【摘要】 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。更多还原

【Abstract】 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 EcoRⅠand XhoⅠ.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-N1-TFF3 was successfully constructed and the expression of its fusion protein was in the nucleus and cytoplasm of COS 7 cells.更多还原