泛素连接酶E6AP敲减及过表达稳转细胞株的构建与鉴定  

Construction and identification of E6AP knockdown and overexpression stable cell lines

作　　者：彭康莉 赵博[1] PENG Kangli;ZHAO Bo(School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学药学院,上海200240

出　　处：《石河子大学学报（自然科学版）》2020年第2期233-239,共7页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(31770921,31971187)。

摘　　要：目的旨在筛选出能稳定敲减及过表达泛素连接酶E6AP的细胞株,在这两种情况下比较已知底物Arc的泛素化修饰区别,以期进一步阐明E6AP通过调控底物的泛素化进而影响底物下游功能的机理。方法从人胚肾293细胞中提取RNA再反转录成cDNA,以c DNA为模板通过PCR扩增得到E6AP目的条带,将E6AP基因克隆至p LVXIRES-mCherry质粒,构建过表达质粒;同时,合成可特异性结合E6AP mRNA的shRNA,并克隆至GIPZ质粒,构建敲减质粒。结果通过嘌呤霉素筛选得到敲减及过表达稳转细胞株,通过免疫共沉淀实验比较底物的泛素化修饰区别。结论成功构建E6AP敲减及过表达稳转细胞株,并能相应影响HEK-293细胞中底物Arc的泛素化修饰水平。Objective We aim to screen the stable cell lines that can either knock down or overexpress the expression of ubiquitin ligase E6-associated protein E6 AP.In these two cases,the ubiquitination levels of an identified substrate Arc were compared to confirm the construction of the cell lines.By using these cell lines,we hope to further elucidate the mechanisms on how E6 AP regulates the ubiquitination of the substrates and downstream functions of the substrates.Methods mRNA encoding the E6AP gene was extracted from human embryonic kidney 293 cells,c DNA was obtained through reverse transcription PCR to amplify the full-length E6AP gene. The E6AP gene was cloned into p LVX-IRES-mCherry vector to generate the overexpression plasmid.shRNAs which specifically bind to E6AP mRNA were synthesized and cloned into GIPZ vector to construct the knockdown plasmids.Results The stable cell lines were obtained by puromycin selection.Protein expression and ubiquitination of Arc were detected by western blotting and immunoprecipitation.Conclusion E6AP knockdown and overexpression stable cell lines were successfully constructed and the ubiquitination of Arc in these cell lines can be influenced by the expression of E6AP.

关 键 词：E6AP ARC 泛素化 

分 类 号：Q291[生物学—细胞生物学] Q51