【摘要】 目的构建真核表达载体pcDNA3.1-MORC2并瞬时转染人胃癌细胞SGC-7901,观察融合蛋白在细胞内表达及定位。方法以人MORC2cDNA为模板,PCR扩增MORC2全长编码基因,亚克隆至pcDNA3.1-hisA表达载体。将构建的重组质粒测序并转染到胃癌细胞SGC-7901中,提取细胞蛋白进行Westernblot检测。用共聚焦激光扫描显微镜观察pcDNA3.1-MORC2在SGC-7901细胞内定位。结果 MORC2全长基因序列克隆到了表达载体pcDNA3.1-hisA中,酶切鉴定片段为3000bp。Westernblot检测到了融合蛋白pcDNA3.1-MORC2在胃癌细胞系SGC-7901中表达,分子量约为122kDa,主要定位于细胞核。结论成功构建了真核表达载体pcDNA3.1-MORC2,并检测到融合蛋白表达主要定位于胃癌细胞核内。更多还原

【Abstract】 Objective To construct the recombinant expression plasmid of microchidia2(MORC2) gene and identify its protein expression and localization in SGC-7901 cells.Methods The MORC2 coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into pcDNA3.1-hisA vector.After the target region was sequenced,the plasmid was transfected into SGC-7901 cell lines.The expression of the recombinant plasmid in SGC-7901 cells was proved by Western blot and the localization of pcDNA3.1-morc2 was observed by using laser scanning confocal microscopy.Results MORC2 was constructed into expressing vector pcDNA3.1-hisA successfully,the length of the fragment was 3000bp,identified by restriction enzymes digestion.The expression of pcDNA3.1-MORC2 fusion protein was detected by Western blot in SGC-7901 cells with a molecular weight 122kDa,and localized in the nucleus of SGC-7901 cells.Conclusions The recombinant plasmid of pcDNA3.1-MORC2 was constructed successfully and the fusion protein was localized in nucleus of SGC-7901 cells.更多还原