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.
出处
《山东医药》
CAS
北大核心
2007年第24期18-20,共3页
Shandong Medical Journal
基金
国家自然科学基金(30571838)。
