HSV-TK与IL-2共表达真核载体的构建及其在A549细胞中的表达  

Construction of Eukaryotic Expression Vector Containing HSV-TK and IL-2 Double Genes and Its Expression in Human Pulmonary Adenocarcinoma Cell A549

作　　者：宋青凤[1] 方慧娟[1] 熊维宁[1] 徐永健[1] 熊盛道[1] 曹勇[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院呼吸内科,武汉430030

出　　处：《肿瘤防治研究》2009年第3期194-198,共5页Cancer Research on Prevention and Treatment

基　　金：湖北省自然科学基金资助项目(2003ABA144)

摘　　要：目的构建含有自杀基因单纯疱疹病毒胸苷激酶(HSV-TK)和人类细胞因子白细胞介素-2(IL-2)的联合表达质粒载体并观察其在人类肺腺癌细胞系A549细胞中的表达。方法设计、合成多克隆位点(MCS)插入到质粒TPNAV中,然后分别从其他不同的质粒剪切片段HSV-TK、内部核糖体进入位点(IRES)、IL-2并依次接入到MCS中,最终得到目的质粒TPNAV-TK-IRES-IL2(pMTⅡ)。目的质粒经酶切、DNA测序鉴定正确后,转染到A549细胞中。用RT-PCR方法、ELISA法检测转染后细胞目的基因的转录和表达;进一步倒置显微镜观察、MTT法检测更昔洛(GCV)对转然后细胞的杀伤作用。结果经酶切、DNA测序、RT-PCR法和ELISA法鉴定,双基因真核表达载体pMTⅡ序列正确并能有效表达。进一步倒置显微镜观察和MTT法检测表明HSV-TK/GCV自杀基因系统对A549细胞有明显的杀伤作用。结论成功构建了pMTⅡ真核表达载体,并观察了其对A549细胞的杀伤效应,为进一步研究HSV-TK/GCV联合IL-2基因治疗肺癌提供实验基础。Objective To construct the eukaryotic expression vector containing Herpes simplex virus thy midine kinase （HSV-TK） and interleukin2（IL-2） double genes and observe its expression in human pulmonary adenocarcinoma cell A549. Methods The sequences of multiple clone site （MCS） was synthesized and inserted to the vector pSNAV. HSV-TK, internal ribosome entry site （IRES）, IL-2 fragments were respectively obtained from different plasmids, then they were inserted into the MCS using restriction en zyme to get the recombinant plasmid pSNAV-TK-IRES-IL2（pMTII）. After identified by restriction enzyme digestion analysis and DNA sequencing, the recombinant plasmid was transfected into A549 （A549/ pMTⅡ ）. The transcription of HSV-TK and IL-2 genes in the A549/pMTll cells were detected by RTPCR. The secretion of IL-2 in the A549/pMT II cells was deteded by ELISA; the cytotoxicity of GCV （gancyclovir） on A549/pMTⅡ cells was observed by light microscope and MTT assay. Results Restriction enzyme digestion analysis, DNA sequencing, RT-PCR amplification and ELISA indicated that the recombinant plasmid pMTⅡ was constructed successfully and the target genes linked by IRES can express in cell A549. The A549/pMT Ⅱ cells administered gancielovir （GCV） demonstrated obvious lethal effect by light microscope and MTT assay. Conclusion The eukaryotic expression vector pMT Ⅱ containing HSV-TK and IL-2 double genes was constructed successfully and provided a basis for further study of the double genes therapy for lung carcinoma.

关 键 词：肺癌 基因治疗 单纯疱疹病毒胸苷激酶(HSV-TK) IL-2 

分 类 号：R73-36[医药卫生—肿瘤] R734.2[医药卫生—临床医学]