CAJ | 학술논문

目的：构建小鼠微小RNA miR-21的真核表达载体,在人胚肾293细胞中验证其活性表达,为进一步以此载体研究miR-21的功能打下基础。方法根据小鼠miR-21的成熟序列及其上下游约170 bp的序列设计引物,利用PCR技术从小鼠基因组DNA扩增含有miR-21前体的片段382 bp,克隆到质粒pRc/CMV,对重组质粒经双酶切及测序分析,鉴定完全正确后转染人胚肾293细胞,G418(1000 mg/L)筛选后获得miR-21稳定表达细胞系, Northern blot验证其在293细胞的过表达；同时构建pmiR-21-Luc reporter荧光素酶报告质粒,与miR-21重组质粒共转染293细胞进行荧光素酶活性分析,验证其调控活性。结果成功构建小鼠miR-21的真核表达载体,其可以在293细胞中稳定高效表达,荧光素酶活性实验证实其有生物活性。结论成功构建小鼠miR-21的真核表达载体,为进一步探讨miR-21的生物学功能奠定了实验基础。
목적：구건소서미소RNA miR-21적진핵표체재체,재인배신293세포중험증기활성표체,위진일보이차재체연구miR-21적공능타하기출。방법근거소서miR-21적성숙서렬급기상하유약170 bp적서렬설계인물,이용PCR기술종소서기인조DNA확증함유miR-21전체적편단382 bp,극륭도질립pRc/CMV,대중조질립경쌍매절급측서분석,감정완전정학후전염인배신293세포,G418(1000 mg/L)사선후획득miR-21은정표체세포계, Northern blot험증기재293세포적과표체；동시구건pmiR-21-Luc reporter형광소매보고질립,여miR-21중조질립공전염293세포진행형광소매활성분석,험증기조공활성。결과성공구건소서miR-21적진핵표체재체,기가이재293세포중은정고효표체,형광소매활성실험증실기유생물활성。결론성공구건소서miR-21적진핵표체재체,위진일보탐토miR-21적생물학공능전정료실험기출。
Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and identification its expression activ-ity in 293 cells. Methods The genomic sequence containing pre-miR-21 was amplified from mouse genomic DNA by PCR and cloned into the pRC/CMV plasmid. The constructed recombinant plasmid pRC/CMV-mmu-miR-21 was transfected to 293 cells by lipofectamine 2000, and the stably transfected cells were screened with G418,from which total RNA was extracted for detecting the expression of mature miR-21 by northern blot. In the meantime,a luciferase report plasmid examing the activity of miR-21 named pmiR-21-Luc reporter was also construc-ted,and luciferase activity analysis indicated the product of pRC/CMV-mmu-miR-21 indeed had biological activity. Results Both restriction enzyme digestion analysis and sequencing proved the recombinant plasmids were constructed correctly. The miR-21 was highly expressed in the screened clones of 293 cells and it had good biological activity. Conclusion The eukaryotic expression plasmid of mouse miR-21 was successfully constructed,which laid the foundation of further investigation of the role of miR-21 during skin wound healing.