靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK构建的初步研究  

A Preliminary Study on Construction of Targeting Double Suicide Gene Therapy Vector pcDNA3.1(-) Cp-CD-TK

作　　者：罗琪[1] 熊宇[1] 卢毅卓[1] 

机构地区：[1]厦门大学附属中山医院福建医科大学厦门中山教学医院普外科,厦门361004

出　　处：《四川大学学报（医学版）》2009年第6期983-987,共5页Journal of Sichuan University(Medical Sciences)

基　　金：厦门市卫生局重点项目基金(WSK0509)资助

摘　　要：目的构建由癌胚抗原启动子(CEA promoter,Cp)驱动的靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,并观察其在CEA阳性大肠癌细胞中专一性表达和对增殖的影响。方法采用PCR分别扩增出Cp、CD、TK三种目的基因并采用双酶切、连接依次将Cp、CD、TK基因插入pcDNA3.1(-)质粒;将靶向性载体pcDNA3.1(-)Cp-CD-TK分别转染CEA阳性的人大肠癌SW480细胞和CEA阴性的Hela细胞,采用RT-PCR检测CD-TK基因的表达。用MTT法检测转染pcDNA3.1(-)Cp-CD-TK后的SW480细胞对化疗前药5-氟胞嘧啶(5-Fc)和丙氧鸟苷(GCV)的敏感性。结果Cp基因片段、CD基因片段和TK基因均克隆正确。靶向性基因治疗载体pcDNA3.1(-)Cp-CD-TK经凝胶电泳和DNA测序证实构建正确。转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞经过RT-PCR鉴定证实有CD-TK基因的表达,CEA阴性Hela细胞则没有CD-TK基因的表达。细胞培养试验中,转染了靶向性载体pcDNA3.1(-)Cp-CD-TK的SW480细胞对前药5-Fc和GCV敏感。结论正确构建了靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK,靶向性双自杀基因治疗载体pcDNA3.1(-)Cp-CD-TK能够使CD-TK基因在CEA阳性细胞中专一性表达,达到靶向杀伤大肠癌细胞目的。Objective To construct the targeting double suicide gene therapy vector pcDNA3.1（-） Cp-CD-TK driven by carcino-embryonic antigen promoter（CEA promoter,Cp）,and to investigate whether the vector could control the expression of CD-TK gene specificity and impact on proliferation of CEA positive colon cancer cells.Methods Three kinds of target gene,Cp,CD,and TK were obtained by PCR,the products were double digested and inserted into pcDNA3.1（-）,then the targeting vector pcDNA3.1（-） Cp-CD-TK was transfected into CEA positive human colon cancer SW480 cells and CEA negative Hela cells,the expression of CD-TK was examined by RT-PCR.The sensitivities of SW480 cells transfected with pcDNA3.1（-） Cp-CD-TK to pro-drug 5-Fluorocytosine（5-Fc） and Ganciclovir（GCV） were detected by MTT analysis.Results Recombinants with Cp,CD and TK insert were obtained.Constructed targeting gene therapy vector pcDNA3.1（-） Cp-CD-TK was confirmed by gel electrophoresis and sequencing.RT-PCR analysis further confirmed CD-TK gene was expressed in SW480 cells and was not expressed in CEA negative Hela cells.MTT assay demonstrated that SW480 cells transfected with targeting vector pcDNA3.1（-） Cp-CD-TK were sensitive to pro-drug 5-Fc and GCV.Conclusion Targeting double suicide gene therapy vector pcDNA3.1（-） Cp-CD-TK was constructed correctly.The vector could make CD-TK gene express specifically in CEA positive cells for the purpose of targeting killing colon cancer.

关 键 词：双自杀基因 癌胚抗原基因启动子 CD TK 大肠癌 

分 类 号：R735.34[医药卫生—肿瘤]