Recombinant Marek's disease virus co-expressing Newcastle disease virus F gene and infectious bursal disease virus supervirulent strain VP2 gene and application of recombinant Marek's disease virus

Abstract

The invention provides a recombinant Marek's disease virus co-expressing a Newcastle disease virus F gene and an infectious bursal disease virus supervirulent strain VP2 gene and application thereof, and belongs to the field of genetic engineering vaccines. According to the invention, by using a recombinant cloning technology, an expression framework containing a Newcastle disease virus gene VII type strain F gene and an infectious bursal disease virus super virulent strain VP2 gene is inserted into a spacer region between Marek's disease virus genome UL45 and UL46 genes; according to the method, a recombinant cosmid is constructed and obtained, wherein a Newcastle disease virus F gene and an infectious bursal disease virus super virulent strain VP2 gene expression framework are inserted between Marek's disease virus genome UL45 and UL46 genes, and a recombinant virus C20101 strain co-expressing the Newcastle disease virus F gene and the infectious bursal disease virus super virulent strain VP2 gene is rescued and obtained by the recombinant cosmid. The vaccine prepared from the recombinant Marek's disease virus strain provided by the invention can prevent Newcastle disease, chicken infectious bursal disease and 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 a Newcastle disease virus F gene and an infectious bursal virus super-virulent strain VP2 gene and an application thereof. Background Art

[0002] Newcastle disease (ND) is an acute, highly contagious disease of chickens caused by Newcastle disease virus (NDV). The disease is currently one of the most serious diseases that endanger the poultry industry, and it is also a poultry disease that is given priority prevention and control and focused on prevention in my country's medium- and long-term animal disease prevention and control plan. The F protein encoded by NDV is a glycoprotein on the NDV envelope. It is the main virulence gene and host protective antigen of NDV, and can induce the production of neutralizing antibodies. Since the 1980s, large-scale chicken farms in my country have adopted a high-frequency and intensive immunization program for ND. However, traditional ND vaccines are easily interfered by maternal antibodies and the immunity duration is not long, so that local ND vaccines must be immunized multiple times to achieve the immune effect, thereby increasing the immune pressure and breeding burden of the chicken flock. At present, there is an urgent need to develop a new ND vaccine that is not interfered by maternal antibodies, has a long immunity duration, and has a good immune effect on the prevalent genotype VII NDV.

[0003] Infectious bursal disease (IBD) in chickens is an acute, highly contagious disease caused by infectious bursal disease virus (IBDV) that mainly harms chicks. The disease was introduced into my country in 1979. By the end of the 1980s, super-virulent strains (vvIBDV) appeared in Europe and my country. The mortality rate of such strains is as high as over 60% and causes more serious immunosuppression, causing huge losses to the world's poultry industry. The VP2 protein encoded by IBDV is the only capsid protein of IBDV, and it is also the main virulence gene and host protective antigen of IBDV, which can induce the production of neutralizing antibodies. At present, the vaccines used to prevent IBD are mainly traditional inactivated vaccines and attenuated live vaccines. The immunity duration of inactivated vaccines is short, and multiple booster immunizations are required; the immune effect of attenuated live vaccines is easily interfered by maternal antibodies, and the side effects of attenuated live vaccines are large, which can cause bursal damage and immunosuppression in vaccinated chickens. At present, it is necessary to develop new IBD vaccines with good safety, no interference from maternal antibodies, and long immunity duration for the prevention and control of super-virulent strains of IBDV.

[0004] Marek's disease virus (MDV) can cause a highly contagious, lymphoproliferative, immunosuppressive tumor disease in chickens. Among them, the serotype 1 MDV attenuated live vaccine CVI988 strain is widely used in the prevention and control of Marek's disease in chickens at home and abroad. MDV has a large genome and many replication-non-essential genes that can be inserted or replaced by exogenous genes. It is an ideal viral vector for constructing recombinant live vector vaccines. Recombinant virus vaccines based on MDV vectors can effectively overcome the defects of inactivated vaccines and attenuated live vaccines that are easily interfered by maternal antibodies. At the same time, MDV carries the virus for life after vaccination, and can continuously express exogenous protein antigens and induce antibodies, thereby giving the body lasting immunity. Summary of the invention

[0005] The object of the present invention is to provide a recombinant Marek's disease virus which co-expresses the Newcastle disease virus F gene and the infectious bursal virus super virulent strain VP2 gene.

[0006] 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 virus super-virulent strain VP2 gene.

[0007] 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 virus super-virulent strain VP2 gene.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: A recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal virus super strain VP2 gene. The recombinant Marek's disease virus is obtained by inserting an expression framework CAGW-F2AV2 comprising a chicken β-actin promoter, the Newcastle disease virus F gene and the infectious bursal virus super strain VP2 gene coding sequence F2AV2, 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 F2AV2 is obtained by connecting the Newcastle disease virus F gene coding sequence and the infectious bursal virus super strain VP2 gene coding sequence with the porcine teschovirus 2A self-cleaving peptide coding sequence. The nucleotide sequence of the coding sequence F2AV2 is shown in SEQ ID NO.1.

[0010] The expression frame CAGW-F2AV2 is obtained by cloning F2AV2 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 F2AV2 sequence and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-F2AV2 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 the genomic DNA fragment of Marek's disease virus CVI988 strain and recombinant cosmid C4-45-F2AV2 containing the Newcastle disease virus F gene and the infectious bursal virus super strain 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-F2AV2 is obtained by inserting the expression frame CAGW-F2AV2 between the UL45 and UL46 genes in the CVI988 genome on the basis of C4, and the recombinant cosmid C4 contains the nucleotide fragment at positions 91415-126182 of the CVI988 genome.

[0012] The invention discloses an application of the recombinant Marek's disease virus which co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus super-virulent strain VP2 gene in the preparation of Newcastle disease, infectious bursal disease and Marek's disease vaccines.

[0013] The present invention utilizes recombinant cloning technology to insert an expression framework containing the Newcastle disease virus gene type VII strain F gene and the infectious bursal disease virus super-virulent strain VP2 gene into the spacer region between the UL45 and UL46 genes of the Marek's disease virus CVI988 strain genome, thereby constructing a recombinant Marek's disease virus vaccine strain C20101 strain that co-expresses the Newcastle disease virus F gene and the infectious bursal disease virus super-virulent strain VP2 gene. After immunizing specific pathogen-free (SPF) chickens with the recombinant virus vaccine strain, the SPF chickens can obtain immune protection against Newcastle disease virus, infectious bursal disease virus and Marek's disease virus, and can be used for the preparation of Newcastle disease, chicken infectious bursal disease and Marek's disease vaccines.

[0014] The vaccine prepared from the recombinant Marek's disease virus strain provided by the present invention can simultaneously provide multiple protections against Newcastle disease virus, infectious bursal virus and Marek's disease virus infection, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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: Figure 1 The figure is a PCR identification result diagram of the recombinant cosmid C4-45-F2AV2 expressing the NDV F gene and the vvIBDV VP2 gene, wherein M is the DL5000 Marker, 1 is the amplification result of the recombinant cosmid C4-45-F2AV2, and 2 is the amplification result of the parental cosmid C4; Figure 2 This is a picture of plaque lesions produced by the recombinant virus C20101 on CEF; Figure 3 The figure is the result of PCR identification of the genomic DNA of the recombinant virus C20101, where M is DL5000 Marker, 1 is the parent virus CVI988, 2 is the recombinant virus 20101, 3 is ddH2O, and 4 is the recombinant cosmid C4-45-F2AV2; Figure 4 The indirect immunofluorescence assay was used to detect the expression of F and VP2 proteins in CEF infected with the recombinant virus C20101; Figure 5 This is a replication kinetic curve of the recombinant virus C20101 on CEF cells; Figure 6 The figure is the result of PCR test of genetic stability of recombinant virus C20101, where M is DL5000 Marker, 1 is ddH2O, 2 is CVI988, 3 is C20101-F5, 4 is C20101-F10, 5 is C20101-F15, and 6 is C20101-F20; Figure 7 This is the result of indirect immunofluorescence test of the 20th generation recombinant virus C20101 expressing F and VP2 proteins; Figure 8 This is the survival curve of chickens immunized with recombinant virus C20101 after being challenged with strong NDV virus; Fig. 9 This is the survival curve of chickens immunized with the recombinant virus C20101 after being challenged with the super-virulent IBDV. DETAILED DESCRIPTION

[0016] 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.

[0017] Unless otherwise specified, the test methods used in the following experimental examples are conventional methods.

[0018] 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.

[0019] Example 1: Construction and identification of recombinant MDV expressing NDV F and vvIBDV VP2 genes 1.1 Establishment of a multi-segment cosmid rescue system for the attenuated live vaccine of serotype 1 MDV strain CVI988 The genomic DNA of serotype 1 MDV attenuated live vaccine CVI988 strain was extracted, and the genomic DNA of CVI988 strain 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 CVI988 strain and could be spliced ​​to cover the complete CVI988 genome were selected. Among them, C1 contains the nucleotide fragment of the CVI988 genome at positions 1-37644, C2 contains the nucleotide fragment of the CVI988 genome at positions 29969-68579, C3 contains the nucleotide fragment of the CVI988 genome at positions 60962-99647, C4 contains the nucleotide fragment of the CVI988 genome at positions 91415-126182, C5 contains the nucleotide fragment of the CVI988 genome at positions 115670-152340, and C6 contains the nucleotide fragment of the CVI988 genome at positions 143712-178311. The GenBank accession number of the genome sequence of the MDV attenuated live vaccine CVI988 strain is DQ530348. The above 6 recombinant cosmids cloned with CVI988 genomic DNA fragments were extracted, and the 6 recombinant cosmids were 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.

[0020] 1.2 Construction and identification of recombinant cosmids expressing NDV F and vvIBDV VP2 genes According to the F gene coding region sequence of NDV genotype VII strain GM strain (GenBank accession number DQ486859), the target gene NDF was synthesized after chicken codon optimization design, and the porcine Teschovirus 2A (P2A) self-cleavage peptide sequence was added to its C-terminus. According to the VP2 gene sequence of IBDV super strain UPM766 strain (GenBank accession number MT505341), the target gene IBDV2 was synthesized after chicken codon optimization design. The target gene F2AV2 (SEQ ID NO.1) containing the F gene and VP2 gene coding sequences was obtained by fusion PCR amplification using primers NDFP1F, NDV2P1R, NDV2P2F and V2P2R, in which the F gene and VP2 gene coding sequences were 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 F2AV2 and the rabbit β-globulin polyadenylation sequence to obtain the recombinant plasmid pCAGW-F2AV2 that co-expresses the NDV F gene and the IBDV VP2 gene. The recombinant plasmid was sequenced using primers CAGF and CAGR, and it was found that the inserted target gene sequence was correct.

[0021] According to the instructions of the Counter Selection BAC Modification Kit, the F2AV2 gene expression framework CAGW-F2AV2 (SEQ ID NO.2) was cloned into the spacer between the UL45 and UL46 genes of the MDV genome in the recombinant cosmid C4 using the Red / ET recombination method to construct the recombinant cosmid C4-45-F2AV2 that co-expresses the NDV F gene and the vvIBDV VP2 gene. The target gene primer vvIBDVP2 and the target gene downstream homology arm primer MDV46R were used to identify whether the recombinant cosmid C4-45-F2AV2 was inserted into the target gene expression framework, and the PCR product was sequenced and identified. The results showed that the length of the obtained PCR product was 2760 bp ( Figure 1 ), the size was consistent with the expectation. 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 negative control sample (parental cosmid C4) had no target gene insertion sequence, and the PCR result was negative. The above results indicate that the recombinant cosmid C4-45-F2AV2 was constructed correctly.

[0022] Table 1 PCR primers used to construct and identify recombinant cosmids containing F and VP2 genes

[0023] 1.3 Rescue and identification of recombinant MDV expressing NDV F and vvIBDV VP2 genes The recombinant cosmid C4-45-F2AV2 and other parental cosmids cloned with MDV CVI988 strain genome fragments were extracted using a plasmid extraction kit. The recombinant cosmid C4-45-F2AV2 and parental cosmids C1, C2, C3, C5, and C6 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 F2AV2 expression framework inserted between the UL45 and UL46 genes of the MDV genome was rescued and named recombinant virus C20101. Among them, the plaque lesions produced by the 5th generation recombinant virus C20101 on CEF are as follows: Figure 2 shown.

[0024] The 5th generation recombinant virus genomic DNA was extracted, and the parent virus CVI988 genomic DNA was used as a control. The recombinant virus was identified by PCR using the target gene specific primer vvIBDVP2F 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 recombinant virus amplification was 2760 bp in length, which was consistent with the expected size ( Figure 3 ). The sequencing results showed that the PCR product contained the vvIBDV VP2 gene sequence and the downstream homology arm sequence of the target gene expression cassette, and the sequence was correct. However, the parental virus CVI988 had no target gene inserted, and the PCR result was negative. The above results showed that the target gene F2AV2 was correctly inserted into the genome of the serotype 1 MDV attenuated live vaccine CVI988 strain, and the recombinant virus C20101 was correctly constructed.

[0025] Example 2: In vitro biological characteristics analysis of recombinant MDV expressing NDV F and vvIBDV VP2 genes 2.1 Detection of F and VP2 proteins expressed by recombinant virus C20101 The recombinant virus C20101 and the parental virus CVI988 were inoculated into CEF cells, and after plaque lesions appeared after 3-4 days of culture, the cells were fixed with anhydrous ethanol, and the expression of F protein and VP2 protein was detected by indirect immunofluorescence test using NDV positive serum and VP2 protein monoclonal antibody as primary antibodies, FITC-labeled rabbit anti-chicken IgG and FITC-labeled goat anti-mouse IgG as secondary antibodies, respectively. CEF infected with the parental virus was used as a negative control. The results showed that the cells infected with the recombinant virus C20101 were able to react with NDV positive serum and VP2 protein monoclonal antibody, 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. The above results indicate that the recombinant virus C20101 can co-express F protein and VP2 protein in infected cells.

[0026] 2.2 Analysis of in vitro replication characteristics of recombinant virus C20101 The recombinant virus C20101 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 was drawn to analyze the in vitro replication characteristics of the recombinant virus C20101 and the parental virus in CEF. The results showed that the replication titers of the recombinant virus C20101 and the parental virus CVI988 reached the highest peak at 120 hours after infection, which were 9.13×10 4 PFU / ml and 9.89×10 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 replication characteristics of the recombinant virus C20101 in CEF are consistent with those of the parent virus CVI988 strain.

[0027] 2.3 Genetic stability test of recombinant virus C20101 The 5th generation recombinant virus C20101 was continuously propagated on CEF for 20 generations. The 5th, 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 virus genome. At the same time, the above 20th generation recombinant virus was inoculated into CEF cells, and an indirect immunofluorescence test was performed with 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 5th, 10th, 15th, and 20th generation recombinant viruses could be amplified to obtain a target gene band of 2760 bp in size ( 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 inserted target gene sequence was correct. The above 20th generation recombinant virus was taken for indirect immunofluorescence test to detect the expression of target genes F and VP2 during the passage process. The results showed that after the recombinant virus C20101 was continuously propagated on CEF cells to the 20th generation, the target genes F and VP2 could still be stably expressed ( Figure 7). The above results show that the recombinant virus C20101 has good genetic stability.

[0028] Example 3: Safety and immunogenicity testing of recombinant virus C20101 3.1 Safety testing of recombinant virus C20101 The fifth generation recombinant virus C20101 was inoculated into one-day-old SPF chickens at a dose of 2000 PFU / chicken, and the clinical symptoms of the chickens in each group were observed every day after inoculation. 28 days after inoculation, 5 test chickens were randomly selected from each group, and their weights were weighed to evaluate the effect of recombinant virus C20101 on the growth and development of the test chickens; the 5 chickens selected from each group were killed, and the organs such as bursa of Fabricius, thymus, spleen, liver, etc. were collected and weighed to observe whether there were symptoms of atrophy or swelling. The results showed that the recombinant virus C20101 did not cause adverse clinical reactions in the test chickens after inoculation of SPF chickens, and the test chickens were eating and drinking normally. 28 days after inoculation, the weighing and autopsy results showed that there was no significant difference in the weight of the test chickens in the recombinant virus C20101 inoculation group and the normal uninoculated control group; the test chickens were autopsied, and the organs such as bursa of Fabricius, thymus, spleen, liver, etc. were collected. The results showed that the above organs were normal and no obvious clinical lesions were observed. The above results show that the recombinant virus C20101 is not pathogenic to SPF chickens.

[0029] 3.2 Immunoprotective test of recombinant virus C20101 against NDV and IBDV 60 one-day-old SPF chicks were randomly divided into 3 groups, 20 in each group; Group 1 was subcutaneously inoculated with the recombinant virus C20101 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, 10 chickens from Group 1 and Group 2 were respectively challenged with NDV strong toxin, and observed for 14 days after the challenge, the clinical symptoms of the test chickens were recorded, and the survival of the chickens in each group was counted. The remaining 10 chickens in Group 1 and Group 2 were challenged with IBDV super toxin, and observed for 7 days after the challenge, the clinical symptoms of the test chickens were recorded, and the survival was counted; 7 days after the challenge, the test chickens were dissected and the lesions of the bursal lesions were observed. Group 3 was not challenged and served as a healthy control group.

[0030] The results showed that all 10 chickens inoculated with the recombinant virus C20101 survived the observation period after being challenged with NDV, while all 10 chickens inoculated with the parental virus CVI988 died after being challenged with strong NDV ( Figure 8 All 10 chickens inoculated with the recombinant virus C20101 survived the observation period after being challenged with vvIBDV, and there was no obvious lesion in the bursa of Fabricius; 9 out of 10 chickens inoculated with the parental virus CVI988 died after being challenged with vvIBDV ( Fig. 9The above results show that the recombinant virus C20101 has a good immune protection effect against both NDV and IBDV.

[0031] 3.3 Immunoprotective test of recombinant virus C20101 against virulent MDV 40 one-day-old SPF chicks were randomly divided into two groups, 20 in each group. Group 1 was subcutaneously inoculated with the recombinant virus C20101 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 MDV strong toxin. After the challenge, the clinical symptoms of the experimental chickens were observed until 80 days after the challenge, and the incidence and mortality of the chickens in each group were counted. Dead chickens were dissected in time. At the end of the observation period, all experimental chickens were dissected and the lesions of various organs were recorded.

[0032] The results showed that 16 chickens died in the non-immune control group after MDV infection, and no typical clinical symptoms of MD were found in the surviving chickens during the observation period. Autopsy revealed that 18 chickens in this group had MD characteristic lesions such as hepatosplenomegaly and organ tumors, and the MD positive rate was 90%. All 20 chickens in the recombinant virus C20101 vaccination group survived after MDV infection, and no clinical symptoms of MD were found. Autopsy revealed that two chickens in this group had liver enlargement, and the remaining 18 chickens did not have MD characteristic lesions. The MD positive reduction rate in the recombinant virus C20101 vaccination group was 89%. The above results show that the recombinant virus C20101 has a good immune protection effect against MDV.

Claims

1. A recombinant Marek's disease virus that co-expresses the Newcastle disease virus F gene and the infectious bursal virus very virulent strain VP2 gene, characterized in that: The recombinant Marek's disease virus is obtained by inserting the expression framework CAGW-F2AV2 containing chicken β-actin promoter, Newcastle disease virus F gene and infectious bursal virus super virulent strain VP2 gene coding sequence F2AV2, woodchuck hepatitis virus post-transcriptional regulatory sequence, and rabbit β-globulin polyadenylation sequence into the Marek's disease virus genome; The coding sequence F2AV2 is obtained by connecting the coding sequence of the Newcastle disease virus F gene and the coding sequence of the infectious bursal virus super strain VP2 gene with the coding sequence of the porcine Teschovirus 2A self-cleaving peptide, and the nucleotide sequence of the coding sequence F2AV2 is shown in SEQ ID NO.1; The expression frame CAGW-F2AV2 is obtained by cloning F2AV2 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 F2AV2 sequence and the rabbit β-globulin polyadenylic acid sequence. The nucleotide sequence of the expression frame CAGW-F2AV2 is shown in SEQ ID NO.

2.

2. The recombinant Marek's disease virus co-expressing the Newcastle disease virus F gene and the infectious bursal virus very virulent strain VP2 gene 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 the genomic DNA fragment of Marek's disease virus CVI988 strain and recombinant cosmid C4-45-F2AV2 containing the Newcastle disease virus F gene and the infectious bursal virus super strain VP2 gene expression framework, and then rescuing the virus; the recombinant cosmids C1, C2, C3, C5, and C6 contain the 1st to 37644th and 29th positions of the Marek's disease virus CVI988 strain genome, respectively. The nucleotide fragments are nucleotides 969-68579, 60962-99647, 115670-152340, and 143712-178311; the recombinant cosmid C4-45-F2AV2 is obtained by inserting the expression frame CAGW-F2AV2 between the UL45 and UL46 genes of the Marek's disease virus CVI988 strain genome on the basis of C4, and the recombinant cosmid C4 contains the nucleotide fragments 91415-126182 of the CVI988 strain genome.

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

Images