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目的:为建立可回复性永生化肝细胞株,构建带有筛选基因NeoR、报道基因EGFP及重组位点loxP的可回复性永生化逆转录病毒载体.方法:将2.1kbSV40T亚克隆到pIRES2-EGFP的相应位点,再酶切下3.4kbSV40T-IRES-EGFP片段插入逆转录病毒载体pLNCX2的两个相同酶切位点之间,构成新载体pLNCTIG.最后,将线性化pLNCTIG和loxP双链片段混合连接.取连接产物转化高效感受态DH5α细胞.菌落PCR和酶切法鉴定阳性克隆.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.扩大培养稳定转染的细胞,免疫组化染色检测SV40T基因的表达.结果:随机挑取19个克隆,3个电泳分离出约1.1kb阳性条带.取上述阳性克隆进行酶切鉴定,结果仅出现一条约9.5kb的条带,证明该克隆为阳性重组体.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pLNCTIGlox.
OBJECTIVE: To construct a retroviral immortalized hepatocyte cell line and construct a retroviral immortalized retroviral vector with a screening gene NeoR, a reporter gene EGFP and a recombination site loxP.Methods: 2.1kbSV40T was subcloned into pIRES2-EGFP Of the corresponding sites, and then digested 3.4kbSV40T-IRES-EGFP fragment inserted into the retroviral vector pLNCX2 between two identical restriction sites to form a new vector pLNCTIG Finally, the linear pLNCTIG and loxP double-stranded fragments were mixed Then, the ligation products were transformed into competent DH5α cells, and the positive clones were identified by colony PCR and restriction enzyme digestion.The transfected cells were transfected with PT67 and the expression of green fluorescent protein (GFP) was observed under a fluorescence microscope.Expanded cells were stably transfected, The results of electrophoresis showed that only a band of about 9.5 kb was found in the positive clones, Cloned as positive recombinant.Transfection of PT67 cells 24h, fluorescence microscopy showed scattered in many green fluorescence.Immunohistochemical staining showed that the nucleus was positive staining.Conclusion: The successful construction and expression can be recovered Immortalization retroviral vector pLNCTIGlox.