人上皮细胞黏附分子的真核表达及鉴定

目的构建人上皮细胞黏附分子(EpCAM)全基因的真核表达质粒,研究其在HepG2细胞中的表达。方法将EpCAM基因克隆入真核表达载体pcDNA3.1(+),构建真核表达质粒pcDNA3.1(+)-EpCAM,转染人肝癌HepG2细胞。通过免疫荧光、Western Blotting及流式细胞检测对转染后细胞内EpCAM蛋白的表达进行鉴定。结果双酶切鉴定结果表明重组质粒构建成功。免疫荧光、Western Blotting及流式细胞检测结果一致表明,转染后EpCAM基因在HepG2细胞中表达成功。结论成功构建了人EpCAM的真核表达载体,并在HepG2细胞中表达,为研究高表达人EpCAM的肝癌细胞的功能打下基础。

Expression and identification of human EpCAM eukaryotic recombinant plasmid

Objective To construct human EpCAM eukaryotic recombinant plasmid ,and to study the expression of EpCAM pro‐tein in HepG2 cells .Methods The recombinant plasmid ,named pcDNA3 .1(+ )‐EpCAMwas constructed by cloning EpCAM gene into eukaryotic vector pcDNA3 .1(+ ) .Then HepG2 cells were transfected with EpCAMrecombinant plasmid .The eukaryotic ex‐pression of EpCAM protein was verified by immunofluorescence ,Western Blotting and flow cytometry .Results The construction of EpCAM recombinant eukaryotic plasmid was identified by restriction enzyme digestion .The results of immunofluorescence , Western Blotting ,and flow cytometry consistently indicated that EpCAM protein were successfully expressed in HepG2 cells .Con‐clusion Human EpCAM eukaryotic vector is constructed and EpCAM protein could be expressed well in HepG2 cells ,which laid a foundation for further research on the function ofhepatocarcinoma cells highly expressing EpCAM .