DOC-1R基因重组表达载体构建及其在HeLa细胞中的表达  

Construction and expression of human DOC-1R retroviral expression vector in HeLa cells

作　　者：刘琦[1] 刘杏[1] 高锦兰[1] 胡西华[1] 罗阳[1] 

机构地区：[1]中国医科大学医学基因组学教研室卫生部细胞生物学重点实验室暨教育部医学细胞生物学重点实验室,辽宁沈阳110001

出　　处：《解剖科学进展》2012年第5期427-430,434,共5页Progress of Anatomical Sciences

基　　金：国家自然科学基金项目(No.81100419;30971164);辽宁省教育厅创新团队项目(No.2008T194)

摘　　要：目的 构建人DOC-1R基因重组逆转录病毒表达载体,实现该基因在体外培养细胞中的大量表达,从而研究表达蛋白的功能。方法 通过重组技术将含有FLAG标签序列的DOC-1R cDNA编码区全长克隆至逆转录病毒载体pLXSN,菌落PCR鉴定、限制性内切酶双酶切及测序验证重组载体的构建。将重组载体转染至GP-293细胞进行病毒包装并以所包装的病毒感染HeLa细胞,通过Western blot及间接免疫荧光染色检测重组DOC-1R蛋白的表达及细胞内定位。结果 测序结果表明重组载体中插入的重组片段序列及开放阅读框架正确,逆转录病毒表达载体pLXSN-FLAG-DOC-1R成功构建。Western blot及间接免疫荧光染色结果证实,逆转录病毒介导的重组DOC-1R蛋白在HeLa细胞中高效表达,表达效率明显高于真核表达载体介导的重组蛋白表达。结论 DOC-1R基因逆转录病毒表达载体成功构建,重组蛋白在体外培养细胞正确高效表达。Objective To construct retroviral expression vector carrying human DOC-1R gene and explore the recombinant protein expression in HeLa cells. Methods Eukaryotic expression vector pFLAG-DOC-IR was used as template, FLAG tagged-DOC-1R encoding fragment was amplified by PCR and subcloned into retroviral vector pLXSN. The identified recombinant DOC-1R vector was transfected into packaging cells GP-293 by liposome-mediated transfection to generate the retrovirus. The DOC-1R retrovirus was used to infect HeLa cells and overexpression of recombinant DOC-1R protein was demonstrated by SDS-PAGE analysis, Western blot and immunofluorescent stain. Results The clony PCR, double enzyme-digestion and DNA sequencing analysis demonstrated that recombinant retroviral expression vector pLXSN- FLAG-DOC-1R was constructed successfully. In HeLa cells, recombinant DOC-1R protein was detected with higher efficiency by retrovirus-mediated infection compared with that mediated by transfection with eukaryotic expression vector. Conclusion DOC-1R retroviral expression vector was constructed and the recombinant DOC-1R protein could be expressed efficiently in HeLa cells using the DOC-1R retroviral vector.

关 键 词：DOC-1R 逆转录病毒表达载体 外源表达 转染 

分 类 号：Q786[生物学—分子生物学]