中文摘要：

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。

英文摘要：

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.