可分泌性GLP-1重组慢病毒的构建  

Construction of Recombinant Lentivirus Vector Expressing GLP-1

作　　者：娄明武[1] 张明东[1] 梁冰[2] 范义[1] 王全颖 

机构地区：[1]深圳市龙岗中心医院介入影像科,广东深圳518116 [2]北华大学,吉林吉林132013 [3]西安市华广生物工程有限公司,陕西西安710000

出　　处：《生物技术》2008年第6期15-18,共4页Biotechnology

基　　金：广东省科学技术厅项目(2005B36001082);深圳市科技和信息局项目(JH200505260306A)资助

摘　　要：目的:为了探讨使用基因治疗在体内分泌表达胰高血糖素样肽-1(GLP-1)方法治疗糖尿病的可行性,构建可分泌表达GLP-1的重组慢病毒。方法:1.将已构建成功的NT4-GLP-1融合基因插入慢病毒包装质粒pLenti6V5D-TOPO中,构建pLenti6V5D-TOPO/NT4-GLP-1重组慢病毒包装质粒。2.用慢病毒包装辅助质粒plp1、plp2、plp/VSVG,及重组慢病毒包装质粒pLenti6V5D-TOPO/NT4-GLP-1,四质粒磷酸钙共沉淀法转染80%融合的293细胞系,包装慢病毒。3.通过免疫组化法染色确定重组慢病毒效应。结果:重组质粒经BamHⅠ和XhoⅠ联合酶切,10g/L琼脂糖凝胶电泳可见在342bp处有一目的片段。该值与NT4-GLP-1融合基因片段的大小一致,说明NT4-GLP-1融合基因已经成功重组于慢病毒包装质粒pLenti6V5D-TOPO内。免疫组化结果显示,实验组细胞内出现大量棕黄色颗粒,阳性细胞达到70%以上,对照组中没有阳性细胞,所以说明NT4-GLP-1重组慢病毒在细胞中可以正确地分泌表达GLP-1。结论:NT4-GLP-1重组慢病毒包装质粒构建正确,病毒包装成功。Objective： The lentivirus which can secrete and express GLP - 1 by the Glucagon - like pepfide - t （GLP - 1 ） which can be secreted in vivo continually for gene therapy has been constructed to investigate the feasibility to treat diabetes. Method：The fusion gene of NT4 - GLP - 1 which has been construeted previously was inserted the lenfivirus packaging plasmid to generate the lenfivirus packaging plasmid pLenfi6V5D- TOPO/NT4 - GLP - 1.80% fused 293 cells were co - transfeeted, using the Calcium phosphate precipitation method, with aid pae okaging plasmids plpl, plp2, plp/VSVG, and the recombinant lentivirus packaging plasmid pLenfi6V5D - TOPO/NT4 - GLP- 1. Virus effects were confirmed by using the methods of immunohistochemistry at the same time. Result： Recombinant plasmids pLenti6V5D - TOPO/NT4 - GLP - 1 were constructed by ligating the 342bp fragments containing clone NT4 - GLP - 1 and big pLenfi6V5D - TOPO fragments by T4 DNA joinase. The recombinant lentivirus plamid vectors were predicted to be 342 bp, corresponded with the 342bp gene sequence observed on resulting of agarese gel eleetro- phoresis of the sequences obtained from the recombinant planfids. Immunohistochemieal staining showed that many buffy partieals appear in the experimental cells and the positive ceils were accounted for more than 70%, no positive cell was observed on control group. Conclusion：The lentivirus plasmid vectors containing of NT4 - GLP- 1 gene have been constructed, packaged successfully and expressed GLP- 1 effectively.

关 键 词：NT4-GLP-1融合基因 重组慢病毒 基因治疗 胰高血糖素样肽-1 

分 类 号：Q78[生物学—分子生物学] Q578