转化生长因子-β1 shRNA表达载体的构建与鉴定

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。

Construction and Identification of Transforming Growth Factor-β1 shRNA Expressing Vector

Objective To construct transforming growth factor-β1 （ TGF-β1） short hairpin RNA （ shRNA）-expressing vector and provide the basis for further experiments in cancer biology. Methods TGF-β1-specific shRNAs were designed with the Target Finder of Ambion according to mRNA of TGFβ1,and were ligated into the mammalian expression vector pSUPER gfp-neo at the BglII and HindIII cloning sit. The recombinant shRNApSUPER gfp-neo was used to transform Escherichia coli DH5a,which was selected and verified by sequencing. Then,select the optimal site for TGFB1 gene silencing with enzyme-linked immunosorbent assay （ ELISA） and Immunofluorescence. Results ELISA and immunofluorescence staining showed reduced expression of TGF-b1 protein in RD cells transfected with shRNA-a, shRNA-b and shRNA-c. The reduction was most pronounced （ P 0. 05） in cells transfected with shRNA-b. Conclusion These data suggest that the constructed TGF-β1shRNA-expressing vector produces efficient TGF-β1 knockdown in the tumor cell line and may be a method of cancer biological research.