Tie2表达载体的构建及其稳定转染SKOV3细胞系的建立  

Construction of Eukaryotic Expression Vector and establishment of its Stably Transfected Cell line SKOV3

作　　者：陈雅静[1] 许淑茹[1] 付达华[1] 许秀宽[1] 舒伟[2] 

机构地区：[1]福建漳州卫生职业学院科研科,漳州市363000 [2]广西医科大学细胞与遗传教研室,南宁市530021

出　　处：《内科》2015年第5期604-606,676,共4页Internal Medicine

基　　金：漳州市科技局自然科学基金项目(ZZ2012J22;ZZ2013J15);福建省卫生厅青年科研课题(2012-1-40);国家自然科学基金项目(81260479)

摘　　要：目的构建人Tie2基因真核表达载体,并建立稳定过表达Tie2基因的SKOV3细胞系。方法采用RT-PCR方法从人肝癌细胞BEL-7402细胞系中扩增Tie2基因编码区(c DNA),将产物克隆至p EGFP-N1真核表达载体,构建重组质粒p EGFP-N1-Tie2并测序鉴定。用构建成功的p EGFP-N1-Tie2真核表达载体转染人卵巢癌细胞SKOV3,经G418稳定筛选,分离单克隆,最后获得Tie2稳定表达的SKOV3细胞系,并用实时荧光定量PCR、Western Blot方法鉴定构建结果。结果成功构建p EGFP-N1-Tie2真核表达载体,获得了稳定过表达人Tie2基因的SKOV3细胞株。结论在SKOV3细胞系中稳定过表达人源Tie2基因,为下一步研究Tie2基因的功能和应用奠定了基础。Objective To construct the eukaryotic expression vector of human Tie2 and establish its stable transfected SKOV3 cell line. Methods The CDS of Tie2 was amplified from BEL-7402 cell line by RT-PCR and cloned into pEGFP-N1. The recombinant plasmid pEGFP-N1-Tie2 was sequenced. SKOV3 ceils were transfected with pEGFP-N1-Tie2 and selected with G418 and than isolated monoclonal. The expression of Tie2 was identified by q-PCR and Western Blot. Results The eukaryotie expressing plasmid of pEGFP-N1-Tie2 was successfully constructed and Tie2 stably expressing SKOV3 cell line was established. Conclusions The recombinant eukaryotic expression vector pEGFP-N1-Tie2 and the established SKOV3 cell line stably expressing Tie2 may be used for further study of Tie2 function.

关 键 词：人Tie2基因 载体构建 转染 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]