摘要
目的构建肝脏特异性的微RNA-miR-122真核细胞表达载体并对其表达活性进行鉴定。方法经PCR从人基因组DNA中扩增肝脏特异性的微RNA-miR-122的前体序列,引入酶切位点和PolyT转录终止序列,将其插入siRNA专用真核表达载体pSuper中。将构建载体进行酶切、测序鉴定,再通过共转染含miR-122靶序列的GFP表达质粒后,经荧光显微镜和流式细胞仪及WesternBlot检测,鉴定载体功能性表达。结果miR-122的前体序列成功地克隆到真核表达载体pSuper上,且可表达出具有生物活性的miR-122,将该真核表达载体命名为pHsa-m122。结论成功构建miR-122真核表达载体pHsa-m122,有助于进一步研究miR-122在肝炎病毒复制及肝癌形成中的作用。
Objective To construct and analyze the activity of the expression vector pHsa-m122. Method Human precursor microRNA （miR-122） was obtained from the genomic DNA by PCR, Restriction sites and poly-T were introduced by ligation. The fragment was then inserted into the eukaryotic inducible expression vector pSuper, The insert was verified by restriction endonuclease digestion and sequencing, and the gene expression was evaluated by fluorescence microscopy, flow cytometry and Western Blot. Result miR-122 was successfully cloned into the eukaryotic expression vector pSuper, The expression product is biologically active, Conclusion The eukaryotic expression vector pHsa-m122 was successfully constructed, The expression vector may be used in the future study of hepatitis virus replication and the development of liver cancer.
出处
《热带医学杂志》
CAS
2008年第4期295-298,F0004,共5页
Journal of Tropical Medicine
基金
湖北省自然科学基金(No.2006ABA143)
华中科技大学同济医学院院基金(No.200601510747)。
