细胞色素P4501A1-Wt和Asn~(461)、Val~(462)真核表达载体的构建及人胚肺成纤维细胞转染

目的构建能够高效表达细胞色素P4501A1(CYP1A1)蛋白的细胞株,分析不同基因型蛋白表达的差异。方法首先将已经建立的T载体中CYP1A1-Wt和Asn461、Val462的CYP1A1片断重组至pBABE-neo载体上,转染入人胚肺成纤维细胞(HLF),G418筛选获得阳性细胞,Western-Blot检测CYP1A1蛋白的表达,观察细胞形态,生长曲线,恶变性等生物学特性的改变。结果CYP1A1野生型、变异型转染的HLF细胞均能高效表达CYP1A1蛋白,其生物学特性与正常细胞无显著差异,也不属于恶性细胞。结论通过细胞转染和筛选获得了表达CYP1A1蛋白的细胞株。

Construction of CYP1A1-Wt and Asn~(461),Val~(462) gene eukaryotic expression vector and itstransfection in HLF cells

Objective To establish the cells model with high expression CYP1A1 and analyze the expression of the variable genotype.Methods Clone the target fragment (CYP1A1-Wt, Asn~ 461 , and -Val~ 462 ) in T-Vectors into pBABE-neo, then transfer into the human embryonic lung fibroblast cells (HLF). The positive cells were selected by G418. The expression of CYP1A1 protein was measured by Western-Blot. At the same time, cells shape, growth of curve, and carcinogenicity were observed.Results HLF transferred with wild-CYP1A1 and variant-CYP1A1 expressed CYP1A1 protein higher than normal HLF. No differences of biology were found between the transfected HLF and normal HLF. These cells are not belong to carcinogenesis cells.Conclusion HLF cells with wild-CYP1A1 or variant-CYP1A1 ware established successfully.