Construction and application of a newcastle disease attenuated marker vaccine strain rMG7-HN-G256E of genotype Ⅶ

Abstract

This invention discloses a Newcastle disease virus (NDV) genotype VII attenuated marker vaccine strain, rMG7-HN-G256E, with accession number CGMCC No. 46292. The complete nucleotide sequence of this vaccine strain is shown in SEQ ID No: 1; wherein, the 256th amino acid (G) of the HN protein is replaced with glutamic acid (E). This invention utilizes a Newcastle disease virus reverse genetics platform to mutate different sites on the MG7 Newcastle disease virus strain, rescuing a genotype VII Newcastle disease virus marker vaccine strain, rMG7-HN-G256E, which exhibits low pathogenicity, good growth characteristics, high viral titer, stable passage, and excellent immunogenicity. This marker vaccine strain has good immunogenicity, can induce high levels of protective antibodies, and provides complete protection for immunized chickens. It can be used to control the currently prevalent genotype VII Newcastle disease virus and lays the foundation for differentiating between vaccine immunization and wild-type virus infection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the construction and application of a genotype VII Newcastle disease attenuated live marker vaccine strain rMG7-HN-G256E. Background Technology

[0002] Newcastle disease (ND) is an acute, highly contagious infectious disease of poultry caused by Newcastle disease virus (NDV), resulting in significant economic losses to my country's poultry industry. Commonly used NDV vaccine strains include LaSota and B1, but these strains do not genotype match the predominantly circulating strains in China. While they provide some immune protection, they are ineffective in reducing viral shedding and can easily lead to atypical NDV symptoms in chickens. Genotype VII NDV attenuated vaccine strains, such as A-VII rDHN3-mF and KBNP-C4152R2L, can be used to control prevalent NDV in chickens, effectively reducing viral shedding. The A-VII strain, as an inactivated vaccine, is widely used in the market, but currently there is no attenuated marker vaccine for genotype VII NDV.

[0003] The growth performance of NDV is regulated by multiple viral proteins, among which the receptor-binding domain of the HN protein, the glycosylation site of the F protein, and the functional domain of the L protein all play key roles.

[0004] The genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E claimed in this invention has not been found in the prior art. Summary of the Invention

[0005] Based on this, the present invention provides a genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E, which has the accession number CGMCC No.46292.

[0006] According to another aspect of the present invention, a Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E of genotype VII is provided, the full nucleotide sequence of which is shown in SEQ ID No: 1; wherein the 256th amino acid glycine (G) of the HN protein is replaced with glutamic acid (E). Further, the hemagglutination titer of this vaccine strain is approximately 9 log2 HA, and the viral titer is approximately 10. 9.5 EID 50 / mL.

[0007] According to another aspect of the present invention, a method for constructing the above-mentioned genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E is provided, the method comprising the following steps: (a) using the pCAGGS-MG7-cDNA whole genome plasmid as a template, performing site-directed mutagenesis on the structural protein differential amino acid sites of the MG7 strain to obtain a mutant plasmid; (b) performing reverse genetics on the mutant plasmid to construct the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E.

[0008] Furthermore, in the pCAGGS-MG7-cDNA whole-genome plasmid, the F protein cleavage site of the MG7 strain has been mutated to the F protein cleavage site of the La Sota strain, and 18 amino acids are deleted from positions 443-460 of the NP protein of the MG7 strain. Further, the upstream primer used for this site-directed mutagenesis is 5'-GTGCTCTAAGGTCACAGAAACTGAAGAGGAGGATT-3'. Further, the downstream primer used for this site-directed mutagenesis is 5'-AATCCTCCTCTTCAGTTTCTGTGACCTTAGAGCAC-3'. Further, the site-directed mutagenesis includes the following steps: (1) PCR amplification: amplify the DNA fragments upstream and downstream of the mutation site, recover the upstream and downstream DNA fragments from the gel, mix them in equal molar amounts as a template, amplify the full-length DNA mutation fragment, and obtain the amplification product; (2) ligation: double digest the amplification product with the pCAGGS-MG7-cDNA whole genome plasmid, recover the digested product from the gel, ligate the digested product with T4 DNA ligase in a metal bath, and obtain the ligation product; (3) transformation: transform the ligation product into DH5α competent cells, add LB liquid medium and culture for about 30 min, plate on ampicillin-resistant LB solid medium plates, and culture for 12 hours; and (4) screening: identify whether the MG7 mutant plasmid has been successfully ligated, and screen to obtain the successfully ligated mutant plasmid. Further, the temperature of the metal bath is about 16°C. Further, the LB solid medium is ampicillin-resistant LB solid medium. Further, the culture temperature is about 37°C. Further, the reverse genetics operation includes the following steps: (i) co-transfecting BHK-21 cells with the successfully ligated mutant plasmid and helper plasmid, culturing, freezing and thawing, filtering, and obtaining a cell culture; and (ii) inoculating the cell culture into chicken embryos, incubating, and collecting allantoic fluid from chicken embryos with positive hemagglutination test results to obtain the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E. Further, the helper plasmids are pBSK-NP, pBSK-P, and pBSK-L. Further, the BHK-21 cells are BHK-21 cells pre-infected with recombinant vaccinia virus VVT7. Further, the multiplicity of infection of the recombinant vaccinia virus VVT7 is 0.001–0.1 MOI, for example, about 0.01 MOI. Further, the pre-infection time is 0.5–1.5 h, for example, about 1 h. Further, the culture time is 48–96 h, for example, about 72 h. Furthermore, the culture temperature is approximately 37°C. Furthermore, the culture container is a cell culture incubator containing 5% CO2. Furthermore, the freeze-thaw cycle is once. Furthermore, the filter pore size is 0.22 μm. Furthermore, the chicken embryos are 9–11 day old SPF chicken embryos. Furthermore, the incubation time is 2–4 days, for example, approximately 3 days.

[0009] According to another aspect of the present invention, the use of the above-mentioned genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E in the preparation of a vaccine is provided.

[0010] Furthermore, this vaccine provides protection against Newcastle disease caused by genotype VII. Furthermore, this vaccine is an avian vaccine. Furthermore, the avian species is chicken. Furthermore, the protective effect further includes one or both of the following: preventing the shedding of Newcastle disease virus and inhibiting the replication of Newcastle disease virus in organs.

[0011] The beneficial effects of this invention are:

[0012] This invention utilizes a Newcastle disease virus reverse genetics platform to mutate different sites on the Newcastle disease virus MG7 strain, rescuing a low-pathogenicity, good growth characteristics, high viral titer, stable passage capability, and excellent immunogenicity marker vaccine strain rMG7-HN-G256E of Newcastle disease virus genotype VII. This marker vaccine strain exhibits good immunogenicity, induces high levels of protective antibodies, and provides complete protection for immunized chickens. It can be used to control the currently prevalent genotype VII Newcastle disease virus and lays the foundation for differentiating between vaccine immunization and wild-type virus infection. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0014] Figure 1 A schematic diagram of the genetic stability analysis results for the rescued virus rMG7-F-N471A.

[0015] Figure 2 A schematic diagram of the genetic stability analysis results for the rescued virus rMG7-HN-G256E.

[0016] Figure 3 A schematic diagram of the genetic stability analysis results for the rescued virus rMG7-L-V1291I.

[0017] Figure 4 This is a schematic diagram showing the results of HI antibody level detection at different time points after SPF chickens were immunized with a live attenuated vaccine.

[0018] Figure 5 This is a schematic diagram showing the survival rate of SPF chickens in each group after challenge with a live attenuated vaccine. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0021] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0022] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0023] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0024] As described in the background section, existing Newcastle disease virus vaccine strains do not match the genotypes of the main circulating strains in China. Although they can provide some immune protection for chickens, they cannot effectively reduce viral shedding and easily lead to atypical Newcastle disease symptoms in the flock. To solve the above problems, this invention provides a genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E, with accession number CGMCC No. 46292.

[0025] The strain of this invention was obtained by the inventors after extensive screening. It was identified as a Newcastle disease attenuated marker vaccine strain of genotype VII by morphological and phylogenetic analysis and named rMG7-HN-G256E. It was deposited at the China General Microbiological Culture Collection Center on November 20, 2024, with the accession number CGMCC No. 46292.

[0026] According to another aspect of the present invention, a genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E is provided, the full genome nucleotide sequence of which is shown in SEQ ID No: 1;

[0027] In the HN protein, the 256th amino acid, glycine (G), is replaced with glutamic acid (E).

[0028] The complete genome nucleotide sequence of this vaccine strain, SEQ ID No: 1, is shown below:

[0029] ACCAAACAGAGAATCTGTGAGGTACGATAAAAGGCGAAGAAGC

[0030] AATCGAGATCGTACGGGTAGAAGGTGTGAATCCCGAG

[0031] TGCGAGGCCGAAGCTCGAACCTGAGGGGACCTTCTACCGATATG

[0032] TCGTCTGTTTTCGACGAGTACGAGCAGCTCCTCGCT

[0033] GCTCAGACCCGCCCTAACGGAACTCATGGAGGGGGAGAGAAAG

[0034] GGAGCACTTTAAAAGTTGAGGTCCCAGTATTCACCCT

[0035] AAACAGTGATGATCAGAGGATAGATGGAATTTTGCGGTATTCTG

[0036] TCTTCGGATTGCTGTTAGCGAGGATGCCAACAAAC

[0037] CACTCAGGCAAGGTGCTCTTATATCCCTCTTTATGCTCCCATTCTCA

[0038] GGGTGATGAAACCATGTTGCCCTTGCAGGGAAA

[0039] CAAAATGAGGCCACACTGGCTGTTCTTGAGATCGATGGTTTTGCT

[0040] AACAGTGTGCCCCAGTTCAACAATAGGAGTGGAGT

[0041] GTCTGAGGAAAGAGCACAGAGATTCATGGTAATCGCAGGATCTC

[0042] TCCCTCGGGCGTGCAGCAACGGTACTCCGTTTGTCA

[0043] CGGCTGGGGTTGAAGATGATGCACCAGAAGATATCACTGACACT

[0044] CTGGAAAGAATCCTATCTATCCAAGTTCAGGTATGG

[0045] GTCACAGTAGCAAAGGCCATGACTGCATATGAGACAGCAGATGA

[0046] GTCAGAAACAAGAAGAATAAATAAGTATATGCAGCA

[0047] AGGTCGAGTTCAGAAGAAGTACATCCTTCATCCTGTATGCAGGA

[0048] GTGCAATTCAACTCACAATCAGACATTCTCTGGCAG

[0049] TCCGTATCTTCCTAGTTAGTGAGCTCAAGAGGGGCCGCAATACAG

[0050] CAGGTGGGAGCTCTACATATTACAACTTGGTCGGG

[0051] GATGTAGACTCATACATCAGGAACACCGGGCTTACTGCATTTTTC

[0052] CTAACACTCAAATATGGAATCAATACCAAGACGTC

[0053] AGCCCTCGCACTCAGCAGCCTCACAGGTGATATCCAAAAAATGA

[0054] AACAGCTCATGCGTTTATATCGGATGAAAGGTGAAA

[0055] ATGCACCATACATGACATTGTTAGGTGACAGTGACCAGATGAGCT

[0056] TTGCACCTGCTGAGTATGCACAACTTTATTCTTTT

[0057] GCCATGGGCATGGCATCAGTCTTAGATAAGGGAACTGGCAAGTA

[0058] CCAATTCGCCAGGGACTTTATGAGCACATCATTCTG

[0059] GAGACTTGGAGTAGAGTATGCTCAGGCCCAGGGAAGTAGCATTA

[0060] ATGAGGACATGGCTGCTGAGCTAAAACTAACCCCGG

[0061] CAGCAAGGAGAGGCCTGGCAGCTGCTGCCCAACGAGTATCCGA

[0062] AGAAATCGGCAGCATGGACATTCCCACTCAACAGGCG

[0063] GGAGTCCTCACCGGGCTCAGTGATGAAGGCCCCCGAACTCCACA

[0064] GGGCAGATCAAACAAGCCGCAAGGGCAACCAGATGC

[0065] TGGGGATGCGCCAAATCCTGTACAGAGCACCACCCATCCAGAGC

[0066] CTCCCCCAACCCCTGGGGCATCCCAAGACAACGACA

[0067] CTGACTGGGGGTACTGATCGACAACACCCAGCCTGCCTTCACAG

[0068] GATCACACCAAACCCTCCGACCAAAACCCTCCCACA

[0069] CTCCCTGACCCACAACCCCGCACGACCACACCAACAAAAGCTCC

[0070] CCCCCACCCTCTCCCCCACTCCCAGCCACACGATCC

[0071] CGCCCACCCGGGACAACACAGGCACAGCTCAGTTCGTCGACAAT

[0072] CCGCCCAGAGCCCAAGGTATTAGAAAAAAATACGGG

[0073] TAGAAGAGAGACATCCAGAGACCAGGACGAGTCACCAAGTTCT

[0074] CTGTTCTCCCTTCTACCCAGTGAATTAGGGTGAAGAT

[0075] GGCCACTTTTACAGATGCGGAGATAGATGACATATTTGAGACCAG

[0076] TGGGACTGTCATTGACAGCATAATTACGGCCCAGG

[0077] GCAAATCAGCTGAGACCGTTGGAAGAAGTGCGATCCCGCAGGG

[0078] CAAGACCAAAGCTCTAAGCACAGCATGGGAGAAGCAC

[0079] GGGAGTATCCAGCCACACGCCAGTCAGGACGCCCCTGACCAACA

[0080] AGACAGAATAGAAAAACAGCCATCCACACCTGAGCA

[0081] GGCGACTCTACACAACAATCCGCCGATCGCATCCACTGAACCGC

[0082] CTCCCACTCAGGCCGCAAGCGAGACCAGCGACACAC

[0083] AGCTCAAGACTGGGGCAAGCAACTCCCTTCTGTCCATGCTCGAC

[0084] AAACTGAGTAATAATCGTCCAATGCTAAAAAGGGC

[0085] CCATGGTCGGGATCCAAGAAGGGCATCACCAACCTCCGGCCCA

[0086] ACAACATGGGAACCAGCCGAGCTATGGAAGCAACCA

[0087] GGGAAGACCGCAGCATCAGGCCAAGGCCGTCCCTGGAAACCGG

[0088] GGCACAGACGAGAACACAGCATATCATGGACAGCGGA

[0089] AGGAGTCACAACCATCAGCTGGTGCAACCCCTCATGCGCCCCAG

[0090] TCAGGGCAGAGCCAAGACAATACTCCTGCACCTGTG

[0091] GATCGTGTCCAGCTACCTGCCGACTTTGTGCAGGCGATGATGTCT

[0092] ATGATGGAGGCATTATCACAGAAGGTAAGTAAAGT

[0093] TGATCATCAGCTGGACCTAGTCTTGAAACAGACATCCTCCATTCC

[0094] TATGATGCGATCTGAAATCCAACAGCTCAAGACAT

[0095] CTGTTGCGATCATGGAAGCTAACTTAGGCATGATGAAAATTCTGG

[0096] ACCCTGGTTGTGCTAATGTTTCATCCTTAAGTGAT

[0097] CTCCGGGCAGTAGCCCGATCCCATCCAGTCCTAGTTTCAGGCCCT

[0098] GGAGACCCATCTCCTTACGTGACACAAGGGGGTGA

[0099] AATGACGCTCAATAAACTCTCACAACCGGTGCAGCACCCCTCTG

[0100] AATTGATTAAGTCTGCCACTGCAAGCGGGCCTGACA

[0101] TGGGAGTGGAGAAGGACACTGTCCGCGCATTAATCACCTCGCGC

[0102] CCGATGCATCCAAGCTCCTCGGCTAAGCTCCTGAGC

[0103] AAGCTAGATGCAGCCAGGTCAATTGAAGAGATCAGGAAGATCAA

[0104] ACGCCTTGCGCTGAATGGTTGATGGCCATCACAACT

[0105] CATAACAGGCTCCCGTCACTTTAGCGTCACACGGAATCCCTCGGG

[0106] GGCCCTCCCTCGCAAATCTATGCTTCAACACCCAA

[0107] AACAACAGCCCTCTCTCACCCCCCCCAATCCCTCGAATGACCGC

[0108] ACAACTGCAACCAATCCAGCAGCATTAGAAATTAAG

[0109] AAAAAATACGGGTAGAATCAAAGTGCCTTGATTGCACCAAAATG

[0110] GACTCATCCAGGACAATTGGGCTGTACTTTGATTCT

[0111] GCCCTCCCTTCCAGCAGCCTGTTAGCATTTCCGATTGTCTTACAA

[0112] GACACGGGAGACGGGAAGAAGCAAATCACCCCACA

[0113] ATACAGGATCCAGCGTCTTGATTCGTGGACAGACAGTAAGGAAG

[0114] ACTCGGTATTCATCACCACCTACGGGTTCATCTTTC

[0115] AAATTGGGAATGAAGAAACCACCGTCGGTGTGATCAATGACAAT

[0116] CCCAGGCACGAGCTACTCTCTTCCGCAATGCTCTGC

[0117] TTAGGGAGTGTCCCGAACGACGGAGATCTTGTTGAGCTGGCGAG

[0118] AGCCTGCCTCACCATGGTGGTAACTTGCAAAAAGAG

[0119] TGCAACTAACACTGAGAGAATAGTCTTTTCAGTAGTGCAGGCTC

[0120] CTCGGGTGCTGCAAAGCTGTATGGTTGTGGCAAATA

[0121] GGTACTCATCAGTGAATGCAGTGAAGCATGTGAAGGCGCCAGAA

[0122] AAGATCCCTGGGAGCGGAACCCTAGAGTATAAAGTG

[0123] AATTTTGTCTCTTTGACCGTGGTGCCAAGAAGGGATGTCTACAGG

[0124] ATCCCAACTGCAGTATTGAAAGTGTCTGGCTCAAG

[0125] CCTGTACAATCTTGCGCTCAATGTCACTATTGATGTGGACGTGGA

[0126] TCCGAAGAGCCCGTTAGTCAAATCCCTTTCCAAGT

[0127] CCGATAGCGGATACTATGCGAATCTTTTTCTGCATATCGGGCTTAT

[0128] GTCCACTGTAGATAAGAAGGGAAAGAAAGTGACA

[0129] TTTGACAAGATAGAGGAAAAGATAAGGAGACTCAATCTATCCGT

[0130] CGGGCTCAGTGATGTGCTCGGACCCTCTGTGCTTGT

[0131] GAAGGCGAGAGGTGCACGGACTAAGCTACTTGCTCCTTTTTTCT

[0132] CTAGCAGTGGGACAGCCTGCTATCCTATAGCAAATG

[0133] CCTCTCCCCAGGTTGCCAAGATACTCTGGAGCCAAACTGCGCAC

[0134] CTGCGGAGTGTGAAAGTCATCATTCAAGCCGGCACT

[0135] CAGCGTGCTGTCGCAGTGACCGCTGATCATGAGGTAACCTCCACT

[0136] AAGATAGAGAGGAGGCATGCCATTGCTAAATACAA

[0137] TCCTTTCAGGAAATAAGTTGCATCCCTAAGACTGCAGTTCACCTG

[0138] CTTTCCCGAATCACCATTACACCAGACAATGATCC

[0139] ATCTCGACTGCTTATAGTTAGTTCACCTGTCTAGCAAATTAGAAA

[0140] AAACACGGGTAGAAGAGTCTGGATCCCGACCGGCA

[0141] CACTCAGGACGCAACATGGGCTCCAAACCTTCTACCAGGATCCC

[0142] AGCACCTCTGATGCTGGTCACCCGGATTATGCTGAT

[0143] ATTGGGCTGTATTCGTTCGACAAGCTCTCTTGACGGCAGGCCTCT

[0144] TGCAGCTGCAGGAATTGTAGTAACAGGAGATAAGG

[0145] CAGTCAATGTATACACCTCGTCTCAGACAGGGTCAATCATAGTCA

[0146] AGTTGCTCCCGAATATGCCCAGGGATAAGGAGGCG

[0147] TGTGCGAAAGCCCCATTAGAGGCATATAACAGAACACTGACTAC

[0148] TTTGCTCACTCCTCTTGGCGACTCCATCCGCAAGAT

[0149] TCAAGGGTCTGTGTCCACGTCTGGAGGAGGGAGACAGGGGCGC

[0150] CTTATAGGTGCTGTTATTGGCAGTGTAGCTCTTGGGG

[0151] TTGCAACAGCGGCACAGATAACAGCAGCTGCGGCCCTAATACAA

[0152] GCCAACAAGAATGCTGCCAACATCCTTCGGCTTAAG

[0153] GAGAGCATTGCTGCAACCAATGAAGCTGTGCATGAAGTCACCGA

[0154] CGGATTATCACAACTATCAGTGGCAGTTGGGAAGAT

[0155] GCAGCAGTTTGTCAATGACCAGTTTAATAATACGGCGCGAGAATT

[0156] GGACTGTATAAAAATCACACAACAGGTTGGTGTAG

[0157] AACTCAACCTATACCTAACTGAATTGACTACAGTATTCGGGCCAC

[0158] AGATCACCTCTCCTGCATTAACTCAGCTGACCATC

[0159] CAGGCACTTTATAATTTAGCTGGTGGCAATATGGATTACTTATTAA

[0160] CTAAGTTAGGTATAGGGAACAATCAACTCAGCTC

[0161] ATTAATTGGTAGCGGCCTGATCACTGGTTACCCTATACTGTATGAC

[0162] TCACAGACTCAACTCTTGGGCATACAAGTGAATT

[0163] TGCCCTCAGTCGGGAACTTAAATAATATGCGTGCCACCTATTTGG

[0164] AGACCTTATCTGTAAGTACAACCAAAGGATATGCC

[0165] TCAGCACTTGTCCCGAAAGTAGTGACACAAGTCGGTTCTGTGAT

[0166] AGAAGAGCTTGACACCTCATACTGTATAGAGTCCGA

[0167] TCTGGATTTATATTGTACTAGAATAGTGACATTCCCCATGTCCCCA

[0168] GGTATTTATTCCTGTTTGAGCGGCAACACATCAG

[0169] CTTGCATGTATTCAAAGACTGAAGGCGCACTCACTACGCCGTATA

[0170] TGGCCCTTAAAGGCTCAGTTATTGCCAATTGTAAG

[0171] ATAACAACATGTAGATGTACAGACCCTCCTGGTATCATATCGCAA

[0172] AATTATGGAGAAGCTGTATCCCTGATAGATAGACA

[0173] TTCGTGCAATGTCTTATCATTAGACGGGATAACTCTGAGGCTCAG

[0174] CGGAGAATTTGATGCAACTTATCAAAAGAACATCT

[0175] CAATACTAGATTCTCAAGTCATCGTGACAGGCAATCTTGATATATC

[0176] AACTGAACTTGGAAACGTCAACAATTCAATCAGC

[0177] AATGCCTTGGATAAGTTGGCAGAAAGCAACAGTAAGATAGAAAA

[0178] AGTCAATGTCAGATTAACCAGCACATCTGCTCTCAT

[0179] TACCTATATTGTTCTAACTGTCATTTCTCTATTTTTCGGTGCACTTA

[0180] GTCTGGGTTTAGCGTGTTACCTGATGTACAAAC

[0181] AGAAGGCACAACAAAAGACCTTGCTATGGCTTGGGAATAATGCC

[0182] CTTGATCAGATGAGAGCCACTACAAGAGCATGAATG

[0183] CAGATAAGAGGTGGATATATACCCAACAGCAGCCTGTGTGTCAAT

[0184] TCCGATAATCTGTCAAGTAGAAGACTTAAGAAAAA

[0185] ACTACTGGGAACAAGCAACCAAAGAGCAATACACGGGTAGAAC

[0186] GGTCAGAGGAGCCACCCTTCAATCGGAAATTAGGCTT

[0187] CACAACATCCGTTCTACCACATCACCATCAACAAGAGTCAATCAT

[0188] GGACCGCGCGGTTAACAGAGTCGTGCTGGAGAATG

[0189] AGGAAAGAGAAGCAAAGAACACATGGCGCCTGGTTTTCCGGAT

[0190] CGCAGTCTTACTTTTAATGGTAATGACTCTAGCTATC

[0191] TCCGCAGCTGCCCTGGCATACAGTATGGAGGCCAGTACGCCGCA

[0192] CGACCTTGCAGGCATATCGACTGTGATCTCTAAGAC

[0193] AGAAGATAAGGTTACGTCTTTACTCAGTTCGAGTCAAGATGTGAT

[0194] AGATAGGATATACAAGCAGGTGGCTCTTGAATCCC

[0195] CGCTGGCGCTACTAAACACTGAATCTACAATTATGAATGCAATAA

[0196] CCTCTCTTTCTTATCAAATTAACGGGGCTGCGAAC

[0197] AATAGCGGATGTGGGGCGCCTGTTCATGACCCAGATTATATCGGG

[0198] GGGATAGGCAAAGAACTCATAGTGGACGACATCAG

[0199] TGATGTCACATCATTTTATCCTTCTGCATATCAAGAACACCTGAAT

[0200] TTCATCCCGGCGCCTACTACAGGATCCGGTTGCA

[0201] CTCGGATACCCTCATTTGACATGAGCACCACCCATTATTGTTATAC

[0202] TCACAATGTAATACTATCCGGTTGCAGAGATCAC

[0203] TCACACTCACATCAATACTTAGCGCTTGGTGTGCTTCGGACATCT

[0204] GCAACAGGGAGGGTATTCTTCTCTACTCTGCGCTC

[0205] CATCAATTTAGATGACACCCAAAATCGGAAGTCCTGCAGTGTGA

[0206] GTGCAACCCCTTTAGGTTGTGATATGCTGTGCTCTA

[0207] AGGTCACAGaaaCTGAAGAGGAGGATTACAAGTCAGCTGCCCCA

[0208] ACATCAATGGTGCACGGAAGGCTAGGGTTTGACGGT

[0209] CAATACCATGAGAAGGACTTAGACACCACGGTCTTATTTAAGGAT

[0210] TGGGTGGCAAATTACCCAGGAGTGGGAGGAGGGTC

[0211] TTTTATTGACGACCGTGTATGGTTCCCAGTTTACGGAGGGCTCAA

[0212] ACCCAATTCACCCAGTGACACTGCACAAGAAGGGA

[0213] AATATGTAATATACAAGCGCCATAACAACACATGCCCCGATGGAC

[0214] AAGATTACCAAATTCGGATGGCTAAATCTTCATAT

[0215] AAACCCGGGCGATTTGGTGGAAAGCGCGTACAGCAAGCCATATT

[0216] ATCCATCAAAGTGTCAACATCCTTGGGTAAGGACCC

[0217] GGTGCTGACTATTCCACCTAATACAATCACACTCATGGGAGCCGA

[0218] AGGCAGAATCCTCACAGTAGGGACATCTCACTTCT

[0219] TGTACCAACGAGGGTCTTCATATTTCTCCCCTGCCTTATTATATCC

[0220] CATGACAGTAAATAACAAAACGGCTACACTCCAT

[0221] AGTCCTTATACGTTTAATGCTTTCACTCGGCCAGGTAGCGTCCCTT

[0222] GCCAGGCATCAGCAAGATGCCCCAACTCATGCAT

[0223] CACTGGGGTCTATACTGATCCATATCCCTTAATCTTCCATAGGAAT

[0224] CATACTCTACGAGGGGTCTTTGGGACGATGCTTG

[0225] ATGATGAACAAGCGAGACTTAACCCCGTATCTGCAGTATTCGACA

[0226] ACATATCCCGCAGTCGTGTCACCCGGGTGAGTTCA

[0227] AGCAGCACCAAGGCAGCATACACGACATCGACATGTTTTAAAGT

[0228] TGTCAAGACCAATAAAGCTTATTGTCTTAGTATTGC

[0229] AGAGATATCCAATACCCTATTCGGGGAATTTAGGATCGTTCCCTTA

[0230] TTAGTTGAGATCCTCAAGGATGATAGAGTTTAAG

[0231] AAGCTAGACTTGGCCGATTGAGCCAATCATAGGATGGTTGGGAA

[0232] GACGACACCACATCAATCATCTCCCACAATGCTTAG

[0233] AGTCAATCTGAATATTAACATAAGCCAGGATCCCATGTTGTTGGG

[0234] CAGCCACAATCAGACAATACTGACATGATCATTCT

[0235] GAGTCCTGCCCACTATCACCTTATTAAGAAAAAATACAAAAAGCA

[0236] TTGAGATATAAGGGGAAACAACCAACAAGAGGGAA

[0237] CACGGGTAGGACATGGCGGGCTCCGGTCCCGAAAGGGCAGAGC

[0238] ACCAGATCATCCTACCAGAGTCACATCTATCCTCTCC

[0239] ATTGGTCAAGCACAAATTGCTATACTACTGGAAATTGACTGGGCT

[0240] ACCGCTTCCTGATGAATGCGACTTTGATCATCTCA

[0241] TTATCAGCAGGCAATGGAAGAGAATACTGGAGTCGGCCACTCCT

[0242] GACACAGAGAGAATGATAAAACTCGGGCGGGCAGTG

[0243] CACCAGACTCTCAACCACAACTCCAAGATAACCGGAGTGCTCCA

[0244] TCCCAGGTGTTTAGAAGAACTGGCTAGTATTGAGGT

[0245] CCCAGATTCAACTAACAAATTCCGGAAGATTGAAAAGAAGATCC

[0246] AGATTCACAACACAAGGTATGGAGACCTGTTCACAA

[0247] AGCTGTGCACGCATGTTGAGAAGAAATTGCTAGGATCATCCCGG

[0248] TCTAATAATGTCCCACGATCAGAGGAATTCAGTAGC

[0249] ATCCGTACAGATCCGGCATTCTGGTTTCACTCAAAATGGTCCAGA

[0250] GCTAAGTTCGCGTGGCTCCATATAAAACAAGTCCA

[0251] AAGGCATCTGATTGTAGCAGCAAGGACAAGGTCTGCAGTCAACA

[0252] AGTTAGTAACATTAAGTCATAAGATAGGCCACGTCT

[0253] TTGTTACTCCTGAGCTTGTCATTGTGACACATACAGACGAGAACA

[0254] AGTTCACATGCCTCACCCAGGAACTTGTATTGATG

[0255] TATGCGGATATGATGGAAGGCAGGGACATGGTCAATATAATATCTT

[0256] CTACAGCAGCACATCTCAGAAACCTATCCGAGAA

[0257] AATTGACGATATTCTGCGGTTAGTAGATGCCCTGGCAAAGGACTT

[0258] AGGTAATCAAGTCTATGACGTTGTAGCATTAATGG

[0259] AGGGATTCGCATACGGTGCTGTTCAGCTGCTTGAGCCATCAGGTA

[0260] CATTTGCAGGAGATTTCTTTGCATTTAACCTACAG

[0261] GAGCTCAAAGACACTTTAATCGAACTTCTCCCAAATAATATAGCG

[0262] GAATCAGTAACTCACGCTATTGCCGCTGTATTCTC

[0263] CGGTTTAGAACAGAATCAAGCAGCTGAGATGTTGTGCTTGCTAC

[0264] GTTTGTGGGGTCATCCTTTGCTTGAGTCTCGTAGTG

[0265] CAGCAAGAGCAGTCAGGAGCCAGATGTGTGCACCGAAGATGGT

[0266] AGACTTCGATATGATCCTCCAGGTACTATCTTTCTTT

[0267] AAAGGAACAATCATCAATGGATACAGAAAGAAGAATTCAGGTGT

[0268] GTGGCCGCGTGTCAAAGTAGATACAATATACGGGAA

[0269] TATCATTGGGCAGCTGTATGCTGATTCAGCAGAGATTCTCACATGAT

[0270] GTCATGTTGAAGGAGTACAAGAGTTTATCTGCTT

[0271] TTGAATTTGAGCCATGTATAGACTATGACCCTGTTACCAATCTAAG

[0272] CATGTTCCTAAGACAAGGCAATCGCACATCCT

[0273] AGTGATAATTGGCTCGCCTCATTTAGGCGGAACCTACTCTCTGAG

[0274] GACCAGAAGAAACAGATAAGATAAGGAGGCAACTTCAAC

[0275] TAACCGCCTCTTGATAGAGTTCTTAGAATCAAATGATTTTGATCCA

[0276] TATAAAGAAATGGAATACCTGACAACCCTCGAGT

[0277] ACCTAAGAGGTGACAGTGTGGCAGTATCGTACTCACTCAAAGAG

[0278] AAAGAGGTAAAAGTGAATGGGCGGATTTTTGCTAAG

[0279] TTAACAAAGAAACTAAGGAACTGCCAGGTAATGGCAGAAGGAAT

[0280] TCTAGCTGACCAGATTGCACCTTTCTTTCAGGGAAA

[0281] TGGGGTCATTCAAGATAGCATATCCTTGACAAAGAGTATGTTAGC

[0282] GATGAGTCAACTGTCCTTTAACAGCAATAAGAAAC

[0283] GTATCACTGACTGCAAAGAGAGGGTTTCCTCGAACCGCAATCAT

[0284] GATCAGAAGAGCAAGAATCGTAGAAGAGTTGCCACT

[0285] TTTATCACGACTGACCTACAAAAGTATTGTCTTAACTGGAGATATC

[0286] AGACAGTCAAACTATTCGCCCATGCTATCAATCA

[0287] GCTGATGGGCCTACCTCATTTCTTCGAGTGGATTCATCTTAGGCTG

[0288] ATGGACACTACAATGTTTGTAGGGGATCCTTTCA

[0289] ATCCTCCAAGTGACCCGACTGACTGTGATCTATCAAGAGTCCCAA

[0290] ACGATGACATATATATTGTCAGTGCTAGAGGGGGC

[0291] ATTGAGGGACTCTGCCAGAAGCTATGGACGATGATCTCAATTGCT

[0292] GCAATCCAACTTGCTGCAGCAAGATCTCATTGTCG

[0293] AGTTGCCTGCATGGTACAAGGTGACAATCAAGTAATGGCTGTAA

[0294] CGAGAGAGGTAAGATCAGATGATTCCCCGGATATGG

[0295] TGTTGACGCAGTTGCATCAAGCTAGTGATAATTTCTTCAAGGAAT

[0296] TGATTCATGTCAATCATTTGATTGGCCATAATCTG

[0297] AAGGATCGTGAAACCATTAGATCAGACACATTCTTCATATACAGC

[0298] AAACGAATATTCAAGGATGGAGCAATACTCAGTCA

[0299] GGTCCTCAAAAATTCATCTAAATTGGTGCTAATATCAGGCGACCTT

[0300] AGCGAAAACACTGTAATGTCCTGTGCCAACATTG

[0301] CATCCACTGTAGCACGACTATGTGAGAATGGGCTTCCAAAGGATT

[0302] TCTGTTACTATTTGAACTACCTAATGAGTTGTGTG

[0303] CAGACATACTTTGATTCGGAGTTCTCTATTACCCACAGCTCGCAA

[0304] TCAGATTCCAACCAGTCCTGGATCGAGGATATCTC

[0305] TTTCGTACACTCATACGTGTTAACCCCTGCCCAGCTGGGGGGACT

[0306] GAGCAACCTTCAATACTCAAGGCTCTACACAAGGA

[0307] ATATTGGTGACCCAGGGACCACTGCTTTCGCAGAGGTCAAGCGA

[0308] CTGGAAGCAGTGGGGTTGCTGAGTCCCAGCATCATG

[0309] ACTAACATCTTAACCAGGCCACCTGGCAATGGAGACTGGGCCAG

[0310] CCTATGCAACGACCCATACTCTTTTAATTTTGAAAC

[0311] TGTTGCAAGCCCAAATATTGTCCTCAAGAAACATACACAGAAAG

[0312] TCCTATTTGAGACATGTTCAAACCCCTTATTATCCG

[0313] GGGTACATACAGAGGACAATGAGGCAGAGGAGAAAGCATTGGC

[0314] TGAATTCTTACTCAATCAAGAAGTGATTCACCCACGT

[0315] GTCGCACATGCTATCATGGAAGCAAGCTCTGTGGGTAGGAGAAA

[0316] GCAAATTCAAGGGCTTGTTGACACAACGAACACTGT

[0317] GATTAAGATTGCACTGACTAGGAGGCCCCTCGGTATCAAAAGGC

[0318] TGATGCGGATAATCAATTACTCGAGCATGCATGCAA

[0319] TGTTGTTCAGAGATGATATTTTCTTATCCAATAGATCCAACCACCC

[0320] ATTAGTTTCTTCCAATATGTGCTCGCTGACGCTA

[0321] GCAGATTATGCCCGAAACAGAAGCTGGTCACCTCTGACAGGGGG

[0322] CAGGAAAATACTGGGTGTATCCAACCCTGATACCAT

[0323] AGAACTTGTGGAGGGAGAGATTCTCAGCGTCAGTGGAGGGTGC

[0324] ACAAAATGTGACAGCGGAGATGAGCAGTTTACTTGGT

[0325] TCCATCTTCCAAGCAATATAGAGCTGACTGATGACACCAGAAAA

[0326] AATCCCCCGATGAGAGTGCCATATCTTGGGTCGAAG

[0327] ACTCAAGAGAGGAGAGCTGCCTCGCTTGCGAAAATAGCCCACAT

[0328] GTCACCACATGTGAAAGCAGCACTAAGGGCATCATC

[0329] CGTGTTAATCTGGGCTTATGGGGACAACGAAGTGAACTGGACTG

[0330] CTGCTCTTAATATTGCAAGGTCTCGATGCAACATAA

[0331] GCTCAGAGTACCTTCGGCTATTGTCACCCCTGCCCACGGCTGGGA

[0332] ATCTCCAACATAGATTGGATGATGGCATAACCCAG

[0333] ATGACATTTACCCCTGCATCTCTCTACAGAGTGTCGCCTTACGTTC

[0334] ACATATCCAATGATTCTCAAAGGCTATTCACCGA

[0335] AGAAGGGGTCAAAGAGGGAAACGTGGTTTACCAACAAATTATGC

[0336] TCTTGGGTTTATCCCTAATTGAATCACTCTTCCCAA

[0337] TGACAGCAACCAGAACATATGATGAGATCACATTACACCTCCACA

[0338] GTAAATTTAGCTGCTGTATCCGAGAAGCGCCTGTT

[0339] GCGGTTCCCTTCGAGCTCCTCGGGCTGGCACCGGAATTAAGAAT

[0340] GGTAACCTCAAATAAGTTCATGTATGATCCTAGCCC

[0341] TATATCAGAGAGAGATTTCGCGAGACTTGACTTAGCTATCTTCAA

[0342] GAGTTACGAGCTTAATTTGGAATCATATTCCACGC

[0343] TGGAGCTAATGAACATTCTTTCAATATCTAGCGGGAAGTTGATTG

[0344] GCCAATCTGTGGTTTCTTATGATGAAGACACCTCT

[0345] ATAAAGAATGATGCTATAATAGTGTATGACAACACACGAAATTGG

[0346] ATTAGTGAGGCGCAGAACTCAGATGTGGTCCGCTT

[0347] GTTTGAGTATGCAGCACTCGAAGTGCTTCTTGACTGTGCTTATCA

[0348] ACTCTACTATCTGAGGGTAAGGGGTCTAAACAACA

[0349] TCGTCCTATACATGAATGACTTATATAAGAACATGCCAGGGATCCT

[0350] ACTCTCCAATATTGCGGCCACGATATCCCACCCC

[0351] ATCATTCACTCAAGGTTGAATGCAGTAGGCCTAATTAACCATGAC

[0352] GGGTCACACCAGCTCGCAGATATAGACTTCGTCGA

[0353] GGTGTCTGCAAAATTGTTAGTCTCTTGCACTCGACGCGTGGTCTC

[0354] AGGCTTATATGCAGGGAATAAGTACGATCTGCTGT

[0355] TCCCATCTGTCTTAGATGATAACCTGAATGAGAAGATGCTCCAAC

[0356] TGATTTCCCGGTTATGCTGTCTGTACACAGTGCTC

[0357] TTTGCTACAACAAGAGAAATCCCAAAAATAAGGGGCCTATCAGC

[0358] AGAAGAGAAATGCTCAATACTCACTGAGTATCTACT

[0359] GTCAGATGCTGTAAAACCATTGCTTAGGCCCGAACAAGTGAATTC

[0360] TATCATGTCTCCCAACATAATCACGTTCCCAGCCA

[0361] ATCTATATTACATGTCTAGGAAGAGCCTTAATTTGATCAGAGAACG

[0362] AGAGGACAGAGATACTATCTTGTCATTGTTGTTC

[0363] CCTCAGGAACCACTGCTTGAGCTTCGCCCAGTACGGGACATTGG

[0364] TGCTCGAGTGAAAGACCCGTTTACCCGGCAACCCGC

[0365] ATCATTCATACAAGAGCTAGATCTGAGTGCCCCAGCAAGGTACGA

[0366] CGCATTTACACTGAGTAAGGCCTGCTTCGAGCATA

[0367] CATTACCGAACCCAAAGGAGGATTACCTAGTACGGTACTTGTTCA

[0368] GAGGAATAGGGACTGCTTCATCTTCTTGGTATAAG

[0369] GCATCTCATCTTCTATCCGTACCTGAGGTCAGGTGTGCAAGACAT

[0370] GGGAACTCCTTATACTTAGCGGAAGGAAGCGGAGC

[0371] CATCATGAGTCTTCTTGAATTGCATATACCACATGAGACTATCTATT

[0372] ACAATACACTTTTCTCGAATGAGATGAACCCTC

[0373] CACAGCGACATTTCGGACCTACACCAACACAGTTTCTAAACTCG

[0374] GTCGTTTATAGGAATCTACAAGCGGAAGTGCCATGT

[0375] AAAGATGGATATGTCCAGGAGTTCTGCCCATTATGGAGAGAGAAT

[0376] GCAGAAGAAAGTGACCTGACCTCAGATAAAGCAGT

[0377] TGGATATATCACATCCGTGGTACCCTACAGGTCTGTATCATTACTA

[0378] CATTGTGACATTGAGATTCCTCCAGGGTCCAATC

[0379] AAAGCTTATTAGATCAACTGGCTACTAATTTATCCCTGATTGCCAT

[0380] GCATTCTGTGAGGGAGGGCGGGGTAGTGATCATC

[0381] AAAGTACTGTATGCAATGGGGTACTACTTCCATTTACTCATGAATT

[0382] TATTCACTCCATGTTCCACAAAAGGATATATACT

[0383] CTCCAATGGCTATGCCTGTAGAGGGGATATGGAGTGTTACCTGAT

[0384] ATTCGTTATGGGCTACTTGGGCGGGCCCACCTTCG

[0385] TGCACGAAGTGGTAAGGATGGCAAAAACTCTAATACAACGACAC

[0386] GGTACACTCCTATCTAAATCAGATGAAATTACATTG

[0387] ACTAAGCTATTTACCTCACAGCAGCGTCATGTAACAGATATCCTAT

[0388] CCAGTCCTTTACCGAAGCTAATGAAGCTCTTGAG

[0389] AGAAAATATTGATGCCGCACTAATTGAAGCTGGGGGACAGCCCG

[0390] TCCGTCCATTCTGTGCGGAAAGTTTGGTGAGCACAC

[0391] TAACAGATATGACTCAGACAGCTCAGATCATTGCCAGCCACATTG

[0392] ACACAGTCATTCGGTCTGTTAATTTACATGGAGGCT

[0393] GAGGGTGACCTCGCCGACACAGTGTTCTTATTACTCCTTACAAT

[0394] CTATCCACAGACGGTAAAAGGAGAACATCACTTAA

[0395] GCAGTGCACCAAACAGATCTTGGAAGTCACAATACTGGGTCTCA

[0396] GAGCCAAAGATATCATAAAGTAGGTGATGTAATCA

[0397] GTTTAGTACTCAGAGGTGCGGTTCTCTAGAGGACCTCCATCCCAT

[0398] TAAGGACATACCTGAAGCGCAGTACCTGCACTAAA

[0399] TACCTGAAAGCGGTCCTAGGTATTACTAAACTCAAAGAATGTTC

[0400] ACAGATACCTCATTACTGTACTTGACTCGTGCTCA

[0401] ACAAAAATTCTACATGAAAACCATAGGTAATGCTGCCAAGGGATA

[0402] TTACTGTAATAATGACTCTTAAAGGCAATCGTACG

[0403] CCAATCAGTTATTCTTCCTAACTGATGACTCCCTCATTGACTCAATT

[0404] ATACCAGATTAGAAAAAAGTTAAATTCCGACTCT

[0405] TTGGAACTCGTATTCGGATTCAGTTAGTTAACTTTAAGCAAGAGTGCGCAAAGTCATCCCTAATTATAGTGATGTCATTC

[0406] ACCAAATCTCTGTTTGGT.

[0407] In a preferred embodiment, the vaccine strain has a hemagglutination titer of approximately 9 log2HA and a viral titer of approximately 10. 9.5 EID 50 / mL.

[0408] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 9" includes ±5% of 9, or from 8.55 to 9.45; "about 10" includes ±5% of 9, or from 8.55 to 9.45; 9.5 "Including 10" 9.5 ±5%, or from 0.95×10 9.5 Up to 1.05×10 9.5 .

[0409] According to another aspect of the present invention, a method for constructing the above-mentioned genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E is provided. The method comprises the following steps: (a) using the pCAGGS-MG7-cDNA whole-genome plasmid as a template, site-directed mutagenesis is performed on the differentially expressed amino acid sites of the structural proteins of the MG7 strain to obtain a mutant plasmid; (b) the mutant plasmid is then subjected to reverse genetics to construct the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E. In a preferred embodiment, in the pCAGGS-MG7-cDNA whole-genome plasmid, the F protein cleavage site of the MG7 strain has been mutated to the F protein cleavage site of the La Sota strain, and 18 amino acids are deleted from positions 443 to 460 of the NP protein of the MG7 strain. In a preferred embodiment, the upstream primer used for this site-directed mutagenesis is 5'-GTGCTCTAAGGTCACAGAAACTGAAGAGGAGGATT-3'. In a preferred embodiment, the downstream primer used for the site-directed mutagenesis is 5'-AATCCTCCTCTTCAGTTTCTGTGACCTTAGAGCAC-3'.

[0410] In a preferred embodiment, the site-directed mutagenesis includes the following steps: (1) PCR amplification: amplify the DNA fragments upstream and downstream of the mutation site, and after gel recovery of the upstream and downstream DNA fragments, mix them in equal molar amounts as a template to amplify the full-length DNA mutation fragment and obtain the amplification product; (2) ligation: double digest the amplification product with the pCAGGS-MG7-cDNA whole genome plasmid, and gel recover the digested product to obtain the digested product. The digested product is ligated in a metal bath using T4 DNA ligase to obtain the ligation product; (3) transformation: transform the ligation product into DH5α competent cells, add LB liquid medium and culture for about 30 min, plate on ampicillin-resistant LB solid medium plates, and culture for 12 hours; and (4) screening: identify whether the MG7 mutant plasmid is successfully ligated, and screen to obtain the successfully ligated mutant plasmid.

[0411] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0412] In a preferred embodiment, the temperature of the metal bath is about 16°C.

[0413] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 16" includes ±5% of 16, or from 15.2 to 16.8.

[0414] In a preferred embodiment, the LB solid medium is an ampicillin-resistant LB solid medium.

[0415] In a preferred embodiment, the culture temperature is approximately 37°C.

[0416] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 37" includes ±5% of 37, or from 35.15 to 38.85.

[0417] In a preferred embodiment, the reverse genetics operation includes the following steps: (i) co-transfecting BHK-21 cells with the successfully ligated mutant plasmid and helper plasmid, culturing, freezing and thawing, filtering, and obtaining a cell culture; and (ii) inoculating the cell culture into chicken embryos, incubating, and collecting the allantoic fluid of chicken embryos with positive hemagglutination test results to obtain the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E. In a preferred embodiment, the helper plasmid is pBSK-NP, pBSK-P, and pBSK-L. In a preferred embodiment, the BHK-21 cells are BHK-21 cells pre-infected with recombinant vaccinia virus VVT7.

[0418] In this invention, when the number of infections, time, or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “0.001 to 0.1” is disclosed, the described range should be interpreted as including ranges “0.001 to 0.1”, “0.001 to 0.08”, “0.001 to 0.06”, “0.001 to 0.04”, “0.001 to 0.02”, “0.02 to 0.1”, “0.02 to 0.08”, “0.02 to 0.06”, “0.02 to 0.04”, “0.04 to 0.1”, “0.04 to 0.06”, “0.06 to 0.1”, “0.06 to 0.08”, “0.08 to 0.1”, etc. When a range of values ​​is described in this document, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0419] In a preferred embodiment, the multiplicity of infection of the recombinant vaccinia virus VVT7 is 0.001 to 0.1 MOI, for example, about 0.01 MOI.

[0420] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.01" includes ±5% of 0.01, or from 0.0095 to 0.0105.

[0421] In a preferred embodiment, the pre-infection time is 0.5 to 1.5 hours, for example, about 1 hour.

[0422] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0423] In a preferred embodiment, the culture time is 48 to 96 hours, for example, about 72 hours.

[0424] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 72" includes ±5% of 72, or from 68.4 to 75.6.

[0425] In a preferred embodiment, the culture temperature is approximately 37°C.

[0426] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 37" includes ±5% of 37, or from 35.15 to 38.85.

[0427] In a preferred embodiment, the culture container is a cell culture incubator containing 5% CO2. In a preferred embodiment, the freeze-thaw cycle is once. In a preferred embodiment, the filter has a pore size of 0.22 μm. In a preferred embodiment, the chicken embryo is a 9-11 day old SPF chicken embryo. In a preferred embodiment, the incubation time is 2-4 days, for example, about 3 days.

[0428] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 3" includes 3 ± 5%, or from 2.85 to 3.15.

[0429] According to another aspect of the present invention, the use of the above-mentioned genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E in vaccine preparation is provided. In a preferred embodiment, the vaccine is a protective vaccine against Newcastle disease caused by genotype VII. In a preferred embodiment, the vaccine is an avian vaccine. In a preferred embodiment, the avian is chicken. In a preferred embodiment, the protective effect further includes one or both of the following: preventing the shedding of Newcastle disease virus and inhibiting the replication of Newcastle disease virus in organs.

[0430] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0431] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0432] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0433] Example

[0434] 1. Materials and Methods

[0435] 1.1 Plasmids and Cells

[0436] The pCAGGS-MG7-cDNA whole-genome plasmid (the F protein cleavage site of the MG7 strain has been mutated to the F protein cleavage site of the La Sota strain, and 18 amino acids are deleted from positions 443 to 460 of the NP protein); the three helper plasmids pBSK-NP, pBSK-P, and pBSK-L, and the BHK-21F cells were all preserved in our laboratory.

[0437] 1.2 Viruses, bacteria, and laboratory animals

[0438] Recombinant poxvirus VVT7 was amplified and preserved in our laboratory; Escherichia coli DH5α competent cells were purchased from Dalian Takara Bio Inc.; SPF chicken embryos and 1-day-old SPF chickens were purchased from Boehringer Ingelheim Ltd., Beijing.

[0439] 1.3 Primer Design and Synthesis

[0440] Based on the pCAGGS-MG7-cDNA whole genome plasmid, corresponding mutant primers were designed using biological software such as Oligo7 (as shown in Table 1).

[0441] Table 1 Primer Information

[0442] Primer name Primer sequences (5′-3′) rMG7-F-N471A-F GAACTTGGAAACGTCGCGAATTCAATCAGCAATGCC rMG7-F-N471A-R GGCATTGCTGATTGAATTCGGCGACGTTTCCAAGTTC rMG7-HN-G256E-F GTGCTCTAAGGTCACAGAAACTGAAGAGGAGGATT rMG7-HN-G256E-R AATCCTCCTCTTCAGTTTCTGTGACCTTAGAGCAC rMG7-L-V1291I-F CAGAGTGTCGCCTTACATACACATATCCAATGATT rMG7-L-V1291I-R AATATTGGATATGTGTATGTAAGGCGACACTCTG

[0443] 1.4 Construction of MG7 mutant plasmid

[0444] Using the pCAGGS-MG7-cDNA whole-genome plasmid as a template, site-directed mutagenesis was performed on differentially expressed amino acid sites in structural proteins of strain MG7. The specific method is as follows: First, PCR amplification of the upstream and downstream DNA fragments at the mutation site was performed. After gel recovery of the upstream and downstream DNA fragments, equimolar amounts were mixed and used as template. Overlap PCR amplification of the full-length DNA mutation fragment was then performed (Tables 2 and 3). The full-length DNA mutation fragment and the pCAGGS-MG7-cDNA whole-genome plasmid were double-digested with enzymes. The double-digested plasmid and the target fragment were recovered by gel electrophoresis. The double-digested plasmid and fragment were ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α competent cells, and 500 μL of LB liquid medium was added. After incubation at 37°C for 30 min, an appropriate amount of culture medium was plated onto LB solid medium plates (ampicillin-resistant) and incubated overnight at 37°C. Single colonies were picked from the solid plates and inoculated into 5 mL of LB liquid medium (ampicillin-resistant) and incubated overnight at 37°C. After culturing, a small amount of MG7 mutant plasmid was extracted, and nucleic acid electrophoresis was used to identify whether the MG7 mutant plasmid was successfully ligated. Suspected successfully ligated MG7 mutant plasmids were sent to the company for sequencing, and positive plasmids were used for MG7 mutant virus rescue.

[0445] Table 2 PCR reaction system

[0446] reagents Volume (50 μL) PrimeStar DNA Polymerase 0.5μL 5×PrimeStar buffer 10μL dNTP solution 4μL upstream primer 1.5μL Downstream primer 1.5μL DNA template (25 ng / μL) 1μL <![CDATA[ddH2O]]> Add to 50μL

[0447] Table 3 Site-directed mutagenesis PCR program

[0448]

[0449] 1.5, MG7 Mutant Virus Rescue

[0450] When the BHK-21F cell density in the cell plate reached approximately 70%, the cell culture supernatant was discarded, and 1 mL of Opti-MEM was added. BHK-21F cells were pre-infected with recombinant vaccinia virus VVT7 (0.01 MOI) for 1 hour. The MG7 mutant plasmid was co-transfected with three helper plasmids (pBSK-NP, pBSK-P, and pBSK-L) into BHK-21 cells. The transfected cell plates were cultured at 37°C in a 5% CO2 incubator for 72 hours. After a freeze-thaw cycle, the cell culture was filtered through a 0.22 μm filter and completely seeded into 9–11 day old SPF chicken embryos. The wax-sealed SPF chicken embryos were incubated at 37°C for 3 days. After 3 days of incubation, allantoic fluid from the chicken embryos was aspirated for an allantoic acid (HA) test. Allantoic fluid from chicken embryos with positive HA test results was collected and sequenced for identification.

[0451] 1.6 Detection of the genetic stability of the MG7 mutant virus

[0452] The MG7 mutant virus was diluted 10% with sterile PBS. 5 The virus was inoculated into 9-11 day old SPF chicken embryos and passaged continuously to the 15th generation. RNA from the MG7 mutant virus was extracted at generations 5, 10, and 15 according to the viral RNA extraction kit instructions. The DNA fragment containing the mutation site was amplified by RT-PCR, and the target fragment was recovered by gel electrophoresis and sent to the company for sequencing. The sequencing results were analyzed using SeqMan software to verify the genetic stability of the MG7 mutant virus.

[0453] 1.7 Virulence determination of MG7 mutant virus

[0454] Referring to the WOAH standard, the virulence of the MG7 mutant virus was determined using two indicators: MDT and ICPI.

[0455] MDT assay: First, the allantoic fluid of the MG7 mutant virus was serially diluted 10-fold, then 10 -9 ~10 -6 Gradual-diluted virus solutions were inoculated into 9-11 day old SPF chicken embryos, which were then incubated at 37°C for 7 days. Embryos were candled twice daily, and embryos that died within 24 hours were discarded. The time of death for each embryo was recorded, and the MDT value of the mutant virus was calculated. The virulence criteria for NDV are as follows: attenuated strains have an MDT greater than 90 hours, moderately virulent strains have an MDT between 60 and 90 hours, and highly virulent strains have an MDT less than 60 hours.

[0456] ICPI determination: First, the allantoic fluid of the MG7 mutant virus was diluted 10-fold. Then, the virus fluid was inoculated intracranially into 1-day-old chicks, 50 μL / chick, 10 chicks per group. The chicks' condition was observed and scored daily (normal chicks: 0 points; sick chicks: 1 point; dead chicks: 2 points) for a total of 8 days. Finally, the ICPI value of the mutant virus was calculated. The virulence criteria for NDV are as follows: attenuated strains ICPI less than 0.7, moderately virulent strains ICPI between 0.7 and 1.5, and virulent strains ICPI greater than 1.60.

[0457] 1.8 Protection test of SPF chickens immunized with attenuated live vaccine and challenge test

[0458] Four-week-old SPF chickens were randomly divided into 6 groups of 16 birds each. SPF chickens were immunized with MG7 strain, three MG7 mutant viruses, and the La Sota strain via nasal or ocular drops (10 chickens per group). 7 EID 50 SPF chickens were vaccinated once with 100 μL / bird, while the PBS control group was vaccinated with 100 μL of sterile PBS via the same route. Blood was collected from the subwing vein of SPF chickens at 1, 2, and 3 weeks post-immunization, and serum was separated. On day 21 post-immunization with a live attenuated vaccine, SPF chickens were vaccinated with NDV-97 strain at a dose of 10 μL / bird. 5 EID 50The chickens were challenged with a dose of 100 μL via intramuscular injection in the leg. They were observed for 14 consecutive days, and the physiological condition and mortality rate of the SPF chickens were recorded (as shown in Table 4).

[0459] Table 4 Immunization and Infection Challenge Protocols

[0460] strain Inoculation dosage route of vaccination Blood collection time Infectious drug dosage rMG7-F-N471A <![CDATA[10 7 EID 50 / 100μL / bird Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]> rMG7-HN-G256E <![CDATA[10 7 EID 50 / 100μL / bird Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]> rMG7-L-V1291I <![CDATA[10 7 EID 50 / 100μL / bird Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]> MG7 <![CDATA[10 7 EID 50 / 100μL / bird Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]> LaSota <![CDATA[10 7 EID 50 / 100μL / bird Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]> PBS Nose and eye drops Weeks 1, 2, and 3 <![CDATA[10 5 EID 50 / only]]>

[0461] 1.9 Detection of the replication capacity of attenuated strains in vivo after immunization of SPF chickens

[0462] Throat and cloacal swabs were collected from SPF chickens in each vaccine-immunized group on days 2, 4, 6, 8, and 10 after immunization. The swabs were placed in 500 μL of PBS containing penicillin and antibiotics, vigorously shaken to mix, and then allowed to stand for 30 min. The supernatant from the swab extract was centrifuged and inoculated into 9-11 day old chicken embryos, with 3 swabs inoculated at 100 μL / swab for each dilution. After incubation at 37℃ for 48 h, the HA titer of the allantoic fluid from the chicken embryos was measured. If the HA titer was ≥1:16, the swab was considered to be shedding the virus, and the virus shedding rate was calculated.

[0463] 1.10 Detection of HI antibody titer after immunization of SPF chickens

[0464] First, add 25 μL of PBS to the hemagglutination plate, then add 25 μL of the detection antigen and serially dilute it 2-fold, then add another 25 μL of PBS, and finally add 25 μL of 1% chicken red blood cell suspension. Shake well and let stand for 15 minutes, then read the HA titer. Prepare 4 units of antigen according to the HA titer of the detection antigen. First, add 25 μL of PBS to the hemagglutination plate, then add 25 μL of the serum to be tested and serially dilute it 2-fold, then add 25 μL of 4 units of the detection antigen. Shake well and let stand for 15 minutes, then add 25 μL of 1% chicken red blood cell suspension. Shake well and let stand for 15 minutes, then read the HI titer of the serum to be tested.

[0465] 1.11. Detection of virus shedding level in SPF chickens after challenge

[0466] Swabs were collected from SPF chickens in each vaccine-immunized group on days 3, 5, 7, 9, and 11 after challenge with the virus. The swabs were placed in 1 mL of PBS containing penicillin and antibiotics, vigorously shaken to mix, and allowed to stand for 30 min. The supernatant from the swab extract was centrifuged and serially diluted 10-fold, then inoculated into 9-11 day old chicken embryos, with 3 swabs per dilution (100 μL / swab). After incubation at 37°C for 48 h, the allantoic fluid (HA) titer was measured. If the HA titer was ≥1:16, the swab was considered to be shedding the virus, and the viral load was calculated.

[0467] 1.12. Detection of virus carriage in various organs of SPF chickens after challenge.

[0468] On days 3 and 5 after SPF chicken immunization and challenge, three SPF chickens from each group were randomly and painlessly sacrificed. Six organs (liver, spleen, kidney, bursa of Fabricius, pancreas, and small intestine) were collected. PBS containing penicillin and antibiotics was added, followed by two autoclaved steel balls. The tissues were homogenized at low temperature for 10 minutes using a high-throughput tissue homogenizer. After homogenization, the supernatant was serially diluted 10-fold and inoculated into 9-11 day old chicken embryos, with three embryos inoculated at 100 μL per embryo for each dilution. After incubation at 37°C for 48 hours, the allantoic fluid (HA) titer was measured. If the HA titer was ≥1:16, the organ was considered to contain the virus. The viral load in the organs was then calculated.

[0469] 2. Results and Analysis

[0470] 2.1 Analysis of the rescue and growth characteristics of the MG7 mutant virus

[0471] Using the pCAGGS-MG7-cDNA whole-genome plasmid as a template, the full-length DNA fragment containing the mutation site was amplified by PCR. After double enzyme digestion, ligation, transformation and sequencing identification, this study successfully constructed multiple MG7 mutant plasmids.

[0472] Chicken embryo allantoic fluid with positive HA test results was collected, and rescued viral RNA was extracted. The target fragment containing the amino acid site mutation was amplified by RT-PCR and sent to the company for sequencing. Sequencing results showed that the amino acid site mutation of the rescued MG7 mutant virus was successful.

[0473] The MG7 mutant virus was continuously passaged in chicken embryos aged 9–11 days. By measuring the HA titer and viral titer of the MG7 mutant virus, this study finally screened out three mutant viruses with superior growth performance to the MG7 strain and named them rMG7-F-N471A, rMG7-HN-G256E and rMG7-L-V1291I (as shown in Table 5).

[0474] Table 5. Analysis of growth characteristics of the rescued MG7 mutant virus in chicken embryos.

[0475] Virus <![CDATA[Hemagglutination titer (log2HA)]]> <![CDATA[Virus titer (lgEID 50 / mL)]]> MG7 9 9 rMG7-F-N471A 9 9.5 rMG7-HN-G256E 9 9.5 rMG7-L-V1291I 9 9.5 rMG7-HN-S269R 7 8.75 rMG7-HN-K293G 8 9.16 rMG7-HN-P315S 9 8.75 rMG7-L-S954N 7 8 rMG7-L-K997R 9 8.75 rMG7-L-K1159R 6 8.38 rMG7-L-R1183S 3 7

[0476] 2.2 Analysis of the genetic stability of the MG7 mutant virus

[0477] RNA was extracted from the allantoic fluid of the 5th, 10th, and 15th generations of the rMG7-F-N471A, rMG7-HN-G256E, and rMG7-L-V1291IMG7 mutant viruses. The mutant fragments were amplified by RT-PCR and sequenced. Sequencing results showed that the three MG7 mutant viruses, rMG7-F-N471A, rMG7-HN-G256E, and rMG7-L-V1291I, were stably inherited at the mutant sites in 9–11 day old SPF chicken embryos up to the 15th generation, without any reversion mutations (e.g., ...). Figure 1 , Figure 2 and Figure 3 (As shown).

[0478] 2.3 Virulence Analysis of MG7 Mutant Virus

[0479] The MDT and ICPI values ​​of the three MG7 mutant viruses were determined according to the WOAH standard. The experimental data showed that the MDT and ICPI values ​​of the three MG7 mutant viruses rMG7-F-N471A (MDT≥120h, ICPI=0.13), rMG7-HN-G256E (MDT≥120h, ICPI=0.02), and rMG7-L-V1291I (MDT≥120h, ICPI=0) all met the requirements for attenuated NDV strains (as shown in Table 6).

[0480] Table 6. Virulence Analysis of MG7 Mutant Virus

[0481]

[0482] 2.4 Analysis of the replication capacity of MG7 mutant virus in vivo after immunization of SPF chickens

[0483] The replication ability of the MG7 mutant virus in SPF chickens was investigated. Results showed that all attenuated strains could replicate in SPF chickens, with the highest shedding rates in the larynx and cloaca on days 4 and 6 post-immunization. The shedding rate in the rMG7-F-N471A vaccine immunization group was higher than that in the MG7 vaccine immunization group; therefore, the rMG7-F-N471A mutant strain exhibited superior replication ability in SPF chickens compared to the MG7 strain (as shown in Table 7).

[0484] Table 7. Results of virus shedding in SPF chickens after immunization with attenuated live vaccine.

[0485]

[0486]

[0487] 2.5 Detection of HI antibody titer after immunization of SPF chickens

[0488] The HI antibody titer in the serum of SPF chickens immunized with each vaccine was determined at different time points after immunization using the MG7 strain as the detection antigen. The results showed that the serum HI antibody titer of SPF chickens immunized with each vaccine increased continuously after the start of immunization, reaching its highest level at week 3. The HI antibody titers of the MG7 and its mutant virus vaccine immunization groups ranged from 1:16 to 1:32. There was no significant difference in HI antibody titers between the rMG7-F-N471A, rMG7-HN-G256E, and rMG7-L-V1291I vaccine immunization groups and the MG7-F-N471A, rMG7-HN-G256E, and rMG7-L-V1291I vaccine immunization groups compared to the MG7 vaccine immunization group. Figure 4 ).

[0489] 2.6 Detection of virus shedding level and virus carriage status in various organs of SPF chickens after challenge.

[0490] SPF chickens were challenged with the NDV-97 strain on day 21 after immunization with a live attenuated vaccine. Results showed that all SPF chickens in the PBS control group died within 6 days, while the protection rate of SPF chickens immunized with the MG7 vaccine was 90%, and the protection rate of SPF chickens immunized with other vaccines was 100% (e.g., ...). Figure 5 (As shown in Table 8). Swab titration results showed that, compared with the MG7 and La Sota vaccine immunization groups, no virus was detected in the throat and cloacal swabs of SPF chickens immunized with rMG7-F-N471A and rMG7-HN-G256E vaccines, while the viral shedding rate of the swabs in the rMG7-L-V1291I vaccine immunization group was 20%. This indicates that the rMG7-F-N471A mutant strain and the rMG7-HN-G256E mutant strain, as attenuated vaccines, can provide 100% protection for SPF chickens and effectively prevent viral shedding in SPF chickens (as shown in Table 8).

[0491] Virus titration was performed on the collected organs. The organ titration results showed that the virus was detectable in all organs of the PBS control group; in the rMG7-F-N471A and rMG7-HN-G256E vaccine immunization groups, the virus could only be isolated from the bursa of Fabricius; in the rMG7-L-V1291I vaccine immunization group, the virus could be isolated from the spleen, pancreas, and bursa of Fabricius; in the MG7 vaccine immunization group, the virus could be isolated from the pancreas, bursa of Fabricius, and kidney; and in the La Sota vaccine immunization group, the virus was distributed in the liver, spleen, pancreas, and bursa of Fabricius. These results indicate that, compared with the MG7 and La Sota vaccine immunization groups, the rMG7-F-N471A mutant and rMG7-HN-G256E mutant strains, as attenuated vaccines, effectively inhibited viral replication in the organs of SPF chickens (as shown in Table 9).

[0492] Table 8. Results of virus shedding after challenge with NDV-97 strain in SPF chickens 21 days after immunization with attenuated live vaccine.

[0493]

[0494] In this case, "-" indicates that all SPF chickens died.

[0495] Table 9. Distribution of virus in different tissues in SPF chickens 21 days after challenge with NDV-97 strain following attenuated live vaccine.

[0496]

[0497]

[0498] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A genotype VII Newcastle disease attenuated live marker vaccine strain rMG7-HN-G256E, characterized in that, Its accession number is CGMCC No.46292.

2. A method for constructing the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E as described in claim 1, characterized in that, The construction method includes the following steps: (a) Using the pCAGGS-MG7-cDNA whole genome plasmid as a template, site-directed mutagenesis was performed on the structural protein differential amino acid sites of the MG7 strain to obtain mutant plasmids; (b) The mutant plasmid was reverse genetically modified to construct the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E.

3. The construction method according to claim 2, characterized in that, In the pCAGGS-MG7-cDNA whole genome plasmid, the F protein cleavage site of the MG7 strain has been mutated to the F protein cleavage site of the La Sota strain, and 18 amino acids are deleted from positions 443 to 460 of the NP protein of the MG7 strain.

4. The construction method according to claim 2, characterized in that, The upstream primer used for the site-directed mutagenesis is 5'- GTGCTCTAAGGTCACAGAAACTGAAGAGGAGGATT-3'.

5. The construction method according to claim 2, characterized in that, The downstream primer used for the site-directed mutagenesis was 5'-AATCCTCCTCTTCAGTTTCTGTGACCTTAGAGCAC-3'.

6. The construction method according to claim 2, characterized in that, The site-directed mutagenesis includes the following steps: (1) PCR amplification: DNA fragments upstream and downstream of the mutation site are amplified. After the upstream and downstream DNA fragments are recovered by gel, they are mixed in equal molar amounts as templates to amplify the full-length DNA mutation fragments and obtain the amplification products. (2) Ligation: The amplification product and the pCAGGS-MG7-cDNA whole genome plasmid were double digested with enzymes, and the digested products were recovered by gel to obtain the digested products. The digested products were ligated in a metal bath using T4 DNA ligase to obtain the ligation products. (3) Transformation: The ligation product was transformed into DH5α competent cells, and LB liquid medium was added for 28.5-31.5 min. The cells were then spread on ampicillin-resistant LB solid medium plates and cultured for 12 hours. as well as (4) Screening: Determine whether the MG7 mutant plasmid has been successfully ligated, and screen out the mutant plasmids that have been successfully ligated.

7. The construction method according to claim 6, characterized in that, The temperature of the metal bath is 15.2–16.8 °C.

8. The construction method according to claim 6, characterized in that, The LB solid medium is an ampicillin-resistant LB solid medium.

9. The construction method according to claim 6, characterized in that, The culture temperature is 35.15–38.85℃.

10. The construction method according to claim 2, characterized in that, The reverse genetics operation includes the following steps: (i) The successfully ligated mutant plasmid and helper plasmid were co-transfected into BHK-21 cells and cultured, frozen and thawed, and filtered to obtain cell culture; and (ii) The cell culture was inoculated into chicken embryos, incubated, and the allantoic fluid of chicken embryos with positive hemagglutination test results was collected to obtain the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E.

11. The construction method according to claim 10, characterized in that, The helper plasmids are pBSK-NP, pBSK-P, and pBSK-L.

12. The construction method according to claim 10, characterized in that, The BHK-21 cells were BHK-21 cells pre-infected with recombinant vaccinia virus VVT7.

13. The construction method according to claim 12, characterized in that, The multiplicity of infection (MOI) of the recombinant vaccinia virus VVT7 is 0.001 to 0.

1.

14. The construction method according to claim 13, characterized in that, The multiplicity of infection (MOI) of the recombinant vaccinia virus VVT7 was 0.0095–0.0105.

15. The construction method according to claim 12, characterized in that, The pre-infection time is 0.5 to 1.5 hours.

16. The construction method according to claim 15, characterized in that, The pre-infection time is 0.95–1.05 h.

17. The construction method according to claim 10, characterized in that, The incubation time is 48–96 hours.

18. The construction method according to claim 17, characterized in that, The culture time was 68.4–75.6 h.

19. The construction method according to claim 10, characterized in that, The culture temperature is 35.15–38.85℃.

20. The construction method according to claim 10, characterized in that, The culture container is a cell culture incubator containing 5% CO2.

21. The construction method according to claim 10, characterized in that, The freeze-thaw cycle is 1 time.

22. The construction method according to claim 10, characterized in that, The filter has a pore size of 0.22 μm.

23. The construction method according to claim 10, characterized in that, The chicken embryos are 9-11 day old SPF chicken embryos.

24. The construction method according to claim 10, characterized in that, The incubation period is 2 to 4 days.

25. The construction method according to claim 24, characterized in that, The incubation period is 3 days.

26. The use of the genotype VII Newcastle disease attenuated marker vaccine strain rMG7-HN-G256E as described in claim 1 in the preparation of a vaccine, characterized in that, The vaccine in question is a protective vaccine against Newcastle disease caused by genotype VII Newcastle disease virus.

27. The use according to claim 26, characterized in that, The vaccine in question is a poultry vaccine.

28. The use according to claim 27, characterized in that, The poultry mentioned is chicken.

29. The use according to claim 26, characterized in that, The protective effect further includes one or both of the following: preventing the shedding of Newcastle disease virus and inhibiting the replication of Newcastle disease virus in organs.

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