含TK-IES-ES非融合基因重组腺相关病毒的构建及其功能的初步鉴定  

Construction of non-fusion recombinant of pAAV-TK-IRES-ES and identification of its function

作　　者：潘建刚[1] 周兴[1] 韩瑞发[2] 陈志光[1] 李江婷[1] 

机构地区：[1]广州医学院第二附属医院泌尿外科,广东广州510260 [2]天津市泌尿外科研究所天津医科大学第二医院泌尿外科,天津300211

出　　处：《基础医学与临床》2009年第8期863-866,共4页Basic and Clinical Medicine

基　　金：广州医学院第二附属医院博士启动基金(2008-2);广州市医药卫生科技一般引导项目(2008-YB-167);广州医学院博士启动基金(0706068);天津市科技发展计划基金(06YFSZSF03900)

摘　　要：目的探讨重组腺相关病毒(rAAV)-胸苷激酶(TK)-核糖体插入位点(IRES)-内皮抑素(ES)(简称rAAVTIE)非融合载体的构建及每个目的基因相应生物学效应的初步鉴定。方法(1)用PCR分别扩增IRES、TK、ES序列至pMD-19T simple载体,构建pAAV-TK-IRES-ES;(2)分别采用AAV-Helper Free System包装系统、"氯仿-PEG/NaCl沉淀-氯仿抽提"及冰乙醇沉淀法进行AAV的包装、纯化和浓缩。(3)用T24细胞及人脐静脉内皮细胞(HUVEC)对rAAV-TIE生物学效应进行初步鉴定。结果(1)成功构建pAAV-TIE非融合载体,并经酶切、PCR及测序证实;(2)rAAV病毒颗粒滴度达2×1010v.p/mL,浓缩后可达到2×1011-12v.p/mL;(3)rAAV-TIE可以分泌内皮抑素,诱导T24细胞及HUVEC凋亡。结论成功构建rAAV-TIE腺相关病毒,体外实验表明每个基因片段均能发挥相应的生物学功能。Objective To construct pAAV-TK-IRES-ES and to identify its function. Methods （1）IRES,TK, ES framents from pIRES-MCS, pAAV-TK, pAAV-ES were attained by PCR and then cloned into vector pMD-19T simple to construct pAAV-TK-IRES-ES. （2）Viral particle of purified rAAV was assayed by AVSachTM ELISA. （3） Identify the primary function of r AAV-TK-IRES-ES via T24 cell and HUVEC cell. Results （ 1 ） pAAV-TK- IRES-ES was constructed and tested by sequence indentification and enzyme digestion. （2）We obtained high quality of rAAV after dissociating and purifying. The viral particles titre of rAAV were 2×10^11 -12v. p/mL. （3）rAAV- TK-IRES-ES has dual functions of endostatin and suicide gene by inducing cell apoptosis of T24 and HUVEC cell. Conclusion We successfully constructed rAAV-TK-IRES-ES, it can inhibit tumor induced angiogenesis and suppress both the initiation and the subsequent growth of human bladder cancer.

关 键 词：膀胱肿瘤 腺相关病毒 自杀基因 内皮抑素 

分 类 号：R737.14[医药卫生—肿瘤]