N端标记GFP-TFF3蛋白在非洲绿猴肾成纤维细胞表达和定位

The identification of N-terminal labeled GFP-TFF3 fusion protein in mammalian fibroblasts

目的构建pEGFP-N1-TFF3融合蛋白表达载体,并检测其在非洲绿猴肾成纤维细胞(COS7细胞)内的表达和定位。方法提取HT29细胞的mRNA,反转录为cDNA;以此为模板PCR扩增hTFF3(286bp-462bp)基因;通过EcoRⅠ和XhoⅠ双酶切位点将hTFF3基因定向插入真核表达载体pEGFP-N1中;将构建的重组质粒测序成功后,转染至人肾细胞(HEK293细胞)中,荧光显微镜观察GFP-TFF3融合蛋白的表达,并提取蛋白进行Western Blot检测;利用激光扫描共聚焦显微镜观察GFP-TFF3融合蛋白在COS7细胞内的定位情况。结果酶切及测序结果证明hTFF3基因成功克隆至真核表达载体pEGFP-N1中,Western Blot检测结果显示其融合蛋白分子量约为34kDa,激光扫描共聚焦显微镜观察结果显示GFP-TFF3融合蛋白在COS7细胞内定位以细胞核为主,在细胞质内少量表达。结论成功构建了pEGFP-N1-TFF3真核表达载体,GFP-TFF3蛋白定位于COS7的细胞核和细胞质中。

Objective To construct the expression vector of pEGFP-N1-TFF3 fusion protein and explore the expression and localization of pEGFP-N1-TFF3 fusion protein in COS7 cells. Methods Total RNA was extracted from human epithelial cells. The hTFF3（286bp-462bp） gene sequence was amplified from the cDNA of HT29 cells by polymerase chain reaction（PCR） method and subcloned into pEGFP-N1 vector by EcoR I and Xho I. After DNA sequencing was employed, the recombinant plasmid was transfected into HEK293 cells, and recombinant protein was identified by Western blot. Then the localization of recombinant protein in COS 7 cells was observed by laser scanning confocal microscopy. Results The hTFF3 gene was successfully cloned into pEGFP-N1 vector, then confirmed by enzyme digestion and sequencing. GFP-TFF3 fusion protein was detected by Western blot, corresponding to a molecular weight about 34kDa. GFP-TFF3 fusion protein was more located in the nucleus, less in the cytoplasm of COS 7 cells. Conclusion The eukaryotic expression plasmid of pEGFP-N 1-TFF3 was successfully constructed and the expression of its fusion protein was in the nucleus and cytoplasm of COS 7 cells.