Construction and application of Nluc-labeled recombinant porcine delta coronavirus infectious clone plasmid

Abstract

The invention discloses construction of an Nluc-labeled recombinant porcine delta coronavirus infectious clone plasmid and application of the Nluc-labeled recombinant porcine delta coronavirus infectious clone plasmid. Aiming at the porcine delta coronavirus, a mode of combining a BAC system and a CRISPR / Cas9 technology is adopted, on the basis of successfully constructed PDCoV infectious clone plasmids, the PDCoV infectious clone plasmids are firstly cut through the CRISPR / Cas9, and then recombinant plasmids with Nluc genes replacing NS6 genes are obtained in a homologous recombination mode; the recombinant virus stably expressing Nluc is rescued after cells are transfected, and drug experiments prove that the recombinant virus can provide a very valuable technical tool for screening research of anti-porcine delta coronavirus drugs.

Description

technical field [0001] The invention belongs to the fields of animal infectious disease prevention and control and molecular biology. It specifically relates to the construction and application of an Nluc-marked recombinant porcine delta coronavirus infectious cloning plasmid. Background technique [0002] Porcine deltacoronavirus (PDCoV) belongs to the genus Deltacoronavirus in the family Coronaviridae of the order Neveviridae, and is a single-stranded positive-sense RNA virus with an envelope and a non-segmented genome. In 2012, Hong Kong scholar Woo et al first detected porcine deltacoronavirus (Woo P.C.Y, Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 2012, 86: 3995-4008). In 2014, PDCoV broke out in pig farms in the United States. For the first time...

AI Technical Summary

Problems solved by technology

However, in vitro multiple fragment ligation and RNA transcription have disadvantages such as time-consuming and low efficiency; while the BAC system does not involve in vitro ligation and transcription, and the rescue efficiency of recombinant viruses is high, coupled with mature CRISPR / Cas9 technology , can efficiently transform recombinant plasmids, this system has stronger advantages

At present, there is no good technical platform for anti-PDCoV drug screening. EGFP is introduced as a foreign gene, which has the disadvantages of large protein and insufficient sensitivity.

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