含TK-IRES-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

基础医学与临床 ›› 2009, Vol. 29 ›› Issue (8): 863-866.

含TK-IRES-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

潘建刚周兴韩瑞发陈志光李江婷

广州医学院第二附属医院广州医学院第二附属医院天津市泌尿外科研究所广州医学院第二附属医院广州医学院第二附属医院

收稿日期:2008-09-04 修回日期:2008-12-07 出版日期:2009-08-20 发布日期:2009-08-20
通讯作者: 潘建刚

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

Jian-gang PAN, Xing ZHOU, Rui-fa HAN, Zhi-guang CHEN, Jiang-ting LI

the Second Affiliated Hospital of Guangzhou Medical College Tianjin Institute of Urology

Received:2008-09-04 Revised:2008-12-07 Online:2009-08-20 Published:2009-08-20
Contact: Jian-gang PAN,

摘要/Abstract

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rAAV-TIE）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。

Abstract: Objective To construct pAAV-TK-IRES-ES and identify its function. Methods①IRES,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.②Viral particle of purified rAAV were assayed by AVSachTM ELISA.③Identify the primary function of r AAV-TK-IRES-ES via T24 cell and HUVEC cell Result ①pAAV-TK-IRES-ES was constructed and tested correctly by sequence indentification and enzyme digestion.②We obtained high quality of rAAV after dissociating and purifying. The viral particles title of rAAV were 2×1011-12 v.p/ml. ③ rAAV-TK-IRES-ES has both functions of endostatin and suicide gene by inducing cell apoptosis of T24 and HUVEC cell. Conclusion We successfully constructed rAAV-TK-IRES-ES and it can inhibit tumor induced angiogenesis and suppress both the initiation and the subsequent growth of human bladder cancer.

潘建刚周兴韩瑞发陈志光李江婷. 含TK-IRES-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[J]. 基础医学与临床, 2009, 29(8): 863-866.

Jian-gang PAN; Xing ZHOU; Rui-fa HAN; Zhi-guang CHEN; Jiang-ting LI. Construction of non-fusion recombinant of pAAV-TK-IRES-ES and primary identify of its function[J]. Basic & Clinical Medicine, 2009, 29(8): 863-866.