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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 pEGFP-N1-TFF3 fusion protein expression vector and detect its expression and localization in African green monkey kidney fibroblasts (COS7 cells). Methods The mRNA of HT29 cells was extracted and transcribed into cDNA. The hTFF3 (286bp-462bp) gene was amplified by PCR. The hTFF3 gene was inserted into the eukaryotic expression vector pEGFP-N1 by EcoRⅠand XhoⅠ restriction sites. ; The constructed recombinant plasmid was successfully sequenced and transfected into human renal cells (HEK293 cells), the expression of GFP-TFF3 fusion protein was observed under a fluorescence microscope, and the protein was extracted for Western Blot detection. The expression of GFP-TFF3 was detected by laser scanning confocal microscopy TFF3 fusion protein localization in COS7 cells. Results The results of enzyme digestion and sequencing showed that the hTFF3 gene was successfully cloned into eukaryotic expression vector pEGFP-N1. The results of Western Blot showed that the molecular weight of fusion protein was about 34 kDa. Laser scanning confocal microscopy showed that GFP-TFF3 fusion protein expressed in COS7 cells Internal targeting to the nucleus-based, a small amount of expression in the cytoplasm. Conclusion The eukaryotic expression vector pEGFP-N1-TFF3 was successfully constructed. The GFP-TFF3 protein was localized in the nucleus and cytoplasm of COS7.