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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 a short hairpin RNA (shRNA) recombinant eukaryotic expression plasmid targeting human proto-oncogene c-fos and identify it. Methods shRNA expression vector pSilencer 3.1-sic-fos targeting human c-fos gene was constructed and transfected into breast cancer MCF-7 cells. The mRNA expression of c-fos gene in stable transfected cells was detected by RT-PCR. Results The recombinant plasmid pSilencer 3.1-sic-fos was correctly constructed by PCR, double enzyme digestion and sequencing. Recombinant plasmid significantly inhibited the expression of c-fos gene in MCF-7 cells (P <0.05). Conclusion The shRNA recombinant expression plasmid of human c-fos gene has been successfully constructed, which provides a technical means for further studying the role of c-fos gene in tumorigenesis and progression.