hRNF6基因重组质粒的构建及蛋白表达和定位

Construction of the Recombinant Plasmid of Human RNF6 Gene and the Expression and Localization of Fusion Protein

目的构建环指蛋白6(RNF6)真核表达载体,并证实融合蛋白在细胞内的表达及定位。方法提取工具细胞HEK-293的mRNA,反转录为cDNA。PCR扩增hRNF6全长编码基因,并将其克隆至pCDNA3.1-myc-his A及pEGFP-C1表达载体中。将构建的重组质粒测序并转染到工具细胞HEK-293中,提取细胞蛋白进行Westernblot检测。利用共聚焦激光扫描显微镜观察pEGFP-RNF6在工具细胞CV-1和胃癌SGC-7901细胞内的定位。结果 hRNF6全长基因序列克隆到了真核表达载体pCDNA3.1-myc-his A和pEGFP-C1中,酶切鉴定片段为2058bp。Western blot检测到了融合蛋白表达,分子量约为78kDa。pEGFP-RNF6在CV-1细胞和胃癌SGC-7901细胞内定位以细胞核为主,在细胞质内少量表达。结论成功的构建了hRNF6全长基因真核表达载体,pEGFP-RNF6蛋白主要定位于胃癌细胞核内。

Objective To construct an eukaryotic expression vector of RNF6（ring finger protein 6）gene and identify its recombinant protein expression and localization.Methods Total mRNA was extracted from HEK-293 cells,cDNA was formed by reverse transcripton.The hRNF6 coding sequence was amplified by polymerase chain reaction（PCR） method and cloned into pcDNA3.1-myc-his A vector and pEGFP-C1 vector.After the target region was sequenced,the plasmid was transfected into HEK-293 cell lines.The expression of the recombinant plasmid in HEK-293 cells was proved by Western blot.The localization of pEGFP-RNF6 in CV-1 cell and gastric cancer cell SGC-7901 was observed by using laser scanning confocal microscopy.Results hRNF6 had been constructed into expressing vector pCDNA3.1-myc-his A and pEGFP-C1 successfully.The length of the fragment was 2 058 bp,identified by restriction enzymes digestion.The expression of myc-RNF6 fusion protein was detected by Western blot,with a molecular weight 78 kDa.The pEGFP-RNF6 protein was localized more in the nucleus,less in the cytoplasm.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,the pEGFP-RNF6 fusion protein was expressed mainly in the nucleus.