ACTA VETERINARIA ET ZOOTECHNICA SINICA ›› 2018, Vol. 49 ›› Issue (6): 1282-1290.doi: 10.11843/j.issn.0366-6964.2018.06.021

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Construction of shRNA Library of Chlorocebus Sabaeus Cathepsin Genes and the Establishment of Its Stable Transfection Cell Lines

WEI Yu-shuang, LIU Jiao-yang, HAN Ying-qian, GUO Zhen-zhen, LIU Xiao-he, BA Gen, HAN Li-qiang, WANG Jiang, CHU Bei-bei*, YANG Guo-yu*   

  1. Key Lab of Regulation on Animal Growth and Development of Agricultural Ministry of China, Henan Agricultural University, Zhengzhou 450002, China
  • Received:2017-11-07 Online:2018-06-23 Published:2018-06-22

Abstract:

Up to now, the roles of cathepsins in the life cycle of Porcine reproductive and respiratory syndrome virus (PRRSV) remain unclear. This study was conducted to characterize the mechanisms of cathepsins involved in PRRSV life cycle including adsorption, invasion, uncoating, biosynthesis, assembly and release. The Chlorocebus sabaeus embryonic kidney epithelial cells Marc145 (PRRSV can be propagated) derived from MA-104 was selected. Each cathepsin shRNA clone was constructed within the lentivirus plasmid vector pLKO.1, and the shRNA library consists of sequence verified shRNA lentiviral plasmid DNA against all the cathepsin genes that allow for high throughput loss-of-function screens. Puromycin was used to select inserts in mammalian cells. Finally, there were 45 clones involving 15 cathepsin genes in this cathepsin-shRNA library, and the expression of cathepsin mRNA in the stable cell lines was markedly downregulated. These stable cell lines will lay the foundation for the further study on the roles of cathepsins in PRRSV life cycle.

Key words: cathepsin, shRNA, Marc145, lentivirus, stable knockdown cell line

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