hTERT启动子调控下PE38KDEL表达载体的构建及其在MiaPaCa2人胰腺癌细胞中的表达

摘要

Objective To construct an expressive vector of PE38KDEL under the control of the hTERT promoter and rnto investigate the expression of this gene in human pancreatic cancer cell lines. Methods hTERTp promoter was identified rnby enzyme digested and sequencing. Sub clone the synthetical PE38KDEL gene into MCS of plRES2-EGFP and hTERTprnIRES2 -EGFP, verified by enzyme digested and RT-PCR. Results The plasmids were successfully constructed. Both MiarnPaCa2 and WI-38 could express PE38KDEL after transfected with pPE38KDEL-IRES2-EGFP, but only the MiaPaCa2 cell rnexpressed the PE38KDEL after tranfected with phTERTp-PE38KDEL-IRES2-EGFP plasmid. Conclusions The expressive rnvector of PE38KDEL under the control of hTERT promoter is successfully constructed and the expression of PE38KDEL rngene can be targeted in the MiaPaCa2 human pancreatic cell line by the hTERTp. This can provide some experimental evirndences for the gene-targeted therapy of human pancreatic cancer.%目的 探讨端粒酶逆转录酶(hTERT)启动子调控铜绿假单胞菌外毒素衍生物(PE38KDEL)表达载体的构建及其在MiaPaCa2人胰腺癌细胞中的表达.方法 通过PCR法扩增hTERT启动子,全基因合成PE38KDEL,将其分别亚克隆到pIRES2-EGFP及phTERTp-IRES2-EGFP质粒的多克隆位点中;均经酶切及测序鉴定.通过脂质体法将两质粒分别转染MiaPaCa2及WI-38细胞,观察荧光表达情况,通过RT-PCR检测PE38KDEL的表达.结果 经酶切及测序鉴定证实质粒构建成功,pPE38KDEL-IRES2-EGFP转染后MiaPaCa2和WI-38细胞中均有PE38KDEL基因表达;但phTERTp-PE38KDEL-IRES2-EGFP转染后仅MiaPaCa2中有PE38KDEL基因表达.结论 hTERT调控下PE38KDEL表达载体构建成功,并可在MiaPaCa2人胰腺癌细胞中靶向性表达,为探讨胰腺癌的靶向基因治疗提供了实验依据.