A kind of genotype VII Newcastle disease marker vaccine strain and its preparation method and application

A technology for Newcastle disease and vaccine strains, applied in the field of vaccine genetic engineering, can solve problems that are not involved in the development of Newcastle disease marked vaccines

Active Publication Date: 2021-06-22
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

There are certain reports in the literature about Newcastle disease marker vaccine and reverse genetic manipulation technology, but none of them involve the research and development of Newcastle disease marker vaccine by gene replacement

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  • A kind of genotype VII Newcastle disease marker vaccine strain and its preparation method and application
  • A kind of genotype VII Newcastle disease marker vaccine strain and its preparation method and application
  • A kind of genotype VII Newcastle disease marker vaccine strain and its preparation method and application

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preparation example Construction

[0025] The present invention also provides a preparation method of the gene type VII Newcastle disease marker vaccine strain Paramyxovirus type II rY2-HB strain, comprising the following steps: a. Constructing the transcription plasmid of the serotype 2 avian paramyxovirus Y2 strain; b. The extracellular domains of the F and HN genes in the Y2 strain transcription plasmid were replaced with the F and HN gene extracellular domains of the gene VII type Newcastle disease virus HB0901 strain respectively; c. the transcription plasmid after the extracellular domain replacement was combined with three auxiliary plasmids The host cells were transfected to obtain the genotype VII Newcastle disease marker vaccine strain Paramyxovirus type II rY2-HB strain. The whole genome gene sequence of the avian paramyxovirus type 2 Y2 strain is SEQ ID NO:1.

[0026] The present invention also provides the application of the gene VII type Newcastle disease marker vaccine strain paramyxovirus type I...

Embodiment 1

[0028] The whole genome transcription plasmid construction of embodiment 1 avian paramyxovirus type 2 (APMV-2) Y2 strain

[0029] Download the whole genome sequences of 12 strains of avian paramyxovirus type 2 in GenBank, the accession numbers are KT071756, KT071757, KT071755, HQ896023, HQ896024, EU338414, HM159993, HM159994, HM159995, LC187305, LC187323 and NC039. After comparing and analyzing the nucleotide sequences of these sequences, the relatively conservative F8 strain (HQ896023) was selected as a template, and 10 unique amino acids in the genome of the strain were mutated into a consensus sequence shared by other strains (T 1440 C, C 2142 T,T 2709 C, C 5552 T,A 6397 G,A 6868 G, G 7768 T,A 11753 C, G 14409 A,T 14480C), obtaining the whole genome sequence of a new strain of paramyxovirus Y2 strain. Through the method of gene synthesis and cloning, the whole genome plasmid of Y2 strain was constructed. The plasmid contains the whole genome sequence of Y2 strain,...

Embodiment 2r

[0034] Whole-genome transcription plasmid construction and virus rescue of embodiment 2rY2-HB strain

[0035] Using the recombinant plasmid pY2 as a template, the extracellular domains of the F and HN genes of the Y2 strain were replaced by the extracellular domains of the F and HN genes of the NDV gene type VII HB0901 strain by the In-fusion method. The specific replacement positions are as follows: the ectodomain (1-1506nt) of the F gene of the NDV HB0901 strain replaces the ectodomain (1-1455nt) of the F gene of the Y2 strain, and the ectodomain (139-1713nt) of the HN gene of the NDV HB0901 strain replaces the HN gene of the Y2 strain Extracellular domain of the gene (130-1740nt). See the construction diagram Figure 4 . The specific construction scheme is as follows.

[0036] 2.1 PCR amplification of extracellular domains of F and HN genes of Newcastle disease virus HB0901 strain

[0037] Taking the allantoic fluid of chicken embryos of Newcastle disease virus HB0901 s...

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Abstract

The invention discloses a gene VII type Newcastle disease marker vaccine strain as well as a preparation method and application thereof. The present invention utilizes reverse genetic manipulation technology, takes the whole genome transcription plasmid of serotype 2 avian paramyxovirus Y2 strain as the backbone, and replaces the ectodomains of its F and HN genes with F and HN of gene VII Newcastle disease virus strain respectively The extracellular domain of the gene, through viral rescue, to obtain a recombinant chimeric virus. The vaccine strain has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: 202052. The chimeric virus can be used for the prevention and control of gene VII Newcastle disease, which is mainly prevalent in my country. At the same time, the chimeric virus does not contain the NP protein of Newcastle disease virus, and it will not produce antibodies against the NP protein of Newcastle disease virus after immunizing chickens. Antibody detection of rY2-HB vaccine strain immunized chicken flocks by conventional Newcastle disease virus NP antibody ELISA detection method can realize specific detection of NP antibody produced by clinical wild strains, and eliminate antibody interference produced by vaccine strains, which is beneficial to Newcastle disease monitoring and purification.

Description

technical field [0001] The invention belongs to the field of vaccine genetic engineering, in particular to a gene type VII Newcastle disease marker vaccine strain paramyxovirus type II rY2-HB strain and a preparation method thereof. More specifically, the present invention relates to the use of reverse genetic manipulation technology to replace the ectodomains of the F and HN genes of the serotype 2 avian paramyxovirus Y2 strain with the ectodomains of the F and HN genes of the gene VII Newcastle disease virus strain, Obtain chimeric virus rY2-HB strain, and the application of this strain in gene VII Newcastle disease marker vaccine. Background technique [0002] Newcastle disease (ND) is a highly contagious disease of poultry caused by Newcastle disease virus (NDV), which seriously endangers the global poultry industry. Its typical symptoms are high fever, dyspnea, diarrhea, nervous disturbance, mucosal and serosal hemorrhage. The prevention and control of ND in my countr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/85G01N33/569G01N33/68A61K39/17A61P31/14
CPCA61K39/12A61K2039/5256A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/85C12N2760/18021C12N2760/18122C12N2760/18134G01N33/56983G01N33/6854G01N2333/125G01N2469/20
Inventor 卢琴温国元王国康邵华斌罗青平商雨王红琳罗玲张蓉蓉汪宏才张腾飞张文婷
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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