人源性受磷蛋白稳定表达细胞系的构建  

Construction of a Stable Expression Cell Line of Human Phospholamban

作　　者：陈岚卿 王宇宁 李典一 徐路遥 李廉杰 李泽浩 刘华[2] 刘茜[1] CHEN Lan-qing;WANG Yu-ning;LI Dian-yi;XU Lu-yao;LI Lian-jie;LI Ze-hao;LIU Hua;LIU Qian(Department of Forensic Medicine,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Key Laboratory of Forensic Toxicology,Ministry of Public Security,People’s Republic of China,Beijing Municipal Public Security Bureau,Beijing 100000,China)

机构地区：[1]华中科技大学同济医学院法医学系,湖北武汉430030 [2]法庭毒物分析公安部重点实验室北京市公安局,北京100000

出　　处：《法医学杂志》2021年第5期615-620,共6页Journal of Forensic Medicine

基　　金：法庭毒物分析公安部重点实验室开放基金项目(2019FTDWFX07);高等学校大学生创新创业训练计划资助项目(202010487029)。

摘　　要：目的构建稳定表达人源性受磷蛋白(phospholamban,PLN)的细胞系,并初步探索其在心肌毒性机制研究中的应用。方法采用FastCloning方法将目的基因PLN的开放阅读框序列插入至真核表达载体pcDNA5/FRT/TO(以下简称pDFT),构建pDFT-PLN-Flag质粒,并与pOG44质粒共转染Flp-In^(TM) T-REx^(TM) 293细胞,实现目的基因的表达,通过潮霉素B抗性持续压力筛选出重组成功的单克隆细胞株。应用West⁃ern印迹法和间接免疫荧光法检验重组细胞目的蛋白的表达情况。对构建成功的细胞系进行乌头碱染毒后,通过Western印迹法,检测PLN磷酸化的变化。结果重组质粒PCR扩增及DNA电泳后含有与已知PLN基因片段大小一致的扩增产物,测序后与人源性PLN基因开放阅读框(open reading frame,ORF)序列一致。间接免疫荧光法和Western印迹法提示构建的可增殖细胞系,诱导调控下可稳定且较高水平表达人源性PLN。乌头碱浓度为1μmol/L、染毒2 h时,PLN的Thr17位点磷酸化水平降低。结论该人源性细胞株可以稳定表达PLN,并可用于乌头碱在分子水平上对细胞影响机制的研究。Objective To construct a cell line that can stably express human phospholamban(PLN)and initially explore its application in the study of myocardial toxicity mechanism.Methods FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expres-sion vector pcDNA5/FRT/TO(hereinafter referred to as pDFT)to construct the pDFT-PLN-Flag plas-mid.The Flp-In^(TM) T-REx^(TM) 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene.Successfully recombined monoclonal cell lines were screened by hygromycin B resistance.Western blot and indirect immunofluorescence(IFA)were used to examine the expression of the target protein in recombinant cells.After the cell line was exposed to aconitine,it was verified by Western blot to detect changes in PLN protein phosphorylation.Re⁃sults After PCR amplification of the recombinant plasmid and DNA electrophoresis,the length of the amplified product is the same as the known PLN gene fragment,which is consistent with the open reading frame(ORF)sequence of the human PLN gene after sequencing.IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation.Preliminary results showed that the phosphorylation level of Thr17-PLN de-creased after two hours of exposure to 1μmol/L aconitine.Conclusion This human cell line can sta-bly express PLN and can be used to study the mechanism of action of aconitine on the cell at mo-lecular level.

关 键 词：法医病理学 法医毒理学 受磷蛋白 人源性细胞系 乌头碱 生物化学 分子生物学 

分 类 号：D919.1[医药卫生—法医学]