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目的构建转化生长因子(TGF)-β1短发卡RNA(shRNA)表达载体,为肿瘤生物学研究奠定方法基础。方法根据TGF-β1的mRNA序列,运用Ambion公司的Target Finder软件筛选TGF-β1shRNA的候选点,在BglⅡ和HindⅢ位点克隆到表达载体pSUPER gfp-neo上。重组的shRNA pSUPER gfp-neo在大肠杆菌DH5a上筛选和鉴定。分别用酶联免疫吸附法(ELISA)测定和免疫荧光筛选最佳TGF-β1shRNA靶点序列。结果 ELISA和免疫荧光染色结果显示,shRNA-a、shRNA-b和shRNA-c均在不同程度上降低了靶细胞TGF-β1蛋白质表达,以shRNA-b所引起的下降最为显著(P<0.05)。结论构建的TGF-β1shRNA在肿瘤细胞中产生较好的TGF-β1沉默效果,为肿瘤生物学研究提供了一种方法。
Objective To construct short hairpin RNA (shRNA) expression vector of transforming growth factor (TGF-β1) and lay the foundation for the study of tumor biology. Methods According to the mRNA sequence of TGF-β1, the target point of TGF-β1 shRNA was screened by Ambion’s Target Finder software and cloned into the expression vector pSUPER gfp-neo at BglII and HindIII sites. Recombinant shRNA pSUPER gfp-neo was screened and identified in E. coli DH5a. The optimal TGF-β1 shRNA target sequence was screened by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence respectively. Results The results of ELISA and immunofluorescence staining showed that shRNA-a, shRNA-b and shRNA-c all reduced the TGF-β1 protein expression to a certain extent, and the most significant decrease was shRNA-b (P <0.05) . Conclusion The constructed TGF-β1 shRNA produces better effect of silencing TGF-β1 in tumor cells and provides a method for the study of tumor biology.