解整合素金属蛋白酶10基因慢病毒载体的构建及鉴定  

Construction of Lentiviral Vector of RNA Interference of ADAM10 Gene

作　　者：李小鸥[1] 黄巍[2] 何兵[1] 周丽荣[2] 

机构地区：[1]武汉大学人民医院儿科,湖北武汉430060 [2]武汉市医学科学研究所,湖北武汉430030

出　　处：《武汉大学学报（医学版）》2013年第1期6-8,23,共4页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:81000094);武汉大学自主青年基金资助(编号:4101016);武汉市卫生局科研项目(编号:WX10B13)

摘　　要：目的:构建解整合素金属蛋白酶10(ADAM10)基因RNA干扰(RNAi)重组慢病毒载体,为进一步体内外实验研究提供基础。方法:筛选确定ADAM10基因RNAi有效靶序列,合成靶序列的Oligo DNA,与LentiLox3.7载体连接,PCR筛选阳性克隆,测序鉴定。用重组慢病毒转导传代心肌细胞(H9C2),通过Western blotting检测心肌细胞ADAM10的蛋白表达。结果:获得的载体插入ADAM10的shRNA核苷酸链序列正确,包装的慢病毒颗粒转导心肌细胞,与对照组比较ADAM10 shRNA转染成功抑制ADAM10蛋白表达。结论:成功构建AD-AM10 shRNA慢病毒载体,为其在扩张型心肌病(DCM)中的生物学功能研究奠定基础。Objective： To construct a lentiviral vector of RNA interference （RNAi） of ADAM10-gene and provide the basis for further experiment. Methods： The effective sequence of siRNA targeting ADAM10 gene was confirmed. Both sense and antisense Oligo DNA of the targeting sequence were designed, synthesized and coloned into the LentiLox 3. 7 vector, and were confirmed by PCR and sequencing. Then, ADAM10 shRNA was transfected into H9C2 cells. The expression of ADAM10 was detected by Western-blot. Results： PCR and DNA sequencing identified the inserted sequences. Western blotting showed that the expression of ADAM10 was negative in the ADAM10 siRNA transfected H9C2 cells. Conclusion. The lentiviral RNAi vector of ADAM10 producing ADAM10 shRNA was constructed successfully.

关 键 词：RNA干扰 解整合素金属蛋白酶10 慢病毒 扩张型心脏病 

分 类 号：Q782[生物学—分子生物学]