靶向人原癌基因c-fos的shRNA表达质粒的构建及鉴定

Construction and Identification of Short Hairpin RNA Expression Vector Targeting to Human Proto-oncogene c-fos

目的构建靶向人原癌基因c-fos的短发夹RNA(Short hairpin RNA,shRNA)重组真核表达质粒,并进行鉴定。方法构建靶向人c-fos基因的shRNA重组表达质粒Psilencer 3.1-sic-fos,转染乳腺癌MCF-7细胞,采用RT-PCR法检测稳定转染细胞中c-fos基因mRNA的表达。结果重组表达质粒Psilencer 3.1-sic-fos经PCR、双酶切及测序证明构建正确;重组表达质粒在mRNA水平明显抑制了MCF-7细胞c-fos基因的表达(P<0.05)。结论已成功构建了人c-fos基因shRNA重组表达质粒,为深入研究c-fos基因在肿瘤发生、发展过程中的作用提供了技术手段。

Objective To construct and identify a short hairpin RNA（shRNA） expression vector targeting to human proto-oncogene c-fos.Methods Recombinant shRNA expression vector Psilencer3.1-sic-fos targeting to human c-fos gene was constructed and transfected to breast cancer MCF-7 cells.The expression of c-fos mRNA in stably transfected cell was determined by RT-PCR.Results PCR,restriction analysis and sequencing proved that recombinant plasmid Psilencer3.1-sic-fos was constructed correctly.The recombinant plasmid inhibited the expression of c-fos gene at mRNA level in MCF-7 cells significantly.Conclusion The recombinant shRNA expression vector for human c-fos gene was successfully constructed,which provided a technical tool for further study on the role of c-fos gene in genesis and progress of tumors.