l-Caldesmon腺病毒的构建及其在ECV304细胞中的表达  

Construction of l-Caldesmon adenovirus vector and its expression in ECV304 cells

作　　者：褚志刚[1] 张家平[1] 张琼[1] 向飞[1] 宋华培[1] 黄跃生[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2007年第12期1145-1147,共3页Journal of Third Military Medical University

基　　金：国家重点基础研究发展规划项目("973"项目)(2005CB522601);国家自然科学基金重点项目(30430680)~~

摘　　要：目的构建含有l-Caldesmon基因的重组腺病毒表达载体。方法以含有l-Caldesmon全长编码序列的pCGN-CaD为模板,PCR扩增l-Caldesmon,T-A克隆,酶切亚克隆到穿梭质粒pAdtrack-CMV上,在BJ5183菌内和pAdeasy同源重组,筛选阳性克隆,酶切鉴定,线性化后脂质体法转染HEK293细胞进行包装、扩增,得到含有l-Caldesmon基因的重组腺病毒,根据报告基因GFP测定病毒滴度。将收集的病毒液感染ECV304细胞,Westernblot检测l-Caldesmon蛋白的表达。结果成功构建了含有l-Caldesmon基因的重组腺病毒载体,病毒滴度可达1×109U/ml。重组病毒感染后可使ECV304细胞l-Caldesmon表达较对照组增加5倍。结论该重组腺病毒载体的构建为研究l-Caldesmon蛋白在内皮细胞中的生物学功能提供了一定的工作基础。Objective To construct the recombinant adenovirus vector containing L-Caldesmon. Methods The L-Caldesmon fragment was amplified by PCR, taking pCGN-CaD as templates, then was cloned to PMD- 18T vector and subcloned to pAdtrack-MV. The linearized shuttle plasmid was homogenously recombined with pAdeasy in B J5183. The candidate clone was further analyzed by restriction endonuclease digestion. Then the recombined plasmid was transfected into HEK293 cells for packaging and amplifying to get recombinant adenovirus pAd-CaD. The titer was determined by green fluorescent protein （GFP） expression. The harvested virus infected ECV304 cells. After infection, the expression of L-Caldesmon was proved by Western blot. Results The recombinant adenovirus vector containing L-Caldesmon was constructed successfully and the titer was 1 × 10^9 U/ml. The expression of L-Caldesmon protein in ECV304 cells infected by the virus was five times much higher than the control. Conclusion The constructed recombinant adenovirus vector containing L-Caldesmon provides a potent tool to investigate its biological function in endothelial cells.

关 键 词：腺病毒载体 1-Caldesmon ECV304细胞 

分 类 号：R322.123[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]