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VII type Newcastle disease virus L gene mutation attenuated vaccine strain and preparation method thereof

A technology of Newcastle disease virus and attenuated vaccine, which is applied in the field of attenuated vaccine strains and its preparation, can solve the problems of incomplete protection efficiency and inability to effectively prevent virus transmission, and achieve high antigen matching, high virus titer, genetic The effect of stable performance

Active Publication Date: 2015-10-14
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and invent the attenuated vaccine strain of the L gene mutation of VII type Newcastle disease virus and its preparation method, thereby solving the problem of NDV vaccine strains (gene I and gene II) used in the current market in my country. ) is not consistent with the genotype of the current NDV epidemic strain (gene type VII), the protection efficiency is not complete, and the actual problem of not being able to effectively prevent the spread of the virus

Method used

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  • VII type Newcastle disease virus L gene mutation attenuated vaccine strain and preparation method thereof
  • VII type Newcastle disease virus L gene mutation attenuated vaccine strain and preparation method thereof
  • VII type Newcastle disease virus L gene mutation attenuated vaccine strain and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0066] Embodiment 1. Construction of transcription vector:

[0067] The BHK-21 cells used by the inventor are hamster kidney cells; the culture medium is containing 10% fetal bovine serum (Gibco); the NDV VII-97 strain is preserved by the Poultry Disease Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; SPF chicken embryo and SPF All chicks were purchased from Beijing Meria Verton Biotechnology Co., Ltd.

[0068] The reverse genetic operation platform of NDV Ⅶ-97 strain is transformed on the basis of pBR322 plasmid, which contains T7 promoter and T7 ribozyme transcription termination sequence at both ends of the whole NDV genome, and inserts it with self-cutting function The hepatitis D virus ribozyme sequence. The DNA fragment cloned between the T7 promoter and the ribozyme can be transcribed under the action of T7 RNA polymerase, and due to the autocatalytic function of the hepatitis D virus ribozyme, it can ensure that the 3' en...

Embodiment 2

[0069] Construction of embodiment 2.NDV genome full-length cDNA:

[0070] Five cDNA clone fragments covering the whole genome were constructed, and a complete cDNA clone of 15192 nt was obtained by ligation and assembly of the transcription vector plasmid pBR322 by using the restriction sites in the overlapping parts of each fragment. The T7 RNA polymerase promoter is prefixed at the 5' end of the full-length cDNA fragment, and the hepatitis D virus ribozyme with autocatalytic function and T7 transcription termination signal are added after the cDNA fragment. The constructed plasmid was named prNDV. To eliminate 14056bp Hind Ⅲ Restriction site, synonymously mutate the 14056th base of the L protein coding region in the genomic cDNA from A to G through PCR genome, and as a molecular marker for saving the virus, we simultaneously mutate the T7 polymerase promoter in the genomic cDNA Two redundant Gs were introduced at the 5' end of , which may help the virus rescue of paramyxov...

Embodiment 3

[0095] Example 3. NDV Ⅶ-97 strain L gene carries out site-directed amino acid mutation and L mutant full-length cDNA connection

[0096] The 1756, 1917 and 1954 amino acids in the L protein domain VI of the NDV VII-97 strain were single-point mutated by using the primers respectively by Overlapping PCR (Table 1), and the amplified target fragment and the prNDV-97 vector were subjected to single-point mutations. Hind III and Xba Ⅰ Double digestion with restriction endonucleases, nucleic acid electrophoresis, gel recovery of the target fragment and vector prNDV-97, and then ligation. The obtained mutant plasmids were named pK1756A, pK1917A and pE1954Q respectively.

[0097] On the basis of pE1954Q, the F protein cleavage site was mutated, and the F protein cleavage site (RRQKRF) of the strong strain was mutated into the F protein cleavage site (GRQGRL) of the attenuated LaSota strain. The F protein mutation position was amplified by the Overlapping PCR method with primers, and...

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Abstract

The present invention relates to a VII type Newcastle disease virus L gene mutation attenuated vaccine strain produced by using a reverse genetic operation technology, and a preparation method thereof, and belongs to the fields of biological products and biotechnology. According to the present invention, the VII type Newcastle disease virus L gene mutation attenuated vaccine strain has characteristics of significantly reduced virulence, high titer on chicken embryos, genetic stability and high antigen matching with epidemic virus, is suitable for mass production of the vaccine, can be used for vaccine manufacturing, and provides broad application prospects in the Newcastle disease incidence and prevalence control field compared with the conventional vaccine (gene I type and gene II type).

Description

technical field [0001] The invention relates to an attenuated vaccine strain with L gene mutation of type VII Newcastle disease virus produced by reverse genetic manipulation technology and a preparation method thereof, belonging to the fields of biological products and biotechnology. Background technique [0002] Newcastle disease is caused by Newcastle disease virus (NDV), which affects chickens and turkeys, and can also infect other poultry and birds. It is an acute and highly contagious disease. One of the infectious diseases (the other being highly pathogenic avian influenza). NDV belongs to the genus Mumpsvirus of the Paramyxoviridae family, has an envelope structure, and its genome is a single-strand negative-sense RNA virus that is not segmented. It is composed of 6 structural proteins, namely NP, P, M, F, HN and L. Proteins: M, F and HN proteins are related to the formation of the viral envelope; NP protein is the nucleocapsid protein, P protein is an essential fac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/85C12N15/45C12R1/93
Inventor 朱启运吉艳红付钰广
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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