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目的构建包含人的G250真核表达载体,并将该载体转导入293 T细胞检测其表达,为构建以G250为靶点的肾癌模型提供研究基础。方法通过聚合酶链反应获得G250基因,将其连接至PMD18T过渡载体,然后克隆到载体pIRES-neo中。把构建后的重组质粒pIRES-neo-G250转染到293 T细胞,用流式细胞仪、免疫荧光和RT-PCR检测其表达。结果 PCR扩增出的G250基因测序正确,酶切鉴定重组质粒pIRES-neo-G250正确,说明质粒构建成功;流式细胞术、免疫荧光和RT-PCT检测结果表明,重组质粒pIRES-neo-G250在293 T细胞中得到表达。结论该实验成功构建了重组质粒pIRES-neo-G250,并且在293T细胞中能够有效表达,为后续构建转染人G250的基因细胞株工作奠定了基础。
Objective To construct human G250 eukaryotic expression vector and transduce the vector into 293T cells to detect the expression of G250, so as to provide the basis for constructing a G250 targeted renal cell carcinoma model. Methods The G250 gene was obtained by polymerase chain reaction, ligated to the PMD18T transition vector and then cloned into the vector pIRES-neo. The constructed recombinant plasmid pIRES-neo-G250 was transfected into 293 T cells, and its expression was detected by flow cytometry, immunofluorescence and RT-PCR. Results The G250 gene amplified by PCR was correctly sequenced and the correct recombinant plasmid pIRES-neo-G250 was identified by restriction enzyme digestion. The results of flow cytometry, immunofluorescence and RT-PCR showed that the recombinant plasmid pIRES-neo-G250 Expressed in 293 T cells. Conclusion The recombinant plasmid pIRES-neo-G250 was constructed successfully and expressed efficiently in 293T cells, which laid the foundation for the subsequent construction of gene-transfected human G250 cells.