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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。
Objective To construct eukaryotic expression vector pcDNA3.1-MORC2 and transiently transfected human gastric cancer cell SGC-7901 to observe the expression and localization of the fusion protein in the cell. Methods Human MORC2 cDNA was used as a template to amplify the full-length MORC2 encoding gene and subcloned into pcDNA3.1-hisA expression vector. The constructed recombinant plasmid was sequenced and transfected into gastric cancer cell SGC-7901, cell protein was extracted for Western blot detection. Confocal laser scanning microscopy was used to observe the localization of pcDNA3.1-MORC2 in SGC-7901 cells. Results The full-length MORC2 gene was cloned into the expression vector pcDNA3.1-hisA. The restriction fragment was 3000 bp. Western blot showed that the fusion protein pcDNA3.1-MORC2 was expressed in gastric cancer cell line SGC-7901, with a molecular weight of about 122 kDa and mainly located in the nucleus. Conclusion The eukaryotic expression vector pcDNA3.1-MORC2 was successfully constructed and the expression of fusion protein was mainly localized in the nucleus of gastric cancer cells.