慢病毒介导的稳定表达人睾丸组织泛素连接酶TRIM69基因的细胞系的建立  

Establishment of a Stable Cell Line Overexpressing TRIM69,a Novel Testis E3 Ligase,Mediated by Lentivirus

作　　者：吴为[1] 李琴山[1] 宋伟[1] 缪时英[1] 王琳芳[1] 

机构地区：[1]中国医学科学院/北京协和医学院基础医学研究所医学分子生物学国家重点实验室,100005

出　　处：《医学研究杂志》2013年第11期23-26,共4页Journal of Medical Research

基　　金：国家重大研究计划项目(2011CB944302);国家重点实验室专项基金资助项目(2060204)

摘　　要：目的利用改造的慢病毒表达系统构建表达人睾丸组织新的泛素连接酶TRIM69基因的稳定细胞系。方法采用RT-PCR方法从人胚肾细胞系HEK293T cDNA中扩增TRIM69基因及其缺失RING结构域的突变体,将其克隆至改造的plentilox3.7慢病毒载体中。将慢病毒质粒pLenti-TRIM69或其突变体与慢病毒辅助质粒(plp1,plp2,VSVG)共转染HEK293T包装细胞,产生有活性的病毒。包装后的重组慢病毒感染HEK293细胞,Western blot检测确认TRIM69及其突变体蛋白的表达。结果经Western blot检测证实,成功建立了稳定表达TRIM69基因及其突变体的293细胞系。结论利用慢病毒成功制备稳定表达TRIM69基因及其突变体的293细胞系,这些细胞为TRIM69生物学功能的研究提供了平台。Objective To establish a stable cell line overexpressing a novel testis E3 ligase TRIM69 mediated by a modified lentivir- us system. Methods The coding regions of human TRIM69 or TRIM69AR （a deletion of ring finger domain） were amplified by PCR from human embroic kidney 293T cDNA and cloned into the modified plentilox3.7 expression vectors. With three helpful plasmids inclu- ding plpl, plp2 and VSVG, the recombinant plamids were packaged into 293T cells to produce the live viruses. HEK293 cells were infec- ted by the viruses and the positive cells were identified by Western blot. Results The stable cell lines overexpressing TRIM69 or TRIM69AR were established by infected with the viruses produced by packaging the four plasmids into HEK293T cells and verified by Western blot. Conclusion The stable cell lines overexpressing TRIM69 or TRIM69AR were successfully established mediated by a modi- fied lentiviral system and provide a useful cell model for further studies of the function of TRIM69.

关 键 词：TRIM69 慢病毒 稳定细胞株 

分 类 号：R73-362[医药卫生—肿瘤]