猴源cathepsin基因shRNA文库及其稳定敲减细胞系的构建

doi:10.11843/j.issn.0366-6964.2018.06.021

摘要/Abstract

摘要：

目前关于组织蛋白酶（cathepsin）在猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）生活周期中发挥功能的研究较少。为了探究宿主细胞中cathepsin在PRRSV吸附、入侵、脱壳、生物合成、组装以及释放过程中的作用，选取源于MA-104的非洲绿猴胚胎肾上皮细胞Marc145细胞系（对PRRSV易感）；利用RNAi原理，构建了以pLKO.1为载体的cathepsin基因shRNA文库；并用puromycin筛选出稳定敲减cathepsin的细胞株。结果显示：本试验构建的cathepsin-shRNA文库包含45个克隆，涉及15种cathepsin基因，筛选出的稳定细胞株中相应cathepsin基因mRNA的表达量均有显著下降。这些细胞株的构建为进一步研究cathepsin在PRRSV生活周期中发挥的重要功能奠定了基础。

关键词: cathepsin, shRNA, Marc145, 慢病毒, 稳定敲减细胞系

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

中图分类号:

S852.23

魏宇双, 刘姣扬, 韩莹倩, 郭珍珍, 刘晓贺, 巴根, 韩立强, 王江, 褚贝贝, 杨国宇. 猴源cathepsin基因shRNA文库及其稳定敲减细胞系的构建[J]. 畜牧兽医学报, 2018, 49(6): 1282-1290.

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. Construction of shRNA Library of Chlorocebus Sabaeus Cathepsin Genes and the Establishment of Its Stable Transfection Cell Lines[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1282-1290.

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