A recombinant Marek's disease virus co-expressing Newcastle disease virus F gene and infectious bursal disease virus novel variant VP2 gene and its application

Abstract

The present invention provides a recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus new variant VP2 gene and its application, belonging to the field of genetically engineered vaccines. The present invention utilizes recombinant cloning technology to insert an expression framework comprising the Newcastle disease virus gene type VII strain F gene and the infectious bursal disease virus new variant VP2 gene into the serotype 1 chicken Marek's disease virus CVI988 strain genome between nucleotides 105561-105563, constructing a recombinant cosmid in which the Newcastle disease virus F gene and the infectious bursal disease virus new variant VP2 gene expression framework are inserted into the chicken Marek's disease virus genome, and the recombinant virus C20103 strain that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus new variant VP2 gene is rescued by the recombinant Marek's disease virus. The vaccine prepared by the recombinant chicken Marek's disease virus provided by the present invention can simultaneously prevent Newcastle disease, infectious bursal disease and chicken Marek's disease, and has good market application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering vaccines, and specifically relates to a recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene and its application. Background Art

[0002] Marek's disease (MD), Newcastle disease (ND), and infectious bursal disease (IBD) are the three major diseases of chickens, requiring vaccination for all types of chicken flocks. Mixed infections with Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) often occur in the wild. However, a vaccine suitable for early immunization of day-old chicks in hatcheries, independent of maternal antibodies, is currently lacking for combined immunization against these diseases. Since its first report in my country in 1982, IBDV infection has become widespread in most poultry-producing regions of the country. Since 2017, atypical IBD outbreaks have occurred in my country and neighboring countries. Affected chickens exhibit reduced production performance and immune function, and autopsies reveal severe atrophy of the bursa of Fabricius. The pathogen causing these outbreaks has been identified as a novel variant of IBDV (varIBDV). This novel variant of IBDV is currently prevalent in my country, but commercially available IBD vaccines do not provide adequate protection against this variant, resulting in severe immunosuppression in infected chickens and posing a new threat to the health of my country's poultry industry.

[0003] Marek's disease virus (MDV) causes a highly contagious, lymphoproliferative, and immunosuppressive tumor in chickens. The attenuated live serotype 1 MDV vaccine, strain CVI988, is widely used both domestically and internationally for the prevention and control of Marek's disease in chickens. MDV is a herpesvirus with a large genome and numerous replication-non-essential genes that can be inserted or replaced by exogenous genes, making it an ideal viral vector for constructing recombinant live vector vaccines. MDV replication is strictly cell-bound, allowing the virus to spread between cells without interference from maternal antibodies. This makes it suitable for early immunization. The vaccine can be administered in ovo or at day one of age, enabling early antibody production. After infection with MDV, the organism carries the virus for life, continuously expressing exogenous protein antigens to induce antibody production, thereby conferring long-lasting immunity. Furthermore, MDV's only natural reservoir is poultry, making it safe for other animals and humans. Summary of the Invention

[0004] The object of the present invention is to provide a recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene.

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

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

[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 Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene. The recombinant Marek's disease virus is obtained by inserting an expression framework CAGW-F2AA2 comprising a chicken β-actin promoter, the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene coding sequence F2AA2, a woodchuck hepatitis virus post-transcriptional regulatory sequence, and a rabbit β-globulin polyadenylation sequence into the Marek's disease virus genome.

[0009] The coding sequence F2AA2 is obtained by connecting the Newcastle disease virus F gene coding sequence and the infectious bursal disease virus new variant VP2 gene coding sequence with the porcine teschovirus 2A self-cleavage peptide coding sequence. The nucleotide sequence of the coding sequence F2AA2 is shown in SEQ ID NO.1.

[0010] The expression frame CAGW-F2AA2 is obtained by cloning F2AA2 into the chicken β-actin promoter and the rabbit β-globulin polyadenylation sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between the F2AA2 sequence and the rabbit β-globulin polyadenylation sequence. The nucleotide sequence of the expression frame CAGW-F2AA2 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-F2AA2 containing the expression framework of the Newcastle disease virus F gene and the VP2 gene of the new variant of infectious bursal disease virus, and then rescuing the virus; the recombinant cosmids C1, C2, C3, C5, and C6 contain the 1st to 37644th positions of the genome of Marek's disease virus CVI988 strain, respectively. Nucleotide fragments at positions 29969-68579, 60962-99647, 115670-152340, and 143712-178311; the recombinant cosmid C4-45-F2AA2 is obtained by inserting the expression frame CAGW-F2AA2 between the nucleotides 105561-105563 of the CVI988 strain genome on the basis of C4, and the recombinant cosmid C4 contains the nucleotide fragment at positions 91415-126182 of the CVI988 strain genome.

[0012] The expression frame CAGW-F2AA2 is inserted between nucleotides 105561 and 105563 of the genome of the Marek's disease virus CVI988 strain.

[0013] The invention relates to an application of the recombinant Marek's disease virus co-expressing the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene in the preparation of Newcastle disease, infectious bursal disease and Marek's disease vaccines.

[0014] The present invention utilizes recombinant cloning technology to insert an expression framework containing the F gene of Newcastle disease virus type VII strain and the VP2 gene of a novel variant of infectious bursal disease virus into the genome of Marek's virus strain CVI988 between nucleotides 105561 and 105563. This constructs a recombinant Marek's disease virus vaccine strain C20103 that co-expresses the Newcastle disease virus F gene and the novel variant VP2 gene of infectious bursal disease virus. Immunizing specific pathogen-free (SPF) chickens with this recombinant virus vaccine strain can provide immunity against virulent Newcastle disease virus type VII strains, novel variants of infectious bursal disease virus, and virulent Marek's disease virus. This recombinant virus vaccine strain can be used in the preparation of vaccines for Newcastle disease, infectious bursal disease, and Marek's disease.

[0015] The vaccine prepared from the recombinant Marek's disease virus provided by the present invention can simultaneously provide multiple protections against Newcastle disease virus, new variants of infectious bursal disease virus and virulent 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 provided 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 an undue limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 Figure 1 is the PCR identification result of the recombinant cosmid C4-45-F2AA2 that co-expresses the NDV F gene and VP2 gene, where M is the DL5000 Marker, 1 is the amplification result of the recombinant cosmid C4-45-F2AA2, and 2 is the amplification result of the parental cosmid C4;

[0018] Figure 2 This is a picture of plaque lesions produced by the recombinant virus C20103 on CEF;

[0019] Figure 3Figure 1 is the PCR identification result of the recombinant virus C20103 genomic DNA, where M is DL5000 Marker, 1 is the recombinant virus C20103, 2 is the parent virus CVI988, 3 is the recombinant cosmid C4-45-F2AA2, and 4 is ddH2O;

[0020] Figure 4 The figure shows the expression of F and VP2 proteins in CEF infected with recombinant virus C20103 detected by indirect immunofluorescence assay;

[0021] Figure 5 This is a graph showing the replication kinetics of the recombinant virus C20103 in CEF cells;

[0022] Figure 6 Figure 1 is the PCR test result of the genetic stability of the recombinant virus C20103, where M is DL5000 Marker, 1 is the 6th generation C20103, 2 is the 10th generation C20103, 3 is the 15th generation C20103, 4 is the 20th generation recombinant virus C20103, 5 is the recombinant cosmid C4-45-F2AA2, and 6 is ddH2O;

[0023] Figure 7 This is the result of indirect immunofluorescence test of F and VP2 proteins expressed by the 20th generation recombinant virus C20103;

[0024] Figure 8 This is the survival curve of chickens immunized with recombinant virus C20103 after being challenged with virulent NDV;

[0025] Figure 9 This is the statistical result of the bursal index of chickens immunized with the recombinant virus C20103 and challenged with the new variant strain of IBDV. 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 solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection 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 principles of the present invention should be included in the scope of protection of the present invention.

[0029] Example 1: Construction and identification of recombinant MDV co-expressing NDV F and varIBDV VP2 genes

[0030] 1.1 Establishment of a multi-segment cosmid rescue system for the attenuated live vaccine CVI988 strain of serotype 1 MDV

[0031] Genomic DNA from the attenuated live vaccine strain CVI988 of serotype 1 MDV was extracted and cloned into the pCC1Fos vector in fragments according to the CopyControlFosmidLibraryProductionKit instructions. Based on the results of terminal sequencing of the recombinant cosmids, six recombinant cosmids (C1, C2, C3, C4, C5, and C6) containing cloned CVI988 genomic DNA fragments and capable of being assembled to cover the entire CVI988 genome were selected. C1 comprises a fragment of nucleotides from positions 1 to 37,644 of the CVI988 genome; C2 comprises a fragment of nucleotides from positions 29,969 to 68,579 of the CVI988 genome; C3 comprises a fragment of nucleotides from positions 60,962 to 99,647 of the CVI988 genome; C4 comprises a fragment of nucleotides from positions 91,415 to 126,182 of the CVI988 genome; C5 comprises a fragment of nucleotides from positions 115,670 to 152,340 of the CVI988 genome; and C6 comprises a fragment of nucleotides from positions 143,712 to 178,311 of the CVI988 genome. The genome sequence of the attenuated live MDV vaccine strain CVI988 has a GenBank accession number of DQ530348. The six recombinant cosmids containing cloned CVI988 genomic DNA fragments were extracted and co-transfected into chicken embryo fibroblasts (CEFs) using the calcium phosphate transfection method. Cytopathic effects were observed 4-5 days after transfection, and the parental virus strain CVI988 was rescued.

[0032] 1.2 Construction and identification of recombinant cosmids co-expressing NDV F and varIBDV VP2 genes

[0033] The target gene NDF was synthesized based on the F gene coding region sequence of the NDV genotype VII strain GM (GenBank accession number DQ486859) after chicken codon optimization. The porcine Teschovirus 2A (P2A) self-cleaving peptide sequence was added to its C-terminus. The target gene IBDA2 was synthesized based on the VP2 gene sequence of the varIBDV FJ-18 strain (GenBank accession number OK167034) after chicken codon optimization. The target gene F2AA2 (SEQ ID NO. 1) containing the F and VP2 gene coding sequences was amplified by fusion PCR using primers NDFP1F, NDA2P1R, NDA2P2F, and A2P2R. The F and VP2 gene coding regions were connected by the self-cleaving peptide P2A coding sequence. The purified PCR product was cloned into the pCAGGS vector downstream of the chicken β-actin promoter via the EcoRI and ClaI restriction sites. To enhance the transcription and translation efficiency of the target gene, a woodchuck hepatitis virus posttranscriptional regulatory sequence was inserted between the target gene F2AA2 and the rabbit β-globulin polyadenylation sequence. This resulted in the recombinant plasmid pCAGW-F2AA2, which co-expresses the NDV F gene and the varIBDV VP2 gene. Sequencing of the recombinant plasmid using primers CAGF and CAGR confirmed the correct sequence of the inserted target gene.

[0034] According to the instructions of the Counter Selection BAC Modification Kit, the F2AA2 gene expression framework CAGW-F2AA2 (SEQ ID NO. 2) was cloned into the recombinant cosmid C4 between nucleotides 105561-105563 of the CVI988 genome using the Red / ET recombination method to construct the recombinant cosmid C4-45-F2AA2 that co-expresses the F gene and VP2 genes. PCR was performed using the target gene primer varIBDVP2F and the target gene downstream homology arm primer MDV46R to detect whether the recombinant cosmid C4-45-F2AA2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that the obtained PCR product was 2760 bp in length ( Figure 1 ), the size was consistent with expectations. Sequencing results showed that the PCR product contained the VP2 gene sequence and downstream homology arms of the target gene expression cassette, indicating the correct sequence. The negative control sample (parental cosmid C4) lacked the target gene insert sequence and the PCR result was negative. These results indicate that the recombinant cosmid C4-45-F2AA2 was correctly constructed.

[0035] Table 1 PCR primers used for construction and identification of F gene and VP2 gene recombinant cosmids

[0036]

[0037] 1.3 Rescue and identification of recombinant MDV co-expressing NDV F and varIBDV VP2 genes

[0038] Recombinant cosmid C4-45-F2AA2 and other parental cosmids cloned with genomic fragments of the MDV CVI988 strain were extracted using a plasmid extraction kit. Using the calcium phosphate transfection method, recombinant cosmid C4-45-F2AA2 was co-transfected with parental cosmids C1, C2, C3, C5, and C6 into CEF cells. After 4-5 days of culture, when plaque lesions appeared, the virus was harvested and serially passaged and stored in CEF cells. A recombinant virus with the F2AA2 expression framework inserted between nucleotides 105561-105563 of the CVI988 strain genome was rescued and named recombinant virus C20103. The plaque lesions produced by the recombinant virus C20103 on CEF cells are shown in Figure 2. Figure 2 shown.

[0039] The genomic DNA of the sixth-generation recombinant virus was extracted, and the genomic DNA of the parental virus CVI988 was used as a control. The recombinant virus was identified by PCR using the target gene-specific primer varIBDVP2F and the target gene downstream homology arm primer MDV46R. The PCR product was further sequenced and analyzed. The PCR results showed that the PCR product obtained by amplifying the recombinant virus was 2760 bp in length, which was consistent with the expected size ( Figure 3 Sequencing results showed that the PCR product contained the correct sequence, including the varIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette. However, the parental virus, CVI988, lacked the target gene insertion and the PCR result was negative. These results indicate that the target gene F2AA2 was correctly inserted into the genome of the serotype 1 attenuated live MDV vaccine strain CVI988, and that the recombinant virus C20103 was correctly constructed.

[0040] Example 2: In vitro biological characteristics analysis of recombinant MDV co-expressing NDV F and varIBDV VP2 genes

[0041] 2.1 Detection of recombinant virus C20103 co-expressing F protein and VP2 protein

[0042] The recombinant virus C20103 and the parental virus CVI988 were inoculated into CEF cells, cultured for 3-4 days to develop plaque lesions, and then fixed with anhydrous ethanol. NDV-positive serum and VP2 protein monoclonal antibodies were used as primary antibodies, and FITC-labeled rabbit anti-chicken IgG and FITC-labeled goat anti-mouse IgG were used as secondary antibodies. Indirect immunofluorescence assay was used to identify the expression of F protein and VP2 protein, respectively. CEF infected with the parental virus was used as a negative control. The results showed that cells infected with the recombinant virus C20103 were able to react with NDV-positive serum and VP2 protein monoclonal antibodies, showing green fluorescence signals ( Figure 4 No fluorescence was observed in cells infected with the MDV parent virus CVI988 strain and in control cells that were not infected ( Figure 4 The above results indicate that the recombinant virus C20103 can co-express F protein and VP2 protein in infected cells.

[0043] 2.2 Analysis of in vitro replication characteristics of recombinant virus C20103

[0044] Recombinant virus C20103 and parental virus CVI988 were inoculated onto CEFs in 6-well plates at a dose of 100 plaque-forming units (PFU). Virus-containing cells were harvested every 24 hours after infection until 144 hours post-infection. Viruses collected at each time point were inoculated onto CEFs, and the number of plaques in the viral fluid at each time point was measured. In vitro replication kinetics were plotted to analyze the in vitro replication characteristics of recombinant virus C20103 and parental virus in CEFs. The results showed that the replication titers of both recombinant virus C20103 and parental virus CVI988 reached their peak at 120 hours post-infection, at 1.36×10 5 PFU / ml and 1.29×10 5 PFU / ml, the titer of the recombinant virus at each time point after infection with CEF was not significantly different from that of the parental virus (P>0.05) ( Figure 5 The above results indicate that the replication characteristics of the recombinant virus C20103 in CEF are consistent with those of the parental virus CVI988 strain.

[0045] 2.3 Genetic stability testing of recombinant virus C20103

[0046] The 6th generation recombinant virus C20103 was continuously passaged on CEF for 20 generations. The 6th, 10th, 15th and 20th generation cytotoxic cells were selected, and the viral genomic DNA was extracted for PCR identification and sequencing to detect the genetic stability of the target gene sequence in the recombinant viral genome. At the same time, the above-mentioned 20th generation recombinant virus was inoculated into CEF cells, and an indirect immunofluorescence test was performed using NDV positive serum and VP2 monoclonal antibody to detect the expression stability of the target genes F and VP2. The PCR identification results showed that the target genes F and VP2 were stably present in the MDV genome, and the 6th, 10th, 15th and 20th generation recombinant viruses could all be amplified to obtain a target gene band of 2760 bp ( Figure 6), which was consistent with expectations. The genomic DNA of the 20th generation recombinant virus was amplified using primers CAGF and CAGR and the PCR products were sequenced, revealing that the inserted target gene sequence was correct. The 20th generation recombinant virus was subjected to an indirect immunofluorescence assay to detect the expression of the target genes F and VP2 during the passage process. The results showed that after the recombinant virus C20103 was continuously passaged on CEF cells for the 20th generation, the target genes F and VP2 were still stably expressed ( Figure 7 ). The above results show that the recombinant virus C20103 has good genetic stability.

[0047] Example 3: Safety and immunogenicity testing of recombinant virus C20103

[0048] 3.1 Safety testing of recombinant virus C20103

[0049] One-day-old SPF chickens were inoculated with the sixth-generation recombinant virus C20103 at a dose of 3000 PFU / bird. Clinical symptoms were observed daily after inoculation. Twenty-eight days after inoculation, five birds were randomly selected from each group and weighed to evaluate the effects of recombinant virus C20103 on growth and development. Five birds from each group were euthanized, and organs such as the bursa of Fabricius, thymus, spleen, and liver were collected, weighed, and observed for signs of atrophy or swelling. The results showed that inoculation of SPF chickens with recombinant virus C20103 did not cause adverse clinical reactions, and the birds' food and water intake remained normal. Twenty-eight days after inoculation, weight and necropsy results showed no significant difference in weight between the C20103-inoculated groups and the uninoculated control group. Necropsy of the bursa of Fabricius, thymus, spleen, and liver revealed normal results, with no obvious clinical lesions. These results demonstrate that recombinant virus C20103 is safe for SPF chickens.

[0050] 3.2 Immunoprotective test of recombinant virus C20103 against novel NDV and IBDV variants

[0051] Sixty one-day-old SPF chicks were randomly divided into three groups, 20 each. Group 1 was inoculated subcutaneously with the recombinant virus C20103 at a dose of 3000 PFU / bird, while Group 2 was inoculated with the parental virus CVI988 at the same dose. Group 3 remained unimmunized and served as a blank control. Twenty-eight days after immunization, 10 chickens from each group were challenged with virulent NDV. The chickens were observed for 14 days after challenge, clinical symptoms were recorded, and survival was calculated. The remaining 10 chickens from groups 1 and 2 were challenged with a novel IBDV variant. They were observed for 7 days after challenge, clinical symptoms were recorded, and survival was calculated. Seven days after challenge, the chickens were necropsied to observe bursal lesions and calculate the bursal weight ratio (F / B) and bursal index (BBIX). 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 bursa atrophy; when BBIX ≥ 0.7, it was judged as normal bursa.

[0052] The results showed that all 10 chickens inoculated with the recombinant virus C20103 survived the observation period after being challenged with NDV; all 10 chickens inoculated with the parental virus CVI988 died after being challenged with strong NDV. Figure 8 The results of the IBDV new variant challenge test showed that all 10 test chickens inoculated with the recombinant virus C20103 survived the challenge with no obvious lesions in the bursa of Fabricius and BBIX greater than 0.7. The parent virus CVI988 inoculation group developed atrophic lesions in the bursa of Fabricius after the challenge with the IBDV new variant ( Figure 9 The above results show that the recombinant virus C20103 has a good immune protection effect against both the virulent NDV and the new variant of IBDV.

[0053] 3.3 Immunoprotective test of recombinant virus C20103 against virulent MDV

[0054] Forty one-day-old SPF chicks were randomly divided into two groups, 20 in each. Group 1 was inoculated subcutaneously with the recombinant virus C20103 at a dose of 3000 PFU / chicken at the nape of the neck, while Group 2 was uninoculated and served as the control group. Seven days after inoculation, each group of chickens received an intraperitoneal injection of virulent MDV. Clinical symptoms of the chickens were observed until 80 days after challenge, and morbidity and mortality were recorded in each group. Deceased chickens were promptly necropsied. At the end of the observation period, all chickens were necropsied, and organ lesions were recorded.

[0055] Results showed that 16 chickens in the non-immunized control group died after challenge with MDV, while none of the surviving chickens showed typical clinical signs of MD during the observation period. Autopsies revealed that 18 chickens in this group developed characteristic MD lesions, including hepatosplenomegaly and organ tumors, for a 90% MD-positive rate. All 20 chickens in the C20103-vaccinated group survived after challenge with virulent MDV, with no clinical signs of MD. Autopsies revealed that one chicken developed splenomegaly, while the remaining 19 birds showed no characteristic MD lesions. The C20103-vaccinated group showed a 94% reduction in MD-positive rates. These results demonstrate that recombinant C20103 provides effective immune protection against virulent MDV.

Claims

1. A recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene, characterized in that: The recombinant Marek's disease virus is obtained by inserting the expression frame CAGW-F2AA2 containing the chicken β-actin promoter, the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene coding sequence F2AA2, the woodchuck hepatitis virus post-transcriptional regulatory sequence, and the rabbit β-globulin polyadenylation sequence into the chicken Marek's disease virus genome; The coding sequence F2AA2 is obtained by connecting the coding sequence of the Newcastle disease virus F gene and the coding sequence of the VP2 gene of the new variant of infectious bursal disease virus with the coding sequence of the porcine Teschovirus 2A self-cleavage peptide. The nucleotide sequence of the coding sequence F2AA2 is shown in SEQ ID NO.1; The expression frame CAGW-F2AA2 is obtained by cloning F2AA2 into the chicken β-actin promoter and the rabbit β-globin polyadenylation sequence of the pCAGGS vector, and inserting the woodchuck hepatitis virus post-transcriptional regulatory sequence between F2AA2 and the rabbit β-globin polyadenylation sequence. The nucleotide sequence of the expression frame CAGW-F2AA2 is shown in SEQ ID NO.2; 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-F2AA2 containing the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene expression framework, and then rescuing the virus; the recombinant cosmids C1, C2, C3, C5, and C6 contain the 1st to 37644th positions of the genome of Marek's disease virus CVI988 strain, respectively. Nucleotide fragments at positions 29969-68579, 60962-99647, 115670-152340, and 143712-178311; the recombinant cosmid C4-45-F2AA2 is obtained by inserting the expression frame CAGW-F2AA2 between the nucleotides 105561-105563 of the CVI988 strain genome on the basis of C4, and the recombinant cosmid C4 contains the nucleotide fragment at positions 91415-126182 of the CVI988 strain genome.

2. Use of the recombinant Marek's disease virus co-expressing the Newcastle disease virus F gene and the infectious bursal disease virus novel variant VP2 gene as claimed in claim 1 in the preparation of Newcastle disease, infectious bursal disease and Marek's disease vaccines.

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