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.
出处
《基础医学与临床》
CSCD
北大核心
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)
关键词
膀胱肿瘤
腺相关病毒
自杀基因
内皮抑素
bladder cancer
adeno-associated virus
suicide gene
endostatin
