Survivin启动子介导自杀基因与突变体融合基因真核表达载体的构建  

Construction of a survivin promoter-mediated adeno-associated virus expression vector carrying a fusion gene constituted with cytosine deaminase and survivin mutant

作　　者：孙萍胡[1] 吴云林[1] 董文杰[1] 乔敏敏[1] 朱黎明[1] 涂水平[1] 

机构地区：[1]上海交通大学附属瑞金医院消化科,上海市200025

出　　处：《世界华人消化杂志》2009年第11期1085-1090,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30772518~~

摘　　要：目的:构建Survivin启动子介导自杀基因胞嘧啶脱氨酶(CD)与SurvivinCys84Ala突变体(SurMut)融合基因的重组腺相关病毒质粒(pAM/SurP-CDglySurMut),探讨其在肿瘤细胞中有效表达融合基因的能力.方法:PCR扩增E.coliCD基因和带有甘氨酸(gly)连接体的Survivin突变体片段,亚克隆至具有Survivin启动子的腺相关病毒载体pAM/SurP,获得重组表达载体pAM/Sur P-CDglySurMut.重组载体瞬时转染胃癌细胞,Western blot检测融合蛋白的表达.结果:经PCR扩增成功克隆E.coliCD基因和带有gly连接体的Survivin突变体片段.重组阳性克隆经限制性酶切鉴定和基因测序,证实目的基因CDglySurMut已正确插入pAM/SurP载体中.Westernblot证实Survivin启动子能够驱动CDglySurMut融合基因在胃癌细胞中表达.结论:pAM/SurP-CDglySurMut载体能够在肿瘤细胞中表达融合蛋白,为进一步探讨其靶向治疗肿瘤奠定了实验基础.AIM： To construct a recombinant survivin promoter （SurP）-mediated adeno-associated virus expression vector carrying a fusion gene constituted with cytosine deaminase （CD） gene and glycin-linked survivin mutant （glySurMut）, and to examine the expression of CDglySurMut fusion protein in cancer cells transfected with this vector. METHODS： The CD gene and glySurMut fragments were amplified by polymerase chain reaction （PCR）, and cloned into the adeno-associated virus expression vector PAM with survivin promoter （PAM/SurP）. The recombinant vector was transformed into E.coli DH5α. and the positive clones were selected. The plasmid DNA was identified using restriction enzyme analysis and sequencing. The recombinant vector pAM/SurP-CDglySurMut was transfected into gastric cancer cells MKN45. The expression of fusion protein in cancer cells was detected using Western blot. RESULTS： The CD gene and glySurMut were cloned from E.coli and gastric cancer cells, respectively. The restrictive enzyme analysis and sequencing showed that the fusion gene of CD and glySurMut was successfully inserted into the PAM/SurP vector. Western blot showed that the recombinant vector expressed the CDglySurMut fusion protein in gastric cancer cells. CONCLUSION： Our results show the pAM/SurP-CDglySurMut vector can efficiently express the recombinant CDglySurMut fusion protein in gastric cancer cells, thus provides experiment evidence for targeting cancer gene therapy using this pAM/SurP-CDglySurMut vector.

关 键 词：SURVIVIN启动子 Survivin突变体 胞嘧啶脱氨酶基因 载体构建 

分 类 号：R73-3[医药卫生—肿瘤]