人DC-STAMP真核表达载体的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector Expressing Human DC-STAMP

目的构建和鉴定人DC-STAMP基因的真核表达载体,并分析在293T细胞中的表达情况。方法通过反转录-聚合酶链式反应(RT-PCR)技术从人破骨细胞体外扩增DC-STAMP的cDNA片段,连接入真核表达载体pEGFP-N1-FLAG后转入293T细胞中,Western blot进行表达鉴定。结果成功扩增出人DC-STAMP全长,构建了重组质粒pEGFP-DC-STAMP-FLAG,并在其转染的293T细胞检测到融合蛋白的表达。结论成功构建了DC-STAMP融合基因表达载体pEGFP-DC-STAMP-FLAG。

Objective To construct an eukaryotic expression vector which expresses human DC-STAMP gene,and to confirm its expression in 293T cells.Methods Human DC-STAMP cDNA was amplified with reverse transcription-polymerase chain reaction（RT-PCR） technique and inserted into eukaryotic expression vector pEGFP-N1-FLAG.The recombinant plasmid was then transfected into 293T cells.The expression of fusion protein was tracked under fluorescence microscope and confirmed by Western blot.Results The full length of human DC-STAMP cDNA was amplified successfully using RT-PCR.The recombinant plasmid pEGFP-DC-STAMP-FLAG was successfully constructed and the expression of fusion protein can be detected in 293T cells.Conclusion DC-STAMP fusion gene expression vector has been successfully constructed.