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目的:利用内部核糖体进入位点(IRES)序列,构建含有血管内皮生长因子(VEGF165)和人钠碘同向转运体(NIS)的真核双表达载体pIRES-VEGF165-NIS,并在体外检测NIS蛋白和VEGF165蛋白在HEK293细胞中的表达。方法:采用聚合酶链反应(PCR)扩增得到目的基因VEGF165和NIS,通过酶切将其连接克隆至pIRES载体以构建pIRES-VEGF165-NIS质粒,再通过脂质体将构建好的质粒转染入HEK293细胞,并采用蛋白印迹法和免疫荧光法检测VEGF165和NIS蛋白的表达。结果:成功构建了双基因双表达载体pIRES-VEGF165-NIS,且其受巨细胞病毒(CMV)极早期基因启动子调控。蛋白印迹法检测显示,VEGF165和NIS可同时在HEK293细胞中表达,而免疫荧光显示转染的HEK293细胞可同时表达VEGF165蛋白和NIS蛋白。结论:IRES序列调控VEGF165和NIS双基因可在细胞中同时独立表达,为进一步构建双基因表达重组腺病毒,并利用人NIS作为报告基因,实时监测VEGF165治疗基因的表达和分布奠定基础。
OBJECTIVE: To construct an eukaryotic expression vector pIRES-VEGF165-NIS containing vascular endothelial growth factor (VEGF165) and sodium sodium iodide symporter (NIS) by using the internal ribosome entry site (IRES) sequence and to detect in vitro Expression of NIS protein and VEGF165 protein in HEK293 cells. Methods: The target genes VEGF165 and NIS were amplified by polymerase chain reaction (PCR), cloned into pIRES vector by restriction enzyme digestion to construct pIRES-VEGF165-NIS plasmid, and the constructed plasmid was transfected by liposome HEK293 cells, and Western blotting and immunofluorescence assay VEGF165 and NIS protein expression. Results: The double-gene double-expression vector pIRES-VEGF165-NIS was successfully constructed and regulated by the very early gene promoter of cytomegalovirus (CMV). Western blotting showed that both VEGF165 and NIS were expressed simultaneously in HEK293 cells. Immunofluorescence showed that the transfected HEK293 cells could express VEGF165 protein and NIS protein simultaneously. Conclusion: VEGF165 and NIS double genes regulated by IRES sequence can be independently expressed in cells simultaneously. This study laid the foundation for further construction of recombinant adenovirus expressing double gene and using human NIS as reporter gene to monitor the expression and distribution of VEGF165 gene in real time.