CN117625689B - Subtype B avian metapneumovirus vaccine strain for expressing IBDV VP2 protein - Google Patents
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Abstract
The invention discloses a subtype B avian metapneumovirus vaccine strain for expressing IBDV VP2 protein. The vaccine strain is obtained by carrying out virus rescue on a recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid expressing a novel variant strain VP2 protein of infectious bursal disease virus, wherein the plasmid is named pOK-LN16A-nVarVP2, and the nucleotide sequence of the plasmid is shown as SEQ ID NO. 1. The rLN A-nVarVP2 strain obtained after rescue is immunized with SPF chicken of 2 weeks old and subjected to immunoprotection evaluation, and the rLN A-nVarVP strain can induce the immunized chicken to produce only neutralizing antibodies against nVarIBDV and aMPV simultaneously, can provide 100% immunoprotection for nVarIBDV and aMPV viruses, and can effectively prevent pathological injuries of aMPV and nVarIBDV on target organs, namely turbinates and bursa of Fabricius. Therefore, the invention provides a new technical means for preventing and controlling two virus diseases of subtype B aMPV and nVarIBDV, and has important significance for the healthy development of poultry industry.
Description
Technical Field
The invention relates to a B subtype avian metapneumovirus vaccine strain for expressing infectious bursal disease virus VP2 protein and a preparation method and application thereof, in particular to a recombinant B subtype avian metapneumovirus vaccine strain for expressing a novel infectious bursal disease virus variant VP2 protein and a preparation method and application thereof. The invention belongs to the field of biotechnology.
Background
Avian metapneumovirus (Avian metapneumovirus, aMPV) is a highly infectious pathogen that is transmitted primarily by direct or indirect contact in a horizontal manner. The natural hosts of aMPV are chickens and turkeys, mainly causing turkey rhinotracheitis and turkey syndrome in chickens. The single infection mortality rate of the aMPV is not high, but the aMPV is easy to be mixed with pathogens such as escherichia coli, newcastle disease virus, infectious bronchitis virus and the like to cause the increase of the mortality rate. At the end of the 70 s of the 20 th century, aMPV was first reported separately in south africa and then was widely prevalent in multiple european countries such as the united kingdom, france, spanish, germany. At present, the disease is reported in all parts of the world except the ocean, and huge economic loss is brought to the poultry industry.
The aMPV belongs to the Paramyxoviridae, pneumovirinae, and metapneumovirus genus. The total length of the aMPV genome is about 13.1-14.2 kb, and the aMPV genome is a single-stranded negative-strand RNA which is not segmented, and the gene arrangement sequence is 3'-N-P-M-F-M2-SH-G-L-5'. The G gene has the greatest degree of nucleotide and amino acid sequence variation compared to other genes, and therefore, the gene is often used for molecular epidemiological investigation and identification of virus subtypes. Based on the difference in G gene sequences, aMPV can be classified into A, B, C and D subtypes. Studies have shown that the A, B subtype strains are widely distributed and present in most countries other than north america and australia, with the greatest threat to the poultry industry. In addition, domestic epidemiological investigation shows that subtype B aMPV has become a dominant subtype epidemic strain in china.
Infectious bursal disease (Infectious bursal disease, IBD) is a highly contagious, acute and immunosuppressive infectious disease caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV). IBDV infects mainly 3-6 week old chickens, and the infected chicken flock mainly presents serious damage to bursa of Fabricius and kidneys, with increased susceptibility to other pathogens. IBDV is a member of the family avidinghua of the family Birnaviridae, and is composed of a nucleic acid and a capsid, without a bursa membrane. Wherein, the capsid protein mainly comprises VP2, and a plurality of antigen epitopes exist on the surface of VP2, which can induce the host to generate neutralizing antibodies, thus being the main protective antigen of IBDV.
IBDV has two serotypes, of which only serotype I is pathogenic to chickens, and serotype I strains can be divided into classical (cIBDV), variant (VarIBDV), supervirulent (very virulent IBDV, vvIBDV) and attenuated (attenuated IBDV, aIBDV). In 1957 cIBDV was first separately reported in the united states, after which the epidemic situation is further expanding worldwide. In 1987, jackwood et al first discovered VarIBDV and reported VarIBDV to break through the immune protection of cIBDV. vvIBDV was introduced into China at the end of the 80 s of the 20 th century, and it was more pathogenic and generally more lethal than cIBDV and VarIBDV. In recent years, the prevalence of vvIBDV in China has been gradually controlled due to the rational use and feeding management of vaccines. Since 2017, a novel VarIBDV (nVarIBDV) different from the early VarIBDV genes has been widely used in the immune chicken farm in china. nVarIBDV can not cause death of chickens, but can directly damage immune organs such as bursa of Fabricius, spleen, kidneys and the like of chickens, so that serious immunosuppression is caused, and further weight gain and production performance of chickens are affected. Because of the antigenic differences, the current commercial vaccines against cIBDV and vvIBDV do not provide complete protection for nVarIBDV, and there is therefore a great need to study high-efficiency vaccines against nVarIBDV.
The invention utilizes reverse genetic operation technology to obtain a recombinant B subtype avian metapneumovirus vaccine strain capable of stably expressing the novel variant VP2 protein of infectious bursal disease virus, provides a certain technical support and theoretical guidance for prevention and control of two virus diseases of B subtype aMPV and nVarIBDV, and has important significance for the healthy development of poultry industry.
Disclosure of Invention
One of the purposes of the invention is to provide a recombinant subtype B avian metapneumovirus strain full-length cDNA infectious cloning plasmid for expressing a novel variant VP2 protein of infectious bursal disease virus;
The second object of the invention is to provide a recombinant subtype B avian metapneumovirus vaccine strain expressing the novel variant VP2 protein of the infectious bursal disease virus, which is obtained by carrying out virus rescue on the full-length cDNA infectious clone plasmid of the novel variant VP2 protein of the infectious bursal disease virus and application thereof.
In order to achieve the above purpose, the invention adopts the following technical means:
firstly, the full-length cDNA infectious cloning plasmid of the subtype B recombinant avian metapneumovirus strain expressing the novel variant VP2 protein of the infectious bursal disease virus is obtained by inserting a gene transcription cassette of nVarIBDV-SHG19 strain VP2 protein into a non-coding region between G and L genes of the full-length cDNA infectious cloning plasmid of the recombinant subtype B avian metapneumovirus strain, wherein the nucleotide sequence of the full-length cDNA infectious cloning plasmid of the recombinant subtype B avian metapneumovirus strain is shown as SEQ ID NO. 1.
Wherein, the nucleotide sequence of the gene transcription cassette of the nVarIBDV-SHG19 strain VP2 protein is shown as SEQ ID NO.2, and the insertion site is positioned between 9015-9016 bp of the full-length cDNA infectious cloning plasmid of the recombinant B subtype avian metapneumovirus strain.
Furthermore, the invention also provides the application of the recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid for expressing the novel variant VP2 protein of the infectious bursal disease virus in virus rescue, and the recombinant subtype B avian metapneumovirus for expressing the novel variant VP2 protein of the infectious bursal disease virus is obtained by the plasmid in a virus rescue mode.
Still further, the invention also provides a recombinant subtype B avian metapneumovirus vaccine strain for expressing the novel variant VP2 protein of the infectious bursal disease virus, and the vaccine strain is obtained by subjecting the full-length cDNA infectious cloning plasmid of the recombinant subtype B avian metapneumovirus strain for expressing the novel variant VP2 protein of the infectious bursal disease virus to virus rescue.
Wherein, preferably, the virus rescue comprises the following steps:
The recombinant B subtype avian metapneumovirus strain full-length cDNA infectious clone plasmid pOK-LN16A-nVarVP2 expressing the novel infectious bursal disease virus variant VP2 protein and helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L are co-transfected into BSR-T7/5 cells, after 5 days of transfection, the supernatant is collected to infect Vero cells, and a normal BSR-T7/5 cell supernatant infection group is set to serve as a blank control group. The virus is continuously passaged through Vero cells, 3,5, 10 and 15 generations are selected for genome extraction and reverse transcription, the obtained cDNA is used as a template, PCR amplification and agarose gel electrophoresis are carried out, the recombinant B subtype avian metapneumovirus with stable expression of the VP2 protein of the novel variant strain of infectious bursal disease virus is obtained, and the helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L are pCAGGS vectors for respectively expressing the genes of the B subtype avian metapneumovirus strain LN16-A strain N, P, M-1 and L.
Preferably, the recombinant B subtype avian metapneumovirus strain full-length cDNA infectious clone plasmid pOK-LN16A-nVarVP2 for expressing the novel variant VP2 protein of the infectious bursal disease virus and helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L are combined according to the following formula 4:1:1:1:1 into BSR-T7/5 cells.
Preferably, the helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L are prepared by the following methods: reverse transcription is carried out on the extracted RNA of the aMPV LN16-A strain virus, the obtained cDNA is used as a template, PCR amplification is carried out on the N, P, M-1 of the LN16-A strain and the ORF of the L gene by using a primer, the amplified PCR fragment and a pCAGGS vector are connected after double digestion by EcoR I and Xho I, monoclonal colonies are picked up and sequenced for verification, and four auxiliary plasmids with correct sequencing are named pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L respectively, wherein the primer sequences are as follows:
Furthermore, the invention also provides application of the recombinant subtype B avian metapneumovirus vaccine strain for expressing the novel variant VP2 protein of the infectious bursal disease virus in preparing vaccines for preventing infection of the novel variant infectious bursal disease virus and the subtype B avian metapneumovirus.
Preferably, the infectious bursal disease virus is a novel variant of infectious bursal disease virus (nVarIBDV).
Compared with the prior art, the invention has the beneficial effects that:
The invention utilizes reverse genetic operation technology, takes a B subtype avian metapneumovirus attenuated vaccine strain LN16-A obtained in the laboratory as a framework, inserts VP2 genes of nVarIBDV-SHG19 strain between G and L genes of a B subtype avian metapneumovirus attenuated vaccine strain LN16-A genome, and obtains a recombinant B subtype avian metapneumovirus attenuated vaccine strain which can stably express a novel variant strain VP2 protein of infectious bursal disease virus and is named rLN A-nVarVP2 strain. Replication kinetics measurements indicate that VP2 insertion does not affect viral replication. The rLN A-nVarVP2 strain is immunized with SPF chicken of 2 weeks old and is subjected to immunoprotection evaluation, and the rLN A-nVarVP strain is found to induce the immunized chicken to generate neutralizing antibodies against subtype B aMPV and nVarIBDV simultaneously, can provide 100% immunoprotection for two viruses of subtype B aMPV and nVarIBDV, and effectively prevent pathological injuries of target organs such as turbinates and bursa of Fabricius caused by subtype B aMPV and nVarIBDV. Therefore, the invention provides a new technical means for preventing and controlling two virus diseases of subtype B aMPV and nVarIBDV, and has important significance for the healthy development of poultry industry.
Drawings
FIG. 1 is a schematic diagram of the construction of pOK-LN16A-nVarVP2 full-length cDNA infectious clone plasmid;
FIG. 2 shows the results of PCR identification to rescue viruses;
wherein ,M：Marker DL5000; 1：rLN16A-nVarVP2 F3;2：rLN16A-nVarVP2 F5;3：rLN16A-nVarVP2 F10;4：rLN16A-nVarVP2 F15;5： is a positive control; 6: a negative control;
FIG. 3 is an IFA identification result of rescuing viruses;
FIG. 4 shows the result of Western blotting identification for rescuing viruses;
FIG. 5 shows the replication kinetics of rLN A-nVarVP2 on Vero cells;
FIG. 6 shows the results of neutralizing antibody level measurements;
wherein A: aMPV neutralizing antibody levels; b: nVarIBDV neutralizing antibody levels;
FIG. 7 shows the protective effect of rLN A-nVarVP2 strain on aMPV and nVarIBDV;
wherein A: the results of the calculation of the cyst weight ratio index of each group; b: rLN16A-nVarVP2 strain protection rate to aMPV;
FIG. 8 is a histopathological observation;
wherein A: aMPV challenge control group chicken turbinates; b, immunization of the turbinates of the group; c: the turbinates of the blank control group; d: nVarIBDV challenge control chicken bursa; e: immunization of chicken bursa of Fabricius; f: the control group was chicken bursa.
Detailed Description
The invention is further described below in connection with specific embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way.
EXAMPLE 1 construction of recombinant avian metapneumovirus vaccine Strain (rLN A-nVarVP Strain) expressing infectious bursal disease Virus novel variant VP2 protein and immunopotency experiments
1. Materials and methods
1.1 Cells, viruses, plasmids and animal Vero and BSR-T7/5 cells (expressing T7 RNA polymerase) were kept by the laboratory. The virus for virus attack is subtype B aMPV LN16-F4 strain and infectious bursal disease virus novel variant strain nVarIBDV-SHG19 strain. pOK12 and pCAGGS vectors were both supplied by this laboratory. SPF chickens were purchased from the laboratory animal center of Harbin veterinary research institute, national academy of agricultural sciences.
1.2 Construction of full-Length cDNA infectious clone plasmid of subtype B aMPV rLN A strain for expressing nVarIBDV-SHG19 strain VP2 protein the full-length cDNA infectious clone plasmid pOK-LN16A of the aMPV LN16A strain has been obtained in the early stage of the laboratory, and the nucleotide sequence is shown in SEQ ID NO. 1. As shown in FIG. 1, the open reading frame (open READING FLAME, ORF) of VP2 gene of nVarIBDV-SHG19 strain was placed in the transcription regulatory sequence of aMPV rLN A strain, the initiation signal sequence of G gene was added upstream of VP2 gene of nVarIBDV-SHG19 strain, and the termination signal sequence of G gene was added downstream, so that gene transcription cassette (shown as SEQ ID NO. 2) of VP2 protein of nVarIBDV-SHG19 strain was successfully constructed, the primer sequences being shown in Table 1 below. After linearizing full-length plasmid pOK-LN16A (shown in SEQ ID NO. 1) with SpeI, the gene transcription cassette of the VP2 protein of the nVarIBDV-SHG19 strain, which was successfully constructed, was inserted into the non-coding region between the G and L genes of pOK-LN16A (genome 9015-9016 bp), to obtain a full-length cDNA infectious clone plasmid of subtype B aMPV rLN A strain, designated pOK-LN16A-nVarVP2, which expresses the VP2 gene of the nVarIBDV-SHG19 strain.
1.3 Construction of helper plasmid RNA from aMPV LN16-A strain virus was extracted and reverse transcribed using HISCRIPT II Q RT SuperMix for qPCR (R222-01). The obtained cDNA was used as a template to amplify the ORFs of N, P, M-1 and L genes of LN16-A strain, and the primers are shown in Table 2. The amplified PCR fragment and the pCAGGS vector are connected after double enzyme digestion by EcoR I and Xho I, a monoclonal colony is selected and sequenced for verification, and four auxiliary plasmids with correct sequencing are named pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L respectively.
1.4 Rescue and identification of recombinant subtype B aMPV attenuated vaccine strains expressing nVarIBDV VP protein full-length cDNA infectious clone plasmids pOK-LN16A or pOK-LN16A-nVarVP2 were combined with helper plasmids (pCAGGS-N, pCAGGS-P, pCAGGS-M21, pCAGGS-L) according to 4:1:1:1:1 into BSR-T7/5 cells. The supernatant was collected 5 days after transfection to infect Vero cells, while a normal BSR-T7/5 cell supernatant infection group was set as a blank control group. The virus is continuously passaged through Vero cells, genome extraction and reverse transcription are carried out by selecting 3, 5, 10 and 15 generations, the obtained cDNA is taken as a template, PCR amplification and agarose gel electrophoresis are carried out, and the stability of the exogenous fragment nVarIBDV VP2 in the recombinant virus is identified. Two recombinant viruses were inoculated into Vero cells at an infectious dose of moi=0.1, and when the cells were slightly diseased, whether the recombinant viruses normally expressed the aMPV N protein and the IBDV VP2 protein was identified by indirect Immunofluorescence (IFA) and Western blotting assays. The successfully rescued viruses were designated rLN A and rLN A-nVarVP2, respectively. rLN16-A and rLN-16A-nVarVP 2 were inoculated into Vero cells at an infectious dose of MOI=0.01, and viral titers of the two recombinant viruses were determined at different time periods (12, 24, 48, 72, 96 and 120 hours), respectively, and the in vitro replication kinetics of the viruses were plotted.
1.5 Immune efficacy trial 50 SPF chickens of 2 weeks of age were randomly divided into 5 groups (Table 3), the 5 groups being rLN A-nVarVP2 immune group 1, rLN A-nVarVP2 immune group 2, nVarIBDV challenge control group, aMPV challenge control group and blank group, 10 each. Groups 1 and 2 were immunized rLN a-nVarVP2 by nasal drip, the immunization dose was 5000 tcid50, and the other 3 groups were dropped with the same volume of PBS. After 21 days of immunization, groups 1 and 3 were challenged with 200 μ L nVarIBDV SHG19 strains, respectively, at a challenge dose of 10 TCID 50/dose, groups 2 and 4 were challenged with 200 μL of aMPV LN16-F4 strain, respectively, at a challenge dose of 5000 TCID 50/dose, and the blank group was challenged with the same volume of PBS, as shown in Table 3. 1-7 days after toxin expelling, observing and recording the disease conditions of each group of chickens in the morning and afternoon every day; after the experiment is finished, the weight of the chicken and the weight of the bursa of Fabricius are weighed and recorded.
1.5.1 Neutralizing antibody titer determination non-anticoagulated blood was collected 7, 14 and 21 days after immunization of each group of chickens, the non-anticoagulated blood was placed in a 37 ℃ incubator and a 4 ℃ refrigerator for 1 hour each, centrifuged at 3000 g min to separate serum, and finally complement was inactivated at 56 ℃ for 30min, filtered by a filter and stored for later use. Continuously diluting the filtered serum by a ratio of 2, uniformly mixing the serum with different dilution factors with 200 TCID50 aMPV LN16-F4 strain and 200 TCID50 nVarIBDV-SHG19 strain respectively, inoculating 100 mu L of each well into 96-well plates full of Vero cells and DF-1 cells, continuously observing and recording cell states for 1-7 days, counting pathological changes of each well after 7 days, and calculating neutralizing antibody titers aiming at nVarIBDV and aMPV according to a Reed-Muench method.
1.5.2 After the pathological histology observation experiments of the chickens of each group are finished, tissues such as bursa of Fabricius, turbinates and the like are collected for pathological section observation. Tissue such as the collected turbinate and bursa of Fabricius was fixed with 10% formalin, and histopathological sections were prepared, and pathological lesions of each group were observed and recorded.
2. Results
2.1 Genetic stability analysis recombinant viruses rLN A-nVarVP2 are inoculated on Vero cells and continuously cultured for 15 generations, F3, F5, F10 and F15 genome PCR identification are extracted, agarose gel electrophoresis results are shown in figure 2, all recombinant viruses rLN A-nVarVP2 can amplify a target band with the fragment size of 1967 bp, and the result shows that VP2 in the recombinant viruses rLN A-nVarVP2 is not lost after 15 generations of in-vitro subculture, and rLN A-nVarVP2 has good genetic stability.
2.2 IFA identification results the use of rabbit polyclonal antibody against aMPV N protein and mouse monoclonal antibody against IBDV VP2 protein as primary antibodies, FITC labeled goat anti-rabbit and TRITC labeled rabbit anti-mouse antibody as secondary antibodies, and the identification of the rescue virus, IFA results show that the rLN-A infected group shows specific green fluorescence, rLN A-nVarVP2 infected group shows specific green and red fluorescence, and neither Mock group shows green and red fluorescence, thus the results show that Vero cells infected by recombinant viruses rLN A-nVarVP2 can normally express aMPV N protein and IBDV VP2 protein, and the results show that the virus rescue is successful.
2.3 The Western blotting identification result is to further verify whether the virus is successfully rescued, and the Western blotting method is adopted to identify the rescued virus, and the result is shown in figure 4, wherein only N protein can be detected in Vero cells infected by rLN strain 16-A; the rLN A-nVarVP2 infected group can successfully detect the N protein and the VP2 protein, and the Mock group does not detect the N protein and the VP2 protein, which indicates that the recombinant virus expressing the VP2 protein of the novel variant strain of infectious bursal disease virus is successfully rescued.
2.4 The replication kinetics test result shows that whether the insertion of VP2 gene can influence the replication of viruses is further verified, and the replication levels of two strains of viruses at different time points on Vero cells are tested, and the results are shown in FIG. 5, rLN-A and rLN A-nVarVP2 show similar growth characteristics on Vero cells, and the virus titer of rLN-A and rLN A-nVarVP2 on Vero cells is more than 107 TCID50/mL, which indicates that the insertion of VP2 gene can not influence the replication capacity of viruses.
2.5 Measurement of neutralizing antibody the levels of the neutralizing antibodies against IBDV and aMPV were measured within 21 days after immunization, and as shown in FIG. 6, the levels of the neutralizing antibodies against aMPV and IBDV were gradually increased in 7, 14 and 21 days after immunization, and the neutralizing antibody levels at 21 days reached 8.2 log2 and 8.6 log2, respectively, and the antibody positive rates of chickens in rLN A-nVarVP2 immunized groups were 100%. These results indicate that rLN A-nVarVP2 strain can simultaneously induce immune chickens to produce only neutralizing antibodies against nVarIBDV and aMPV.
2.6 After the toxicity attack protection rate experiment is finished, the chickens of each group are sectioned, the weights of the chickens and the bursa of Fabricius are observed and recorded, the result of calculating the bursa weight ratio index (BBIX) is shown as the graph in fig. 7, and the BBIX values of the rLN A-nVarVP2 immune group and the blank group chickens are higher than 0.7, so that rLN A-nVarVP2 strain can effectively prevent the pathological damage of the bursa of Fabricius. In addition, after the virus attack, the disease condition of each group of chickens is observed and recorded, and the results show that the aMPV virus attack control group has turbidity and viscous nasal liquid on the 3 rd day, and the disease rate reaches 100% on the 6 th day. rLN16A-nVarVP2 and aMPV immunized group had no clinical symptoms and the incidence was 0. These results indicate that rLN A-nVarVP2 strain provides 100% immune protection against both nVarIBDV and aMPV viruses.
2.7 Histopathological results 7 days after toxin challenge, the turbinate bone and bursa tissue of each fixed group of chickens are observed after being dyed, and the result is shown in figure 8, the lymphatic follicles of nVarIBDV toxin-challenged control group chickens are obviously atrophic, the lymphocytes are obviously reduced, and a small amount of heteroeosinophilic infiltration occurs in marrow; the submucosa of the turbinates of the aMPV virus-attacking control group can be infiltrated by multiple inflammatory cells, and the turbinates and bursa of Fabricius of rLN A-nVarVP2 immune group and the blank control group have no obvious pathological changes, which shows that the rLN A-nVarVP2 strain can effectively prevent pathological damage of aMPV and nVarIBDV to target organs of turbinates and bursa of Fabricius.
Claims (8)
1. The recombinant subtype B avian metapneumovirus strain (Avian metapneumovirus, aMPV) full-length cDNA infectious clone plasmid for expressing the VP2 protein of the novel variant strain (Novel variant infectious bursal disease virus, nVarIBDV) of the infectious bursal disease virus is characterized in that the plasmid is obtained by inserting a gene transcription cassette of the VP2 protein of nVarIBDV-SHG19 strain into a non-coding region between G and L genes of the recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid, wherein the nucleotide sequence of the recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid is shown as SEQ ID NO.1, and the insertion site is positioned between 9015 and 9016bp of the recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid.
2. The recombinant subtype B avian metapneumovirus strain full-length cDNA infectious clone plasmid for expressing the novel variant VP2 protein of infectious bursal disease virus according to claim 1, wherein the nucleotide sequence of the gene transcription cassette of nVarIBDV-SHG19 strain VP2 protein is shown as SEQ ID NO. 2.
3. The use of the recombinant subtype B avian metapneumovirus strain full-length cDNA infectious cloning plasmid expressing the novel variant VP2 protein of infectious bursal disease virus according to claim 1 or 2 in virus rescue, characterized in that the recombinant subtype B avian metapneumovirus expressing the novel variant VP2 protein of infectious bursal disease virus is obtained by the way of virus rescue from the plasmid.
4. The recombinant subtype B avian metapneumovirus vaccine strain expressing the novel variant VP2 protein of the infectious bursal disease virus is characterized in that the vaccine strain is obtained by carrying out virus rescue on the full-length cDNA infectious clone plasmid of the recombinant subtype B avian metapneumovirus strain expressing the novel variant VP2 protein of the infectious bursal disease virus according to claim 1 or 2.
5. The recombinant subtype B avian metapneumovirus vaccine strain expressing the novel variant VP2 protein of infectious bursal disease virus of claim 4, wherein the virus rescue comprises the steps of:
The recombinant B subtype avian metapneumovirus strain full-length cDNA infectious cloning plasmid expressing the novel variable strain VP2 protein of the infectious bursal disease virus in the claim 1 or 2 is co-transfected into BSR-T7/5 cells together with auxiliary plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L, the supernatant is collected to infect Vero cells after 5 days of transfection, a normal BSR-T7/5 cell supernatant infection group is set at the same time as a blank control group, the virus is continuously passaged through the Vero cells, 3, 5, 10 and 15 generations are selected for genome extraction and reverse transcription, the obtained cDNA is used as a template, PCR amplification and agarose gel electrophoresis are carried out, and the recombinant B subtype avian metapneumovirus with the novel variable strain VP2 protein of the infectious bursal disease virus is obtained, and the auxiliary plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCGS-L are vectors for expressing the gene of the B subtype avian metapneumovirus strain AGLN 16-A and N, P, M-L1 strain.
6. The recombinant subtype B avian metapneumovirus vaccine strain expressing the novel variant VP2 protein of infectious bursal disease virus according to claim 4, wherein the full-length cDNA infectious cloning plasmid of the recombinant subtype B avian metapneumovirus strain expressing the novel variant VP2 protein of infectious bursal disease virus according to claim 1 is combined with helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21, pCAGGS-L according to 4:1:1:1:1 into BSR-T7/5 cells.
7. The recombinant subtype B avian metapneumovirus vaccine strain expressing the novel variant VP2 protein of infectious bursal disease virus of claim 4 or 5, wherein helper plasmids pCAGGS-N, pCAGGS-P, pCAGGS-M21, pCAGGS-L are prepared by the following method: reverse transcription is carried out on the extracted RNA of the aMPV LN16-A strain virus, the obtained cDNA is used as a template, PCR amplification is carried out on the N, P, M-1 of the LN16-A strain and the ORF of the L gene by using a primer, the amplified PCR fragment and a pCAGGS vector are connected after double digestion by EcoR I and Xho I, monoclonal colonies are picked up and sequenced for verification, and four auxiliary plasmids with correct sequencing are named pCAGGS-N, pCAGGS-P, pCAGGS-M21 and pCAGGS-L respectively, wherein the primer sequences are as follows:
PCAG-N-F：GTCTCATCATTTTGGCAAAGATGTCTCTTGAAAGTATTAGACTCA;PCAG-N-R：AGGGAAAAAGATCTGCTAGCTTACTCAAACTTCTGTGATTTGTCT;PCAG-P-F：GTCTCATCATTTTGGCAAAGATGTCTTTCCCCGAAGGCAAGGA;PCAG-P-R：AGGGAAAAAGATCTGCTAGCCTACATATCAAAGCTGTATATATCA;PCAG-M21-F：GTCTCATCATTTTGGCAAAGATGTCCAGAAGGAATCCCTGCAGA;PCAG-M21-R：AGGGAAAAAGATCTGCTAGCTTAATTACTGCTGTCACCCTTTTGC;PCAG-L-F：GTCTCATCATTTTGGCAAAGATACCTAAAGGATGACTACACTGC;PCAG-L-R：GTCTCATCATTTTGGCAAAGCTATTTTGTGCTCAGTATGTACCC.
8. use of a recombinant subtype B avian metapneumovirus vaccine strain expressing a novel variant VP2 protein of infectious bursal disease virus as claimed in any one of claims 4-7 for the preparation of a vaccine for preventing infection by the novel variant infectious bursal disease virus and subtype B avian metapneumovirus.
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