重组质粒pLMP1-p53mt的构建与表达

Investigation on construction of recombinant plasmid pLMP1-p53mt and its expression in HeLa cells

目的构建含突变型p53基因和EB病毒LMP1基因的双基因真核表达质粒,为研究突变型p53基因和EB病毒LMP1基因在鼻咽癌发生中的相互作用奠定基础。方法通过分子克隆技术,构建含突变型p53基因和EB病毒LMP1基因的真核表达载体pLMP1-p53mt;培养HeLa细胞,采用脂质体lipofectAMINETM2000将该质粒转入HeLa细胞中,转染后48h,利用免疫细胞化学法研究突变型p53基因和EB病毒LMP1基因的表达情况。结果重组质粒限制性内切酶酶切及PCR鉴定结果与预期一致;观察到转染细胞中突变型p53基因和EB病毒LMP1基因的高效表达。结论成功构建了含突变型p53基因和EB病毒LMP1基因的双基因真核表达质粒。

[Objective] To construct eukaryotic expression plasmid containing mutation p53 gene and Epstein-Barr virus latent membrane protein 1 gene (LMP1) so that the reciprocity of mutation p53 with LMP1 in the tumorogenesis process of nasopharyngeal carcinoma could be investigated more easily. [Method] Molecular cloning techniques were used to construct the recombinant plasmid pLMP1-p53 mt containing mutation p53 gene and Epsteins-Barr virus LMP1 gene. Then, the recombinant plasmid pLMP1-p53 mt was transfected into HeLa cells by lipofectamine TM 2000 and the expression of mutation p53 and LMP1 was detected by immunocytochemical method after 48 h following transfection. [Result] Following the recombinant plasmid digested by restricted endoenzymes and identified by PCR procedures, gel electrophoresis showed that the DNA band distribution was basically the same as that of theoretic expect. The expression of mutation p53 and LMP1 were highly active in HeLa cells as well. [Conclusion] It should be successful in the construction of recombinant eukaryotic expression plasmid containing mutation p53 gene and Epstein-Barr virus LMP1 gene.