大鼠β防御素2基因慢病毒载体的构建及功能检测  

Construction and expression of lentiviral vector of rat β-defensin-2 gene

作　　者：雷撼[1] 方路[2] 何翔[1] 

机构地区：[1]同济大学医学院附属东方医院呼吸科,上海200120 [2]上海市杨浦区中心医院

出　　处：《中华实验和临床病毒学杂志》2010年第3期232-234,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（30670932）;上海浦东新区社会发展局基金（pw2005A-10）;上海浦东新区医学领先人才项目基金（CX2004004）

摘　　要：目的 构建大鼠β防御素2（rBD2）基因慢病毒表达载体,并转染培养细胞检测其表达,为rBD2研究及大鼠体内实验奠定基础.方法 提取大鼠上皮细胞总RNA,PCR扩增获得rBD2基因,双酶切PCR产物和慢病毒载体Lentivirus[含H1启动子和绿色荧光蛋白（GFP）],连接构成rBD2基因慢病毒表达载体LV-rBD2,行测序鉴定.用慢病毒包装系统对LV-rBD2进行慢病毒颗粒包装并梯度稀释法测定病毒滴度,病毒液感染培养细胞,RT-PCR和Western Blot检测rBD2表达.结果 凝胶电泳和测序结果表明rBD2基因克隆到慢病毒载体中,序列正确.完成慢病毒颗粒包装,调整病毒滴度至1×105ifu/μl.RT-PCR和Western-Blot显示rBD2基因获得表达.结论 rBD2基因慢病毒表达载体LV-rBD2被成功构建,能转染细胞并获得有效表达.Objective To construct a lentiviral expression vector of rat β-defensin-2（rBD2）gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein（GFP）],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.

关 键 词：基因转染技术 表达的序列标记 逆转录聚合酶链反应 印迹法 蛋白质 防御素类 

分 类 号：R346[医药卫生—基础医学]