可回复性肝细胞永生化载体pCTGTKlox的构建、鉴定、转染与表达

目的构建可回复性永生化逆转录病毒载体,为肝细胞可回复性永生化奠定基础。方法扩增加强型绿色荧光蛋白(EGFP)及胸腺激酶(TK)并采用重叠延伸拼接法(SOE)连接EGFP和TK,将EGFP-TK融合基因亚克隆至pBABE-puro-lox,构建成新载体pBGTKlox,再将永生化基因SV40T连接至pBGTKlox,构成新载体pBTGTKlox,最后将雌激素受体与重组酶融合基因(Cre-ER)连接至pBTGTKlox,构成新载体pCTGTKlox。将pCTGTKlox和pPDF15质粒共转染包装细胞PT67,并在荧光显微镜下观察绿色荧光蛋白的表达。免疫组化染色检测SV40T基因的表达。结果成功构建包含EGFP-TK、SV40T、Cre-ER及重组序列loxP的新载体pCTGTKlox,经酶切鉴定证实为重组阳性体,经测序验证无核苷酸突变。pCTGTKlox转染PT67细胞24h后,荧光显微镜下可见散在多处绿色荧光。免疫组化染色显示细胞胞核呈阳性染色。结论成功构建并表达可回复性永生化逆转录病毒载体pCTGTKlox,为细胞的可回复性永生化提供了一种良好的新型载体。

Construction,characterization,transfection and expression of vector pCTGTKlox for hepatocyte reversible immortalization

Objective To establish a retroviral vector for reversible hepatocyte immortalization.Methods The enhanced green fluorence protein(EGFP) and thymokinase(TK) were amplified and fused.The fusion gene EGFP-TK was cloned to pBABE-puro-lox to construct pBGTKlox,then the immortalized SV40T was connected to the pBGTKlox to establish pBTGTKlox,and finally Cre-ER was connected to pBTGTKlox for establish pCTGTKlox.PT67 cells were co-transfected with pCTGTKlox and pPDF15 and the expression of green fluorescence protein was observed under fluorence microscope.The steadily transfected cells were stained immunohistochemically to detect the expression of SV40 large T antigen.Results A new vector pCTGTKlox containing Cre-ER,SV40T,EGFP-TK and LoxP was constructed and verified by restriction endonuclease digestion.DNA sequencing demonstrated that there was no coding mutation in pCTGTKlox.After co-transfection with pCTGTKlox and pPDF15,PT67 cells emitted green fluorescence under the fluorescence microscope.The nuclei of the transfected cells were positive for immunochemical staining.Conclusion The reversible vector pCTGTKlox is successfully constructed and expressed,thus it provides an ideal vector in the reversible immortalization of hepatocytes and other mammalian cells.