miR-93真核表达载体的构建及其表达验证  

The construction and expression verification of miR-93 eukaryotic expression vector

作　　者：黄军[1] 吴开杰[1] 惠珂[1] 王彬[1] 王新阳[1] 贺大林[1] 

机构地区：[1]西安交通大学第一附属医院泌尿外科,陕西西安710061

出　　处：《现代泌尿外科杂志》2016年第10期799-802,共4页Journal of Modern Urology

基　　金：国家自然科学基金(No.81572516);陕西省国际科技合作与交流计划项目(No.2016KW-021)

摘　　要：目的构建人miR-93真核表达载体,并验证其表达效果,为进一步研究miR-93的生物学功能提供有利工具。方法用PCR方法从293T细胞基因组DNA中扩增miR-93前体序列,引物引入酶切位点和保护碱基,产物用限制性内切酶EcoRⅠ和BglⅡ双酶切后插入pEZX-MR04表达载体中,构建miR-93真核表达载体pEZX-MR04-miR-93。构建好的载体用双酶切、PCR和测序鉴定,同时转染293T细胞,用Real time PCR检测重组pEZX-MR04-miR-93质粒的miR-93表达效率。结果成功构建miR-93真核表达载体pEZX-MR04-miR-93,重组pEZX-MR04-miR-93质粒转染293T细胞后miR-93表达上升约21倍。结论构建的miR-93真核表达载体pEZX-MR04-miR-93可显著上调细胞中miR-93水平,为进一步研究miR-93在泌尿系肿瘤中的生物学功能提供可靠工具,奠定了坚实的实验基础。Objective To construct a eukaryotic expression vector of human miR-93 and test its expression efficiency, in order to offer a good tool to further study the biological functions of miR-93. Methods PCR was used to amplify the precursor sequence of human miR-93 from the genomic DNA of 293T cells, which were then cloned into the pEZX-MR04 expression vector to produce the recombinant pEZX-MR04-miR-93 plasmid. Enzyme double digestion, PCR and sequence analysis were adopted to verify the recombinant pEZX-MR04-miR-93 plasmid. After that, 293T cells were transfected with the recombinant pEZX-MR04-miR-93 plasmid and real time PCR was used to test its expression efficiency of miR-93. Results The eukaryotic expression vector of miR-93 was successfully constructed. After transfection with the recombinant pEZX-MR04-miR-93 plasmid, the expression level of miR-93 in 293T cells could be up-regulated by 21 folds. Conclusion The recombinant pEZX- MR04-miR-93 expression vector was successfully constructed and could significantly increase miR-93 level in cells, which will offer us a convenient tool to further study the biological functions of miR-93.

关 键 词：miR-93 表达载体 pEZX-MR04 293T 

分 类 号：R331[医药卫生—人体生理学]