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 ineffective prevention of virus transmission and incomplete protection efficiency, and achieve high antigen matching, high virus titer, genetic The effect of stable performance

Active Publication Date: 2014-01-22
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • 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

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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Example Embodiment

[0066] Example 1. Construction of transcription vector:

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

[0068] The reverse genetic operation platform of NDV Ⅶ-97 strain is modified on the basis of pBR322 plasmid, which contains T7 promoter and T7 ribozyme transcription termination sequence at both ends of NDV whole genome, and inserts at the same time with self-cutting function. DNA sequence of the hepatitis D virus ribozyme. 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, the 3'end of the tran...

Example Embodiment

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

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

Example Embodiment

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

[0096] Through primers and Overlapping PCR method, the 1756, 1917 and 1954 amino acids in the L protein domain VI of the NDV VII-97 strain were subjected to single point mutations (Table 1), and the amplified target fragment and the prNDV-97 vector were subjected to single point mutations. Hind Ⅲ and Xba Ⅰ Restriction endonuclease double digestion, 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 virulent strain was mutated to the F protein cleavage site (GRQGRL) of the attenuated LaSota strain. The mutation position of F protein was amplified by Overlapping PCR with primers, and the amplified target fra...

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Abstract

The invention relates to a VII type Newcastle disease virus L-gene mutation attenuated vaccine strain produced by using a reverse genetic manipulation technology and a preparation method of the VII type Newcastle disease virus L-gene mutation attenuated vaccine strain, and belongs to the field of biological products and biological technologies. Toxicity of the VII type Newcastle disease virus L-gene mutation attenuated vaccine strain is weakened obviously, and at the same time, the vaccine strain on a chick embryo has high titer, stable heritability and high compatibility to an antigen of a prevalent virus, is suitable for large-scale production of vaccines, and can be used for producing the vaccines. Compared with conventional vaccines (gene I and gene II types), the VII type Newcastle disease virus L-gene mutation attenuated vaccine strain has a wide application prospect in control of incidence and prevalence of the Newcastle diseases.

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 fa...

Claims

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Application Information

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IPC IPC(8): C12N7/04C12N15/45C12N15/63A61K39/17A61P31/14C12R1/93
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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