重组质粒pBud-CD44v6的构建及其在胃癌细胞内的表达  

Construction of recombinant plasmid of pBud-CD44v6 and its expression in gastric adenocarcinoma cells

作　　者：曾波[1] 姜政[1] 张滨[2] 李彦[1] 向廷秀[3] 陶小红[1] 王丕龙[1] 

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016 [2]重庆医科大学公共卫生学院社会医学教研室,重庆400016 [3]重庆医科大学附属第一医院实验研究中心,重庆400016

出　　处：《第三军医大学学报》2007年第22期2164-2167,共4页Journal of Third Military Medical University

基　　金：重庆市科委项目(2005BB5031)~~

摘　　要：目的构建含人的肿瘤侵袭和转移的特异性抗原CD44v6编码基因的真核表达的重组载体,进行核苷酸序列分析,并在肿瘤细胞中表达。方法①以pBud作为真核表达载体,选择CD44v6编码基因进行RT-PCR扩增,构建重组表达质粒,进行酶切鉴定和测序分析。②在Lipofectamine 2000的介导下,将构建成功的pBud-CD44v6重组表达质粒转染到肿瘤细胞中,通过免疫组织化学方法检测CD44v6的表达。结果经酶切鉴定和测序分析表明,插入的目的基因片段为CD44v6的编码基因,长为129bp,与GenBank中登录的cDNA相比较,同源性高达100%,且方向正确;重组质粒pBud-CD44v6转染对数生长期胃癌细胞株SGC-7901,进行免疫组织化学检测结果显示,与空质粒转染组对比,重组质粒组肿瘤细胞的细胞质中可见大量CD44v6基因编码的表达,其蛋白呈棕黄色,而对照组呈阴性。结论成功构建了pBud-CD44v6真核表达的重组载体,并在肿瘤细胞中表达。Objective To construct an eukaryotic gene expression vector for CD44v6 and its expression in tumor cells. Methods （1） CD44v6 gene was amplified by RT-PCR and cloned into plasmid pBud to form a recombinant plasmid pBud-CD44v6, which was then identified by restriction endoenzyme digestion and nucleotides sequencing. （2） The recombinant plasmid of pBud-CD44v6 was transfected into the gastric adenocarcinoma cell line SGC7901 by Lipofectamine 2000, and the expression of CD44v6 was detected by immunohistochemistry. Results （1） the restriction digestion and sequence analysis demonstrated that this 129 bp of CD44v6 was the same as that published in GenBank, and it was inserted into the vector accurately. （2） After the SGC7901 cells were transfected with CD44v6, obvious expression of CD44v6 protein was seen in the cytoplasm, while in the control group no brown protein was found. Conclusion A new plasmid of pBud-CD44v6 has been successfully established and expressed in tumor cells.

关 键 词：胃肿瘤 CIM4v6基因 SGC-7901细胞 

分 类 号：R394-33[医药卫生—医学遗传学] R394.3[医药卫生—基础医学]