构建携带重组人胰岛素基因慢病毒表达载体及其病毒包装  

Construction of recombinant human insulin gene lentiviral expression vector and virus packaging

作　　者：薛美思[1] 刘毅[2] 

机构地区：[1]兰州大学第二临床医学院,甘肃省兰州市730000 [2]解放军兰州军区兰州总医院全军烧伤整形外科中心,甘肃省兰州市730050

出　　处：《中国组织工程研究与临床康复》2010年第33期6133-6137,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30872689);项目名称:胰岛素基因转染脐带间充质干胞构脂肪组织工程的实验研究~~

摘　　要：背景:腺病毒载体作为脂肪组织工程转基因载体,在应用中存在转导细胞免疫排斥及炎症反应等问题。应用慢病毒作为载体转染干细胞尤其是应用含胰岛素基因的慢病毒载体转染干细胞可避免腺病毒载体的诸多问题。目的:实验拟构建含有人重组胰岛素(insulin)与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因慢病毒表达载体pLenti6.3-insulin-IRES-EGFP,并进行病毒颗粒包装。方法:应用聚合酶链反应方法获得目的基因,在目的基因上、下游分别加上BamHⅠ,AscⅠ两个酶切位点,进行T载体克隆,转化入感受态DH5α细胞中,通过筛选获得重组质粒。用限制性内切酶酶切,将目的基因片段和pLenti6.3-IRES-EGFP载体连接,转化入感受态DH5α细胞中,通过筛选获得慢病毒表达载体pLenti6.3-insulin-IRES-EGFP,并进行测序。抽提慢病毒载体,转染293T细胞,包装病毒,测定病毒滴度。结果与结论:通过聚合酶链反应获得长度为347bp带有BamHⅠ和AscⅠ序列的目的基因。pMD18-T载体和慢病毒表达载体pLenti6.3-IRES-EGFP连接,慢病毒表达载体pLenti6.3-insulin-IRES-EGFP构建与预期相匹配,成功包装慢病毒颗粒。BACKGROUND：Adenovirus vector faces many problems,such as immunological rejection and inflammatory reaction,as an adipose tissue engineering of genetically modified vector.These problems can be avoided when using lentivirus as vector,especially using lentivirus carrier containing insulin gene.OBJECTIVE：To construct containing human recombinant insulin and enhanced green fluorescent protein（EGFP） gene lentiviral vector pLenti6.3-insulin IRES-EGFP,and to make virus particles packaging.METHODS：Polymerase chain reaction（PCR） method was used to obtain the target gene and BamHⅠ,AscⅠ two restriction sites were added,after T vector cloning,the gene was transformed into competent DH5α cells.The gene fragment and pLenti6.3-IRES-EGFP vector were transformed into competent DH5α cells by means of enzyme digestion.The lentiviral expression vector pLenti6.3-insulin-IRES-EGFP was obtained after screening followed by sequencing.The lentiviral vector was used to transfect 293T cells and package virus,and the virus titers were determined.RESULTS AND CONCLUSION：A length of 347 bp with BamH Ⅰ and Asc Ⅰ target gene sequences was obtained by PCR.The pMD18-T vector was connected to the lentiviral vector pLenti6.3 IRES-EGFP,and the constructed lentiviral expression vector pLenti6.3-insulin-IRES-EGFP was corresponded to the expected.The lentiviral particles were successfully packaged.

关 键 词：胰岛素 绿色荧光蛋白 克隆 基因表达 慢病毒表达载体 病毒包装 

分 类 号：R318[医药卫生—生物医学工程]