Rab25基因3'UTR荧光素酶报告基因载体及突变体的构建  

Construction of Rab25 gene 3'UTR luciferase reporter vector and mutational vector

作　　者：周建国[1] 张钰[1] 吕水萍 王菲[1] 王怡[1] 柏玉举[1] 苟晓丽[1] 张廷友[1] 沈刚[1] 马虎[1] 

机构地区：[1]遵义医学院附属医院肿瘤医院暨贵州省生物治疗人才基地,贵州遵义563099

出　　处：《遵义医学院学报》2016年第1期30-34,共5页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:81360351);贵州省科技厅攻关项目(NO:黔科字[2013]3003);贵州省科技厅及遵义医学院;遵义市科技局联合基金重点项目(NO:黔科合J字LKZ[2013]07);遵义医学院博士启动基金项目(NO:F-577);遵义医学院研究生社会实践项目(NO:zy-yjs2015004);贵州省高层次创新人才"千"层次人才基金项目

摘　　要：目的针对Rab25基因3＇端非翻译区（untranslated region,3＇UTR）构建Rab25荧光素酶报告基因载体及突变体,为研究miR-125a-5p在肺癌耐药中调控其靶基因RAB25提供有效的工具。方法 PCR扩增包含Rab25的3＇UTR的DNA片段,克隆至荧光素酶载体GV306,构建GV306/Rab25＇UTR载体;通过Trget Scan Release 6.2查找Rab25与miR-125a-5p结合区域并设计突变序列,参照构建GV306/Rab25＇UTR载体方法构建GV306/Rab25＇UTR突变载体。结果通过获得Rab25与miR-125a-5p的种子区域及侧翼部分120~126 nt,并上下延伸150 bp,合成Rab25＇UTR,并且在120~126 nt区域设计突变序列,构建了GV306/Rab25 3＇UTR及其突变载体。结论成功构建了含Rab25基因3＇UTR区的荧光素酶报告基因载体及突变体,为进一步研究Rab25在非小细胞肺癌EGFR-TKIs耐药中的作用打下基础。Objective To construct the dual-luciferase recombinant Rab25 3＇-UTR vector and mutational vector. Methods 3＇-UTR of Rab25 gene was amplified through PCR method,and was inserted in the dual luciferase reporter vector which was digested by enzyme. The sequence of mutated Rab25 gene was searched in Trget Scan Release 6. 2,the mutational vector used the same method. Results we obtained miR-125a-5p with the seed region and the flanking portion of Rab25,and extended vertically 150 bp to synthesize Rab25＇ UTR,and used 120 ~ 126 nt area to design mutated sequence. Furthermore,3＇-UTR of Rab25 gene and mutated gene were successfully cloned into the GV306 vector,which was verified by gel electrophoresis and DNA sequencing methods. Conclusion The luciferase reporter gene vectors containing Rab25 3＇UTR or mutational 3＇UTR area were successfully constructed,which will be used for further functional study of EGFR-TKIs resistance in NSCLC.

关 键 词：RAB25 miR-125a-5p 荧光素酶报告基因质粒 非小细胞肺癌 基因调节 

分 类 号：R734.2[医药卫生—肿瘤]