tPA基因逆转录病毒载体构建及纯包装细胞系培育  

Construction of retroviral vector with tissue type plasminogen activator gene and culture of pure packaging cell line

作　　者：龚永生[1] 张凯伦[1] 蒋雄刚[1] 孙图成[1] 刘金平[1] 陈澍[1] 郭超[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院,湖北武汉430022

出　　处：《山东医药》2007年第24期18-20,共3页Shandong Medical Journal

基　　金：国家自然科学基金(30571838)。

摘　　要：目的构建含人组织型纤溶酶原激活剂（tPA）基因增强型绿色荧光蛋白（EGFP）基因逆转录病毒载体及产高滴度病毒的纯包装细胞系。方法PCR技术扩增目的基因tPA,定向克隆人逆转录病毒载体pLEGFP- N1中,测序鉴定重组逆转录病毒载体pLEGFP-N1-tPA,将其转入包装细胞PT67,培育全EGFP纯包装细胞系;测定病毒滴度。结果证实pLEGFP-N1-tPA中含有tPA基因。转染有pLEGFP-N1-tPA的纯PT67细胞产病毒滴度为1×10^7 CFU/ml。结论成功构建了pLEGFP-N1-tPA载体,并培育了产高滴度病毒纯包装细胞系。[ Objective ] To construct recombinant enhanced green fluorescent protein （EGFP） gene retroviral vector with human tissue type plasminogen activator （tPA） gene and culture pure packaging cell line producing high titer retroviruses. [ Methods] tPA gene was amplificated and cloned into retroviral pLEGFP- N1 plasmid, pLEGFP- N1 -tPA was identified and transferred into packaging cell PT67. PT67/ pLEGFP - N1 - tPA cells were selected. A pure packaging cell line/pLEGFP - N1 - tPA cells expressing EGFP was cultured. The virus titer was assayed. [ Results ] The recombinant pLEGFP - N1 - tPA vector containing tPA gene was confirmed. The virus titer of the pure packaging cell line/ PT67/ pLEGFP - N1 - tPA cells was 1×10^7CFU/ml. [ Conclusion] A kind of recombinant retroviral vector pLEGFP - N1 - tPA as well as a pure packaging cell line producing high virus titer are constructed successfully.

关 键 词：逆转录病毒 纤溶酶原激活剂 遗传载体 包装细胞 

分 类 号：R373.5[医药卫生—病原生物学] R394.33[医药卫生—基础医学]