickens, but also aggravates the mutation of the virus. Therefore, there is an urgent need to develop a safer and more effective new IBD vaccine.

IBDV belongs to the genus Avian BiRNAvirus of the family BiRNAviridae. Its genome consists of two double-stranded RNA segments. The VP2 protein encoded by segment A is the only capsid protein of IBDV and also the main host protective antigen of IBDV.

After the H9N2 subtype avian influenza virus (AIV) infects chickens, it causes a decrease in egg production in laying hens or causes secondary infections, bringing huge economic losses to the breeding industry. The H9N2 subtype AIV is prevalent in terrestrial poultry and has evolved into different genotypes. At present, the dominant strain of H9N2 subtype AIV in chickens in my country is the G57 genotype, which first appeared in 2007 and became the dominant genotype after 2010. The H9N2 subtype AIV of this genotype not only seriously threatens the healthy development of the breeding industry, but also plays an important role as a gene donor in the production of new influenza viruses such as H7N9 and H10N8, bringing huge challenges to the prevention and control of avian influenza viruses. Inactivated vaccine immunization is the main means of preventing and controlling H9N2 subtype AIV, but it takes a long time to stimulate the body to produce antibodies after immunization, and the antibody maintenance time is short, which can easily cause the chickens to miss the best protective agent. Therefore, the results of epidemic surveys in recent years show that the isolation rate of H9N2 subtype AIV has increased year by year, and traditional inactivated vaccine immunization cannot effectively prevent and control the infection of H9N2 subtype AIV. Therefore, there is an urgent need to develop a safer and more effective new H9N2 subtype AIV vaccine.

AIV belongs to the Orthomyxoviridae family, Influenzavirus genus. It is an enveloped RNA virus with a genome consisting of 8 segments. The HA protein it encodes is the main protective antigen of the virus.

Marek’s Disease (MD) is a highly contagious, tumorous disease caused by Marek’s Disease Virus (MDV) that seriously harms the development of the poultry industry. The disease can cause multiple lymphomas in chickens, leading to chicken failure and death, while damaging the immune organs of the chickens, causing severe immunosuppression, and is prone to complications of other diseases. The disease has a long course and often leads to the elimination of the entire flock. Once the disease occurs, the losses are often particularly huge. Vaccines are the main means of controlling the disease. MD vaccines are a must-use vaccine in breeder flocks and laying hens. The use of this vaccine in broiler flocks can significantly improve breeding efficiency.

Recombinant live vector vaccines are live vaccines that use genetic engineering technology to construct a virus or bacteria into a vector, and then insert foreign genes into it for expression. This type of vaccine induces a wide range of immunity in the body, including humoral immunity, cellular immunity, and even mucosal immunity; more importantly, live vector vaccines can express multiple antigens at the same time to make multivalent or multi-unit vaccines, which can prevent multiple diseases with one shot. It is one of the main directions of current and future vaccine research and development. Studies have shown that the herpes virus genome is large and has 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 present, there have been a large number of research reports on herpes virus live vector vaccines. As a member of the herpesviridae family, MDV is also a good live vaccine vector. MDV is immunized at 1 day old in chickens, so immunity occurs early and exogenous proteins can be expressed continuously, thus producing lasting immunity in the body. Secondly, MDV is a cell-bound virus. After vaccination, the virus spreads between cells and is relatively less interfered by maternal antibodies. In addition, MDV exists in cells and is not easy to cause vector effects. Different types of MDV can be used for synergistic immunity or multiple vaccinations. The natural host of MDV is only poultry, and it is safe for other livestock and humans. Compared with HVT vectors, MDV serotype I vaccine vectors can provide better immune protection against MD.

At present, the vaccines used for MD prevention are mainly serotype 1 vaccines, with representative strains being CVI988, 814 and rMSΔMeq, which have good protection against MDV super-virulent. Among them, the CVI988 strain was isolated and identified in the Netherlands in the 1970s, and the 814 strain was isolated and identified by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and the serotype 1 MDV attenuated vaccine strain was successfully cultivated in 1981. The rMSΔMeq strain was isolated and identified by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences and is a new type of Marek's disease gene-deficient live vaccine successfully developed in 2021. The vaccine has good immunogenicity and can resist attacks of MDVs of different virulences. Since it was launched in 2022, it has good safety and no adverse reactions to chickens. The rMSΔMeq strain has the characteristics of the current MDV epidemic strains in my country, and its traits are stable, making it an ideal vector for the study of MDV live vector vaccines.

In previous studies, the inventors of the present invention successfully established a multi-fragment rescue system for the Meq gene-deficient vaccine strain of Marek's disease virus (rMSΔMeq strain). In the present invention, this platform was used to construct a recombinant Marek's disease virus (rMDV-VP2-HA) expressing the IBDV protective antigen VP2 gene and the H9N2 subtype AIV protective antigen HA gene, and its biological activity and immune efficacy were evaluated. The results showed that rMDV-VP2-HA inoculated SPF chickens could induce significant IBDV-specific antibodies and H9N2 subtype AIV-specific antibodies, and provide complete protection against the attacks of IBDV super-virulent strains and H9N2 subtype AIV. The immunized chickens survived completely after the attack and did not excrete toxins, and had no clinical symptoms, and the bursa of Fabricius of the immunized chickens did not atrophy.

Summary of the invention

The object of the present invention is to provide a recombinant Marek's disease virus strain rMDV-VP2-HA expressing IBDV VP2 gene and H9N2 subtype AIV HA gene, and a construction method and application thereof.

In order to achieve the above object, the present invention adopts the following technical means:

The present invention utilizes recombinant cloning technology to insert a gene fragment CMV-VP2 containing a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence into the US2 gene of a Marek's disease virus Meq gene-deficient vaccine strain (rMSΔMeq strain), and replace the nucleotide sequence at positions 156501-157116 of the genome sequence, thereby constructing a recombinant cosmid having a CMV-VP2 expression frame inserted into the US2 gene. Meanwhile, a gene fragment SV40-HA containing an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence is inserted into the UL41 gene of the rMSΔMeq strain, and replaces the nucleotide sequence at positions 96927-97106 of the genome sequence, thereby constructing a recombinant cosmid having an SV40-HA expression frame inserted into the UL41 gene. The two recombinant cosmids are co-transfected with the other three cosmids containing the rMSΔMeq genome to rescue CEF cells to obtain a recombinant cosmid expressing IBDV. Recombinant Marek's disease virus rMDV-VP2-HA with VP2 gene and H9N2 subtype AIVHA gene. Studies have shown that the recombinant Marek's disease virus rMDV-VP2-HA obtained by the present invention has the same in vitro replication ability and good genetic stability as the parent virus rMSΔMeq strain, and rMDV-VP2-HA can provide good protection against both IBDV super strains and H9N2 subtype AIV after immunizing chickens.

On the basis of the above research, the present invention proposes a recombinant Marek's disease virus strain expressing IBDV VP2 gene and H9N2 subtype AIV HA gene. The recombinant Marek's disease virus strain is obtained by using recombinant cloning technology to insert a gene fragment CMV-VP2 containing a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence into the US2 gene of a Marek's disease virus Meq gene-deficient vaccine strain, and at the same time, insert a gene fragment SV40-HA containing an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence into the UL41 gene of a Marek's disease virus Meq gene-deficient vaccine strain, thereby simultaneously expressing the IBDV VP2 gene and the H9N2 subtype AIV HA gene.

Among them, preferably, the Marek's disease virus Meq gene deleted vaccine strain is rMSΔMeq strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and its microbial deposit number is: CGMCC No.4612.

Among them, preferably, a gene fragment containing a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence is inserted into the US2 gene of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain, and replaces the nucleotide sequence at positions 156501-157116 of the rMSΔMeq strain genome sequence. At the same time, a gene fragment SV40-HA containing an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence is inserted into the UL41 gene of the rMSΔMeq strain, and replaces the nucleotide sequence at positions 96927-97106 of the rMSΔMeq strain genome sequence, thereby obtaining a recombinant Marek's disease virus strain that simultaneously expresses the IBDV VP2 gene and the H9N2 subtype AIVHA gene.

Among them, preferably, the H9N2 subtype AIV is the influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) strain, which is deposited in the China Center for Type Culture Collection with the deposit number: CCTCC No. V202333.

Among them, preferably, the nucleotide sequence of the gene fragment CMV-VP2 is shown as SEQ ID NO.12, and the nucleotide sequence of the gene fragment SV40-HA is shown as SEQ ID NO.13.

Furthermore, the present invention also proposes a method for constructing the recombinant Marek's disease virus strain, which comprises the following steps:

(1) Construction of a reverse genetics system for the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq

The reverse genetic operating system comprises 5 cosmids, which are obtained by cloning the gene fragment of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain into pCC1Fos, and the 5 cosmids respectively contain the nucleotide sequences of the genome of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526, respectively, and are named pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5, wherein pMS-3 comprises the UL41 gene of the rMSΔMeq strain, and pMS-4 comprises the US2 gene of the rMSΔMeq strain; the genome sequence of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain GenBank accession number is JQ314003.1;

(2) Construction and identification of recombinant cosmids pMS-VP2 and pMS-HA expressing target genes

pMS-3 cosmid and pMS-4 cosmid were transfected into competent cells containing pRed E/T expression plasmid to prepare corresponding competent cells; rpsl-neo expression cassette DNA was used as template, primers UL41-rpsl F/R and US2-rpsl F/R were used to amplify by PCR to obtain rpsl-neo gene fragments containing homologous arms at both ends of the UL41 insertion site and homologous arms at both ends of the US2 insertion site, which were purified and transfected into competent cells containing pMS-3 and pMS-4 to prepare competent cells; expression plasmid containing IBDV VP2 gene was used as template, primers US2-CMV F/R were used to amplify by PCR to obtain CMV-VP2 gene fragments containing homologous arms at both ends of the US2 insertion site, which were purified and electrotransfected into competent cells containing rpsl-neo; expression plasmid containing H9N2 subtype AIV HA gene was used as template, primers UL41-SV40 F/R was amplified by PCR to obtain the SV40-HA gene fragment containing the homologous arms at both ends of the UL41 insertion site, which was purified and transfected into competent cells containing rpsl-neo. Positive colonies were picked and expanded for culture and then cosmids were extracted. The obtained cosmids were named pMS-VP2 and pMS-HA respectively; the names and sequences of the primers required for construction are as follows:

;

(3) Rescue of recombinant Marek's disease virus rMDV-VP2-HA

Extract the five cosmid DNAs of pMS-1, pMS-2, pMS-HA, pMS-VP2 and pMS-5; co-transfect the five cosmids into the secondary CEF by calcium phosphate transfection method, observe whether the transfected cells show MDV-specific plaque lesions after 4-5 days; harvest the virus after plaques appear, continuously passage and preserve in CEF cells, extract the genomic DNA of the recombinant virus by kit, and set up the parental virus genomic DNA control at the same time, identify by PCR with primers VP2F/R: 5’ATGACAAACCTGCAAGATCA3’, 5’TTACCTTAGGGCCCGAATTA3’ and HA-F/R: 5’ATGGAGACAGTATCACTAAT 3’, 5’TTATATACAAATGTTGCATC 3’, and sequence the identified recombinant virus correctly, and name it rMDV-VP2-HA.

Among them, preferably, the plasmid containing the IBDV VP2 gene is obtained by amplifying the VP2 gene using the IBDV HLJ-0504 strain genome as a template and cloning it into the CMV expression cassette of the vector pEGFP-N1; the expression plasmid containing the H9N2 subtype AIV HA gene is obtained by codon-optimizing the HA gene of type A influenza virus A/chicken/Guangxi/S11583/2019 (H9N2) and cloning it into the SV40 expression cassette of the vector pcDNA3.1.

Among them, preferably, the nucleotide sequence of the gene fragment CMV-VP2 containing the CMV promoter sequence, IBDV VP2 gene and terminator sequence in the expression plasmid containing the IBDV VP2 gene is as shown in SEQ ID NO.12; the nucleotide sequence of the gene fragment SV40-HA containing the SV40 promoter, H9N2 subtype AIV HA gene and terminator sequence in the expression plasmid containing the H9N2 subtype AIVHA gene is as shown in SEQ ID NO.13.

Furthermore, the present invention also proposes the use of the recombinant Marek's disease virus strain in the preparation of a drug for preventing H9N2 subtype AIV and/or IBDV infection.

Among them, preferably, the IBDV is a very strong strain of IBDV or a new variant strain of IBDV.

Among them, preferably, the drug is a recombinant Marek's disease virus live vector vaccine expressing IBDV VP2 gene and H9N2 subtype AIV HA gene.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discloses a recombinant Marek's disease virus strain rMDV-VP2-HA expressing IBDV VP2 gene and H9N2 subtype AIV HA gene and a construction method thereof. Inoculating SPF chickens with the recombinant Marek's disease virus strain rMDV-VP2-HA can induce the chickens to produce IBDV-specific antibodies and H9N2 subtype AIV-specific antibodies, and the immune chickens provide complete protection against the attacks of IBDV super-virulent strains and H9N2 subtype AIV. After the attack, the immune chickens completely survive and do not excrete toxins, and have no clinical symptoms, and the bursa of Fabricius of the immune chickens is not atrophied. The present invention provides a technical means for the development of a recombinant MDV live vector vaccine for preventing IBDV and H9N2 subtype AIV infections at the same time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the results of PCR identification of recombinant cosmids pMS-HA and pMS-VP2;

FIG2 is a diagram showing the cytopathic effect of recombinant Marek's disease virus rMDV-VP2-HA on CEF;

Among them, A is rMDV-VP2-HA; B is Mock;

FIG3 is a diagram showing the PCR identification results of the recombinant Marek's disease virus rMDV-VP2-HA genomic DNA;

FIG4 is a graph showing the results of indirect immunofluorescence assay for detecting the expression of VP2 protein and HA protein in CEF infected with recombinant Marek's disease virus rMDV-VP2-HA and parental virus rMSΔMeq;

FIG5 is a graph showing the replication kinetics of the recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq on CEF;

FIG6 is a graph showing the results of antibody titer detection of IBDV HLJ-0504 strain induced by chickens immunized with recombinant Marek's disease virus rMDV-VP2-HA and parental virus rMSΔMeq 4 weeks after vaccination;

FIG7 is a graph showing the results of the HI antibody titer test induced by chickens immunized with the recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq for 4 weeks;

FIG. 8 is a graph showing the immune protection results of chickens immunized with the recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq against the challenge of IBDV HLJ-0504 strain.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope 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 or replacements all fall within the scope of protection of the present invention.

Example 1 Construction of Marek's disease virus Meq gene deleted vaccine strain rMSΔMeq

The Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq is described in a patent application with publication number CN102363769B and invention name "Chicken Marek's disease virus Meq gene-deficient vaccine strain, construction method and application thereof". The specific construction method is as follows:

1. Construction of recombinant virus transfer plasmid:

1. Test methods

1) Design and synthesis of primers: Referring to the published gene sequence of the virulent GA strain of MDV (No.AF147806), primer pairs A and B were designed, targeting fragments A and B on both sides of the Meq gene as homologous sequences for constructing transfer vector plasmids. The upstream of the primer pair LacZB is on the LacZ gene, and the downstream is on the recombination arm B, which is used to identify whether there is recombination; the upstream of the primer pair ALB is on the recombination arm A, and the downstream is on the recombination arm B, which is used to identify the position and number of deletions. If the amplified size of primer ALB is 660 bp, it indicates that the Meq gene is not deleted. If the amplified size is 4288 bp, it indicates that LacZ is inserted in the predetermined position. The upstream and downstream of primer Meq are located on both sides of the Meq gene, respectively, and are used for PCR identification of the recombinant vector rMS-LacZΔMeq. For the detection of the parental virus MS strain, since the Meq gene is intact, the amplified fragment size is about 1.4Kb. For the recombinant strain rMSΔMeq, it is missing 468bp (shown in SEQ ID NO.1), and the amplification result is about 1kb. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. The primer names and sequences are shown in Table 1:

2) Preparation of DNA template: 9-day-old SPF chicken embryos were used to prepare chicken embryo fibroblasts (CEF) according to conventional methods. The MDV MS strain was inoculated into CEF and cultured in a 37°C 5 mL/L CO ₂ incubator. When the pathological rate reached 80%, the cells were digested and harvested, and total DNA was extracted according to conventional methods and dissolved with an appropriate volume of TE (pH 7.4).

3) Amplification and recovery of homologous fragments: The total viral genome DNA extracted above was used as a template for amplification. The reaction system was 25 μL: 17.5 μL of ultrapure water, 2.5 μL of 10×PCR reaction buffer, 1 μL of dNTP (10 mmol∙L-1), 1 μL of upstream and downstream primers (10 pmol∙L-1), 1 μL of rTaq enzyme (5 U∙μL-1), and 2 μL of DNA (0.5 μg∙μL-1). The two PCR reaction conditions were: denaturation at 94°C for 5 min, then denaturation at 94°C for 1 min, annealing at 57°C (annealing of B fragment at 58°C) for 1 min, extension at 72°C for 1 min, 30 cycles, and finally extension at 72°C for 10 min. The amplified products were identified by 1.0% agarose electrophoresis containing ethidium bromide (EB). The correctly identified products were recovered by fragment gel.

4) Construction of transfer vector plasmid: Using genomic DNA of MDV MS strain as template, homology arm A of upstream fragment of Meq gene was obtained by PCR, digested by SalⅠ and SphⅠ, and connected to pUC19 to obtain plasmid pUCA; fragment B was obtained by PCR amplification, digested by SalⅠ and SacⅠ, and connected to pUCA to obtain plasmid pUCAB. The LacZ expression gene cassette obtained by SalⅠ digestion was connected to pUCAB plasmid to obtain plasmid pALacZB. The plasmid was extracted with Qiagen plasmid midi extraction kit, dissolved in TE with pH 7.4, and stored at -20℃ for later use.

II. Acquisition and identification of recombinant virus - rMS△Meq

First recombination: 1) Co-transfection: The total genomic DNA of MDV MS strain and the transfer vector plasmid pALacZB were co-transfected into CEF using the calcium phosphate precipitation method. The specific method is as follows: chicken embryo fibroblasts were prepared. When CEFs grew into a monolayer within 24 hours, they were digested and subcultured into cell culture dishes with a diameter of 60 mm. The CEF inoculation amount was 9×10 ⁶ cells per dish. First, add 388 μL of nuclease-free deionized water to a clean and sterile 1.5 mL centrifuge tube; then add 10-18 μL of the prepared MS strain infected CEF genome total DNA (0.8 μg∙μL-1) and 2 μL of plasmid pALacZB (1 μg∙μL-1); add 30-36 μL TE (pH 7.4); slowly add 62 μL 2mo1∙L-1 CaCl ₂ and mix slowly; use a 1 mL pipette to slowly add 500 μL of 2×HEPES from the bottom, use a 200 μL micropipette to blow about 20 bubbles from the bottom to mix it, and place it in a 37°C incubator for incubation for 30 minutes, during which it will be packaged into small precipitated particles; gently blow and resuspend the calcium phosphate DNA, evenly add it to the surface of the prepared CEF secondary cells, and gently shake the cell culture dish several times. At this time, the culture medium turns yellow-orange and is placed at 37°C with 5% CO ₂ incubator for 2~3h; pour out the upper culture medium, rinse with serum-free culture medium or sterile PBS buffer 3 times, add 2mL of 15% glycerol shock solution to shock for 90s, and then rinse with serum-free culture medium or sterile PBS buffer 3 times; add 3% fetal bovine serum culture medium to promote cell growth into a monolayer, and culture in a 37℃ 5% CO ₂ incubator. The whole process is operated under sterile conditions. After culturing for about 4~6 days, when typical Marek's plaques appear, proceed to the next step of cloning and purification.

2) Plaque cloning and purification of recombinant virus: After plaques appear, place them under an inverted microscope for observation in sequence to prevent missed detection. Pour out the culture medium and add 2mL of 3% maintenance solution, add 20μL of X-Gal (20mg∙mL-1) to make the total concentration 0.2mg∙mL-1, culture at 37°C, 5% CO ₂ for 2h, select blue plaques for digestion, cloning and purification. Add 0.25% trypsin to the circled plaques, digest for several minutes, scratch with the tip of the gun a few times, suck up the digestion solution, add to the chicken embryo fibroblasts that have grown into a monolayer, aspirate some of the maintenance solution from the plate with monolayer cells, wash the circled and digested plaques, add to the chicken embryo fibroblasts that have grown into a monolayer, continue to culture at 37°C, 5% CO ₂ , and purify again after the plaques grow. After cloning, all plaques are blue.

3) Identification of recombinant virus: The recombinant virus purified from plaques was inoculated on primary CEF. When the plaques grew to 70%, the cells were harvested and the total DNA was extracted according to the molecular cloning method. LacZB primers and ALB primers were used for amplification respectively. The reaction system was 25μL: ultrapure water to 17 μL, 0×PCR reaction buffer 2.5μL, dNTP (10mmol∙L-1) 1 μL, upstream and downstream primers (10pmol) 1μL each, rTaq enzyme (5U∙μL-1) 1 μL, recombinant virus DNA 2μL (0.5μg∙μL-1). The reaction conditions for both PCR were: denaturation at 94℃ for 5min, then denaturation at 94℃ for 1min, annealing at 57℃ (annealing temperature of primer ALB is 58℃) for 1min, extension at 72℃ (extension time of primer ALB is 4min) for 1min, 30 cycles, and finally extension at 72℃ for 10min. The PCR product was connected to the pMD-18T vector, and the bacterial solution was sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing and further identification. The purified virus was named: rMS-LacZΔMeq. The recombinant virus rMS-LacZΔMeq was detected by PCR using primer LacZB, and a band consistent with the expected size (1300bp) was obtained.

The copy number of Meq gene deletion was identified using primer ALB. The amplified size of the parental MS strain was about 656 bp, and the recombinant rMS-LacZΔMeq was about 4388 bp. The results showed that the size was consistent with expectations, indicating that the construction was correct. It also proved that both Meq genes of the recombinant virus rMS-LacZΔMeq were deleted.

Second recombination: The method is the same as the first recombination. The total genomic DNA of the MDV rMS-LacZΔMeq strain and the transfer vector plasmid pUCAB are used for co-transfection to obtain the recombinant virus rMSΔMeq strain lacking Meq and LacZ.

Identification method of rMSΔMeq virus strain: extract the total DNA of rMSΔMeq infected CEF according to the conventional method, and use Meq primers for PCR amplification. The fragment size of the parental virus MS strain F5 detection is 1.4Kb, and the amplification result of the recombinant virus rMSΔMeq strain F25 is 1Kb, without 1.4Kb band. The result is consistent with expectations, proving that both Meq genes of the recombinant virus have been deleted. The sequencing results show that the first 468bp of the Meq gene ORF is deleted, which is consistent with the expected results of the experimental design.

The Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the microbiological deposit number is: CGMCC No.4612.

Example 2 Construction of recombinant Marek's disease virus rMDV-VP2-HA and its biological activity and immune efficacy

1. Materials and Methods

1.1 Plasmids, strains and viruses

The Marek's disease virus rMSΔMeq strain (recorded in the patent application with publication number CN102363769B and invention name "Chicken Marek's disease virus Meq gene deleted vaccine strain, its construction method and application", prepared according to the method of Example 1) and the chicken infectious bursal disease virus HLJ-0504 strain (recorded in the patent application with publication number CN117625688B and invention name "Subtype B avian metapneumovirus reverse genetic operating system and its application") were preserved in this laboratory; influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) (preservation number: CCTCC No. V202333) was isolated, identified and preserved by the National Avian Influenza Reference Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences; the plasmid pRed/ET expressing the recombinase and the plasmid containing the screening gene rpsl-neo were from Counter-Selection BAC Modification Kit, preserved by this laboratory; EPI300-T1 strain and DH10B strain are preserved by this laboratory;

1.2 Main Reagents

Conventional PCR reaction enzymes, restriction endonucleases, and ligases were purchased from TaKaRa; calcium phosphate transfection kits and anti-chicken IgG-FITC and anti-mouse IgG-TRITC were purchased from Invitrogen; plasmid extraction kits were purchased from QIAGEN; cell lysate was purchased from Bio-Time; cell culture medium and fetal bovine serum were purchased from Gibco; dimethyl sulfoxide (DMSO) was purchased from BioFroxx; VP2 monoclonal antibody (MAb) and H9N2 subtype AIV HA polyclonal antibody were prepared and stored by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

1.3 Construction of a reverse genetics system for the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq

The reverse genetics operating system comprises five cosmids containing the nucleotide sequences of the genome of the Meq gene-deficient vaccine strain rMSΔMeq strain of Marek's disease virus at positions 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526, respectively. Each cosmid is produced by using the Copy Control Fosmid library production The kit cloned the gene fragments of the corresponding Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain into pCC1Fos, and named them pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5, respectively, among which pMS-3 contained the UL41 gene of the rMSΔMeq strain, and pMS-4 contained the US2 gene of the rMSΔMeq strain; the GenBank accession number of the genome sequence of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain is JQ314003.1.

1.4 Construction and identification of recombinant cosmids pMS-VP2 and pMS-HA expressing target genes

The pMS-3 cosmid and the pMS-4 cosmid were transfected into competent cells containing the pRed E/T expression plasmid to prepare the corresponding competent cells; the rpsl-neo expression cassette DNA in the Counter-Selection BAC Modification Kit was used as a template, and the primers UL41-rpsl F/R and US2-rpsl F/R were used to amplify the rpsl-neo gene fragment containing the homologous arms at both ends of the UL41 insertion site and the homologous arms at both ends of the US2 insertion site, which were purified and transfected into competent cells containing pMS-3 and pMS-4 to prepare the competent cells; the expression plasmid containing the IBDV VP2 gene was used as a template, and the primers US2-CMV F/R was amplified by PCR to obtain the CMV-VP2 gene fragment containing the homologous arms at both ends of the US2 insertion site, which was purified and then electroporated into competent cells containing rpsl-neo; using the expression plasmid containing the HA gene of influenza A virus A/chicken/Guangxi/S11583/2019(H9N2) as a template, the SV40-HA gene fragment containing the homologous arms at both ends of the UL41 insertion site was amplified by PCR with primers UL41-SV40 F/R, which was purified and then transfected into competent cells containing rpsl-neo, and the positive colonies were picked up for expansion culture and then the clay was extracted, and the obtained clays were named pMS-VP2 and pMS-HA, respectively. Among them, the plasmid containing the VP2 gene of the IBDV HLJ-0504 strain was amplified by using the IBDV HLJ-0504 strain genome as a template and then cloned into the CMV expression cassette of the commercial vector pEGFP-N1, named pCMV-VP2. The plasmid containing the HA gene of the influenza A virus A/chicken/Guangxi/S11583/2019(H9N2) was cloned into the SV40 expression cassette of the vector pcDNA3.1 after codon optimization of the HA gene of the influenza A virus A/chicken/Guangxi/S11583/2019(H9N2), named pSV40-HA. The nucleotide sequence of the gene fragment CMV-VP2 containing the CMV promoter sequence, the IBDV VP2 gene and the terminator sequence in the expression plasmid containing the IBDV VP2 gene is shown in SEQ ID NO. 12; the nucleotide sequence of the gene fragment SV40-HA containing the SV40 promoter, the influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) HA gene and the terminator sequence in the expression plasmid containing the influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) HA gene and the terminator sequence is shown in SEQ ID NO. 13. The names and sequences of the primers required for construction are shown in Table 2.

1.5 Rescue of recombinant Marek's disease virus rMDV-VP2-HA

The five cosmid DNAs of pMS-1, pMS-2, pMS-HA, pMS-VP2, and pMS-5 were extracted using QIAGEN's midi extraction kit. The five cosmids were co-transfected into the secondary CEF by calcium phosphate transfection method. After 4 to 5 days, the transfected cells were observed for MDV-specific plaque lesions. After plaques appeared, the virus was harvested and continuously passaged and stored in CEF cells. The genomic DNA of the recombinant virus was extracted using a kit, and the parental virus genomic DNA control was set up at the same time. The primers VP2F/R (5'ATGACAAACCTGCAAGATCA3', 5'TTACCTTAGGGCCCGAATTA3') and HA-F/R (5'ATGGAGACAGTATCACTAAT3', 5'TTATATACAAATGTTGCATC') were used for PCR identification and sequencing identification. The correctly identified recombinant virus was named rMDV-VP2-HA.

1.6 Expression of target genes VP2 and HA in recombinant Marek's disease virus rMDV-VP2-HA

The recombinant Marek's disease virus rMDV-VP2-HA obtained by the rescue and the parental virus rMSΔMeq strain were inoculated into CEF cells cultured in six-well plates. After 120 h of culture, the cells were collected and fixed with anhydrous ethanol. The VP2 protein monoclonal antibody (1:400) was used as the primary antibody, TRITC-labeled anti-mouse IgG (1:200) was used as the secondary antibody, and the HA polyclonal antibody (1:100) and FITC-labeled anti-chicken IgG (1:200) were used as the secondary antibodies. The expression of VP2 and HA proteins was identified by indirect immunofluorescence assay (IFA). In all the above experiments, CEF inoculated with the parental virus rMSΔMeq was used as a negative control.

1.7 Replication curve of recombinant Marek's disease virus rMDV-VP2-HA

The recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq strain were inoculated into the secondary CEF cells cultured in 6-well plates, and the viruses were collected every 24 hours after infection until 144 hours after infection. The titers of the viruses collected at each time point were measured, and the growth curves were drawn to analyze the in vitro replication characteristics of the recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq in CEF.

1.8 Immune efficacy test of recombinant Marek's disease virus rMDV-VP2-HA

Fifty one-day-old SPF chicks were randomly divided into five groups, with 10 chicks in each group. Groups 1-2 were subcutaneously inoculated with recombinant Marek's disease virus rMDV-VP2-HA at a dose of 2000 PFU/chicken; Groups 3-4 were inoculated with parental virus rMSΔMeq as negative control; Group 5 was not exempted as a blank control. Blood was collected every week after immunization to detect IBDV neutralizing antibodies, and the HI antibody titer was detected by hemagglutination inhibition test. Four weeks after immunization, the experimental chickens in Groups 1 and 3 were nasally infected with H9N2 subtype AIV virus liquid at a dose of 10 CID50. Groups 2 and 4 were infected with the super-virulent HLJ-0504 strain of IBDV by nose drops and eye drops at a dose of 500 ELD50. On the 3rd and 5th day after the virus attack in Group 1 and Group 3, the laryngeal and cloaca swabs of each chicken were collected, added to PBS containing double antibodies, and centrifuged after repeated freezing and thawing three times. The supernatant was taken and inoculated into 11-day-old chicken embryos through the allantoic cavity. The eggs were illuminated every day and incubated for observation for 48 hours to determine the HA titer. If the HA titer ≥ 1:16 (micro-amount method), it can be judged as positive for virus isolation, indicating that the chicken has excreted the virus. Groups 2 and 4 were observed for 7 consecutive days after the virus attack, and the death and morbidity of the experimental chickens in each group were counted. After the observation period, the experimental chickens were dissected, the bursa of Fabricius was collected, and the bursa index (BBIX) was 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.

2. Results

2.1 Construction and identification of recombinant cosmids expressing target genes

The IBDV VP2 gene and expression cassette were amplified, and the recombinant cosmid was constructed using the Red recombination method. The IBDV VP2 gene expression cassette was cloned into the rMSΔMeq recombinant cosmid pMS-4 to construct the pMS-VP2 cosmid. The recombinant cosmid was identified by PCR and sequencing using primers VP2F/R. The results showed that the length of the PCR product obtained was 1359 bp (Figure 1). The sequencing results showed that the PCR product contained the VP2 gene, while the PCR identification result of the negative control sample was negative, with no VP2 gene insertion sequence, indicating that the recombinant cosmid pMS-VP2 was successfully constructed. Similarly, the HA gene and expression cassette were amplified, and the HA gene expression cassette was cloned into the rMSΔMeq recombinant cosmid pMS-3 using the Red recombination method to construct the pMS-HA cosmid. The successfully constructed pMS-HA cosmid was obtained by PCR amplification and sequencing.

2.2 Rescue and PCR identification of recombinant Marek's disease virus rMDV-VP2-HA

The recombinant cosmids pMS-VP2 and pMS-HA were co-transfected into CEF with three other recombinant cosmids pMS-1, pMS-2, and pMS-5 cloned with rMSΔMeq genome fragments. MDV plaque lesions appeared 4-5 days after transfection, and the recombinant Marek's disease virus rMDV-VP2-HA was rescued (Figure 2). The recombinant virus genomic DNA was extracted for PCR identification and sequencing. The results showed that the length of the PCR product was consistent with the expected result (Figure 3), and the sequencing results showed that the insertion sequence was consistent with the expected result. The amplification result of the genomic DNA of the parental virus rMSΔMeq strain was negative. The above results show that the VP2 gene and HA gene are correctly inserted into the rMSΔMeq genome and can exist stably, and the recombinant virus rMDV-VP2-HA is correctly constructed.

2.3 Expression of target genes VP2 and HA of recombinant Marek's disease virus rMDV-VP2-HA

The recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq strain were inoculated into CEF cells, and the cells were collected 120 hours later. The expression of the target genes VP2 and HA was detected by IFA. The results showed that the cells infected with rMDV-VP2-HA reacted with the VP2 monoclonal antibody, and a red fluorescent signal was visible. At the same time, the cells reacted with the HA polyclonal antibody, and a green fluorescent signal was visible. The parental virus rMSΔMeq inoculated and uninoculated cells did not show fluorescence (Figure 4). The above results show that the recombinant virus rMDV-VP2-HA can successfully express the target genes VP2 and HA at the same time.

2.4 Replication curve of recombinant Marek's disease virus rMDV-VP2-HA

The recombinant Marek's disease virus rMDV-VP2-HA and the parental virus rMSΔMeq strain were inoculated into CEF at 100 PFU, and the cells were collected every 24 hours to titrate the virus replication curve. The results showed that the titer of rMDV-VP2-HA at each time point was not significantly different from that of the parental virus rMSΔMeq (P>0.05) (Figure 5). This shows that the replication characteristics of rMDV-VP2-HA in CEF are consistent with those of the parental virus.

2.5 Detection of antibody levels induced by immunization of SPF chickens with recombinant Marek's disease virus rMDV-VP2-HA

The recombinant Marek's disease virus rMDV-VP2-HA was inoculated into one-day-old SPF chickens at a dose of 2000 PFU/chicken. Blood was collected 28 days after immunization, and serum was separated to detect IBDV neutralizing antibodies. The results showed that 28 days after immunization, the average titer of IBDV HLJ-0504 neutralizing antibodies in the serum of chickens immunized with the recombinant Marek's disease virus rMDV-VP2-HA could reach ^28.9 (Figure 6), and the IBDV neutralizing antibodies of chickens immunized with the parental virus rMSΔMeq strain were all negative; the average titer of HI antibodies in the serum of chickens immunized with the recombinant Marek's disease virus rMDV-VP2-HA could reach 8.2, and the HI antibody titers of chickens immunized with the parental virus rMSΔMeq strain were all negative (Figure 7). The above results show that the recombinant Marek's disease virus rMDV-VP2-HA induced a good immune response after immunization of chicks.

2.6 Test on the immune protection efficacy of recombinant Marek's disease virus rMDV-VP2-HA against H9N2 subtype AIV

The recombinant Marek's disease virus rMDV-VP2-HA was inoculated into one-day-old SPF chickens at a dose of 2000 PFU/chicken. Four weeks after immunization, the chickens were challenged with influenza A virus A/chicken/Guangxi/S11583/2019(H9N2) through the nasal infection route. On the 3rd and 5th days after the challenge, laryngeal and cloacal swabs were collected from each chicken to detect virus shedding. The results showed that the recombinant Marek's disease virus rMDV-VP2-HA could completely block the shedding of H9N2 subtype AIV after immunization, and the challenge protection rate was 100% (Table 3).

2.7 Test on the immune protection efficacy of recombinant Marek's disease virus rMDV-VP2-HA against IBDV

The recombinant Marek's disease virus rMDV-VP2-HA was inoculated into 1-day-old SPF chickens at a dose of 2000 PFU/chicken. The IBDV super-virulent strain HLJ-0504 was challenged 4 weeks after immunization. The mortality rate was observed for 7 days after the challenge. The results showed that the experimental chickens in the recombinant Marek's disease virus rMDV-VP2-HA immunization group survived completely after the challenge with the IBDV super-virulent strain HLJ-0504, and no obvious symptoms were observed after the challenge (Figure 8); the parental virus rMSΔMeq strain immunization group showed obvious clinical symptoms after the challenge with the IBDV super-virulent strain HLJ-0504, and 8 out of 10 chickens died; the non-immunized control group died after the challenge with 9 out of 10 chickens. The above results show that the protection rate of chickens immunized with the recombinant Marek's disease virus rMDV-VP2-HA against the attack of the IBDV super-virulent strain HLJ-0504 is 100%.

Claims (10)

1. A recombinant Marek's disease virus strain expressing the VP2 gene of infectious bursal disease virus (IBDV) and the HA gene of avian influenza virus (AIV) of H9N2 subtype, characterized in that the recombinant Marek's disease virus strain is obtained by using recombinant cloning technology to insert a gene fragment CMV-VP2 containing a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence into the US2 gene of a Marek's disease virus Meq gene-deficient vaccine strain, and at the same time, insert a gene fragment SV40-HA containing an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence into the UL41 gene of a Marek's disease virus Meq gene-deficient vaccine strain, thereby simultaneously expressing the IBDV VP2 gene and the H9N2 subtype AIV HA gene. 2. The recombinant Marek's disease virus strain according to claim 1, characterized in that the Marek's disease virus Meq gene deleted vaccine strain is rMSΔMeq strain, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, and its microbiological deposit number is: CGMCC No.4612. 3. The recombinant Marek's disease virus strain as described in claim 2 is characterized in that a gene fragment comprising a CMV promoter sequence, an IBDV VP2 gene and a terminator sequence is inserted into the US2 gene of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain, and replaces the 156501-157116 nucleotide sequence of the rMSΔMeq strain genome sequence, and at the same time, a gene fragment SV40-HA comprising an SV40 promoter sequence, an H9N2 subtype AIV HA gene and a terminator sequence is inserted into the UL41 gene of the rMSΔMeq strain, and replaces the 96927-97106 nucleotide sequence of the rMSΔMeq strain genome sequence, thereby obtaining a recombinant Marek's disease virus strain that simultaneously expresses the IBDV VP2 gene and the H9N2 subtype AIV HA gene. 4. The recombinant Marek's disease virus strain according to claim 3, characterized in that the H9N2 subtype AIV is an influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No. V202333. 5. The recombinant Marek's disease virus strain according to claim 4, characterized in that the nucleotide sequence of the gene segment CMV-VP2 is shown as SEQ ID NO.12, and the nucleotide sequence of the gene segment SV40-HA is shown as SEQ ID NO.13. 6. A method for constructing the recombinant Marek's disease virus strain according to claim 4 or 5, characterized in that it comprises the following steps: (1) Construction of a reverse genetics operating system for the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq: The reverse genetic operating system comprises 5 cosmids, which are obtained by cloning the gene fragment of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain into pCC1Fos, and the 5 cosmids respectively contain the nucleotide sequences of the genome of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain 1-47178, 42801-87422, 87223-125265, 121752-159339 and 159189-177526, respectively, and are named pMS-1, pMS-2, pMS-3, pMS-4 and pMS-5, wherein pMS-3 comprises the UL41 gene of the rMSΔMeq strain, and pMS-4 comprises the US2 gene of the rMSΔMeq strain; the genome sequence of the Marek's disease virus Meq gene-deficient vaccine strain rMSΔMeq strain GenBank accession number is JQ314003.1; (2) Construction and identification of recombinant cosmids pMS-VP2 and pMS-HA expressing target genes: pMS-3 cosmid and pMS-4 cosmid were transfected into competent cells containing pRed E/T expression plasmid to prepare corresponding competent cells; rpsl-neo expression cassette DNA was used as template, primers UL41-rpsl F/R and US2-rpsl F/R were used to amplify by PCR to obtain rpsl-neo gene fragments containing homologous arms at both ends of the UL41 insertion site and homologous arms at both ends of the US2 insertion site, which were purified and transfected into competent cells containing pMS-3 and pMS-4 to prepare competent cells; expression plasmid containing IBDVVP2 gene was used as template, primers US2-CMV F/R were used to amplify by PCR to obtain CMV-VP2 gene fragments containing homologous arms at both ends of the US2 insertion site, which were purified and electroporated into competent cells containing rpsl-neo; expression plasmid containing H9N2 subtype AIV HA gene was used as template, primers UL41-SV40 F/R was amplified by PCR to obtain the SV40-HA gene fragment containing the homologous arms at both ends of the UL41 insertion site, which was purified and transfected into competent cells containing rpsl-neo. Positive colonies were picked and expanded for culture and then cosmids were extracted. The obtained cosmids were named pMS-VP2 and pMS-HA respectively; the names and sequences of the primers required for construction are as follows: ; (3) Rescue of recombinant Marek's disease virus rMDV-VP2-HA: Extract the five cosmid DNAs of pMS-1, pMS-2, pMS-HA, pMS-VP2 and pMS-5; co-transfect the five cosmids into the secondary CEF by calcium phosphate transfection method, observe whether the transfected cells show MDV-specific plaque lesions after 4-5 days; harvest the virus after plaques appear, continuously passage and preserve in CEF cells, extract the genomic DNA of the recombinant virus by kit, and set up the parental virus genomic DNA control at the same time, identify by PCR with primers VP2F/R: 5’ATGACAAACCTGCAAGATCA3’, 5’TTACCTTAGGGCCCGAATTA3’ and HA-F/R: 5’ATGGAGACAGTATCACTAAT 3’, 5’TTATATACAAATGTTGCATC 3’, and sequence the identified recombinant virus correctly, and name it rMDV-VP2-HA. 7. The method of claim 6, characterized in that the expression plasmid containing the IBDV VP2 gene is obtained by amplifying the VP2 gene using the IBDV HLJ-0504 strain genome as a template and cloning it into the CMV expression cassette of the vector pEGFP-N1; the plasmid containing the H9N2 subtype AIV HA gene is obtained by codon-optimizing the HA gene of influenza A virus A/chicken/Guangxi/S11583/2019 (H9N2) and cloning it into the SV40 expression cassette of the vector pcDNA3.1; The nucleotide sequence of the gene fragment CMV-VP2 containing the CMV promoter sequence, IBDV VP2 gene and terminator sequence in the expression plasmid containing the IBDV VP2 gene is shown in SEQ ID NO.12; the nucleotide sequence of the gene fragment SV40-HA containing the SV40 promoter, H9N2 subtype AIV HA gene and terminator sequence in the expression plasmid containing the H9N2 subtype AIV HA gene is shown in SEQ ID NO.13. 8. Use of the recombinant Marek's disease virus strain according to any one of claims 1 to 5 in the preparation of a medicament for preventing infection with H9N2 subtype AIV and/or IBDV. 9. The use according to claim 8, characterized in that the IBDV is a very virulent strain of IBDV or a new variant of IBDV. 10. The use according to claim 8, characterized in that the drug is a recombinant Marek's disease virus live vector vaccine expressing IBDV VP2 gene and H9N2 subtype AIV HA gene.