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Method for efficiently and rapidly constructing porcine intestinal coronavirus recombinant virus

A coronavirus and recombinant virus technology, applied in the field of animal infectious disease prevention and control, can solve the problems of complicated operation and long experimental period, and achieve the effect of simple operation, short experimental period and wide target selection.

Inactive Publication Date: 2019-10-15
HUAZHONG AGRI UNIV
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Problems solved by technology

This method is complicated to operate, and requires suitable enzyme cutting sites, and the experimental period is long

Method used

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  • Method for efficiently and rapidly constructing porcine intestinal coronavirus recombinant virus
  • Method for efficiently and rapidly constructing porcine intestinal coronavirus recombinant virus
  • Method for efficiently and rapidly constructing porcine intestinal coronavirus recombinant virus

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Experimental program
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Embodiment 1

[0057] 1. Formulas of main test materials, vectors, plasmids, reagents and related media or solutions:

[0058] PEDV AJ1102 strain: isolated, identified and preserved from the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Jing Bi, Songlin Zeng, Shaobo Xiao, Huanchun Chen, Liurong Fang. Diarrhea in China. Journal of Virology. 2012, 86(19):10910–10911).

[0059] pBAC-AJ1102 is an infectious clone containing the full-length cDNA of PEDV AJ1102 strain (GenBank accession number: MK584552). PEDV can be rescued by transfecting Vero cells.

[0060] Plasmid pEGFP-C1: preserved by the Virus Division of the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University.

[0061] Vero cells: purchased from China. Wuhan. China Center for Type Culture Collection (CCTCC) of Wuhan University.

[0062] Chloramphenicol (product number: BS049B): purchased from Biosharp Company.

[0063] DMEM medium, SuperScript IV Reverse Transcripta...

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Abstract

The present invention belongs to the technical field of animal infectious disease prevention and control and specifically relates to a method for efficiently and rapidly constructing a porcine intestinal coronavirus recombinant virus. A BAC plasmid containing full-length cDNA of PEDV AJ1102 strain is cleaved by a CRISPR / Cas9 system, then a recombinant BAC plasmid of EGFP gene substituted with PEDVAJ1102 strain ORF3 gene is obtained by a homologous recombination method, and after transfecting cells, the recombinant virus rAJ1102-delta ORF3-EGFP is rescued. Since different porcine intestinal coronaviruses have similar genomic structure and replication strategies, the method is also suitable for construction of other porcine intestinal coronavirus recombinant viruses. Compared with traditional methods, the method has prominent advantages of wide target selection, high specificity, simple operation, high efficiency, short experimental period, etc., can obtain the recombinant virus withinone week, and is the method for efficiently and rapidly constructing the porcine intestinal coronavirus recombinant virus.

Description

technical field [0001] The invention belongs to the technical field of animal infectious disease prevention and control. In particular, it relates to a method for efficiently and rapidly constructing a recombinant virus of porcine enteric coronavirus. Background technique [0002] Coronaviruses are single-stranded positive-sense RNA viruses that mainly cause intestinal and respiratory tract infections. Among them, coronaviruses that cause intestinal infections in pigs mainly include porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, referred to as PEDV), porcine transmissible gastroenteritis Transmissible gastroenteritis virus (TGEV for short), porcine deltacoronavirus (PDCoV for short) and porcine enterical phacoronavirus (PEAV for short), all of these four coronaviruses can cause severe diarrhea in piglets. Threatening the healthy development of the pig industry. [0003] The construction of coronavirus recombinant viruses is often achieved by modifying th...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N15/65C12N15/50C12R1/93
CPCC07K14/005C12N15/65C12N15/85C12N15/907C12N2770/20022C12N2770/20052C12N2800/107
Inventor 肖少波方六荣彭棋王荡方谱县周艳荣刘康
Owner HUAZHONG AGRI UNIV
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