野生型和突变型p16在H460细胞株的表达

应用ＰＣＲ体外定点突为技术，构建了ｐ１６－Ｐ４８Ｌ和ｐ１６－Ｄ７４ｎ突变体。野生型和突变型ｐ１６ｃＤＮＡ克隆地ｐｃＤＮＡ３真核表达载体，导入纯合缺失ｐ１６基因的人肺癌细胞株Ｈ４６０，经ＲＮＡ点杂交初筛吴Ｇ４１８抗性的细胞株，再用Ｎｏｒｔｈｅｒｎ印迹证实外源ｐ１６表达。

Expression of Wild type and Mutant p 16 in H460 Cell Line

Two p 16 mutants, P48L and D74N, were obtained by site directed mutagenesis using two step PCR method. Mutant p 16 cDNAs and wild type p 16 cDNA were cloned into the mammalian expression vector pcDNA3 to construct p 16 expression vector p CMV p 16, p CMV p 16P48L and p CMV p 16D74N, respectively. After the introduction of these expression vectors into human lung cancer cell line H460 in which endogenous p 16 gene was homozygously deleted, exogenous p 16 expression was detected in G418 resistant cells by Northern blotting and immunocytochemistry staining. The results of immunofluorescence and immunocytochemistry staining showed that the P16 protein was located in the cell cytoplasm. The p 16 cDNAs amplificated from the genomic DNA of recombinant H460 cell lines indicated that the plasmid p 16 cDNA was integrated into the chromosome of cell lines. That the over expression of wild type p 16 caused G1 arrest suggested the wild type P16 protein expressed in H460 cell line to be a functional protein.