Rescuing method of gene VII type Newcastle disease virus subjected to codon replacement

Abstract

The invention relates to the technical field of reverse genetic manipulation, and discloses a rescuing method of a gene VII type Newcastle disease virus subjected to codon replacement. The method comprises the steps: respectively cloning the NP, P and L genes of the Newcastle disease virus and the DE3 gene for expressing the T7 RNA polymerase into a pXJ40 vector to obtain plasmids pXJ40-NP, pXJ40-P, pXJ40-L and pXJ40-DE3; cloning the whole genome cDNA of the Newcastle disease virus into a pBR322 plasmid to obtain pBR322-DHN3; replacing part of codons in an NP gene coding region with codons with the highest use frequency in a unified manner, and replacing the codons with pBR322-DHN3 plasmids to form new plasmids pBR322-mNPDHN3; and co-transfecting bHK-21 cells by adopting the five plasmidsso that the rescued virus rDHN3-mNP is obtained. The method provided by the invention is more beneficial to virus rescue and virus pathogenic mechanism research.

Description

technical field [0001] The invention relates to the technical field of reverse genetic manipulation, in particular to a method for rescuing a strain of gene VII Newcastle disease virus undergoing codon substitution. Background technique [0002] Newcastle disease is a highly contagious and fatal disease caused by Newcastle disease virus (NDV) that mainly affects chickens, turkeys, wild birds and ornamental birds, commonly known as fowl plague. It is a highly contagious, acute and severe infectious disease. Humans occasionally become infected, manifesting as conjunctivitis. The World Health Organization (OIE) lists it as an infectious disease that must be notified, and the Ministry of Agriculture of my country also lists it as a class of animal diseases that must be notified. Because of its fast transmission speed and 100% morbidity and fatality rate, once it spreads, it will seriously endanger the poultry industry and cause immeasurable losses. [0003] According to epide...

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Problems solved by technology

This method may lead to: (1) Every time the virus is rescued, a large number of plasmids need to be extracted, and then digested and spliced ​​to obtain the full-length genome of the virus. The base is missing, and finally the virus cannot be obtained

(2) The full-length spliced ​​viral genome is transcribed in vitro, and the environment cannot be completely simulated into the intracellular environment during the in vitro transcription process. Even if the transcript is transfected into the cell again, the working efficiency of the sub-transcription product cannot be estimated

However, the disadvantage of this method is that the virus of fowl pox virus exists in the cells at the same time. Even if the virus is successfully rescued, it still takes a lot of time and energy to purify the virus in the later stage.

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