DOC-1R基因重组表达载体构建及其在HeLa细胞中的表达

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。

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

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.