人PSCA真核表达载体的构建及表达

Construction of eukaryotic expression vector for human prostate stem cell antigen and its expression

目的构建含有信号肽及FLAG标签的人前列腺干细胞抗原(PSCA)真核表达载体,并在人胚肾293T细胞中进行表达。方法通过PCR方法获得SIG-FLAG基因片段及人PSCA基因片段,插入到真核表达载体pIRES-neo中。构建的重组质粒pIRES-neo-sig-FLAG-PSCA转染293T细胞,利用流式细胞仪、免疫荧光及RT-PCR方法检测其表达情况。结果PCR扩增出的SIG-FLAG及PSCA基因测序正确,酶切鉴定证明重组质粒pIRES-neo-sig-FLAG-PSCA构建成功;检测结果显示重组质粒pIRES-neo-sig-FLAG-PSCA在293T细胞中得到表达。结论成功构建了重组质粒pIRES-neo-sig-FLAG-PSCA,且在293T细胞中能有效表达,为后续转人PSCA基因细胞系的构建工作奠定了基础。

Objective To construct the eukaryotic expression plasmid of human prostate stem cell antigen（PSCA）containing a signal peptide and a FLAG tag and to detect its expression in eukaryotic 293T cells.Methods SIG-FLAG and human PSCA gene was constructed by PCR and inserted into a eukaryotic expression vector pIRES-neo.The recombinant plasmid pIRES-neo-sig-FLAG-PSCA was tansfected to the 293T cells,and its expression was detected by FACS,IMF and RT-PCR,respectively.Results The gene sequence of SIG-FLAG and human PSCA amplified by PCR was correct.Enzyme digestion analysis showed that the recombinant plasmid pIRES-neo-sig-FLAG-PSCA was successfully constructed and expressed in 293T cells.Conclusion The recombinant plasmid pIRES-neo-sig-FLAG-PSCA,we constructed,can be effectively expressed in 293T cells,which may lay a foundation for the construction of human PSCA-transfected cell line in the future.