pcDNA3.1-MORC2重组质粒的构建及其在胃癌细胞SGC-7901中的表达和定位  

Construction of pcDNA3.1-MORC2 recombinant plasmid and its expression and localization in SGC-7901 gastric cancer cells

作　　者：刘芙蓉[1] 刘彤[1] 王春玉[1] 李家滨[1] 李丰[1] 

机构地区：[1]中国医科大学细胞生物学教研室卫生部细胞生物学重点实验室,沈阳110001

出　　处：《解剖科学进展》2011年第3期268-270,275,共4页Progress of Anatomical Sciences

基　　金：国家自然科学基金资助项目(No.30370736;No.30570966);教育部博士点基金资助项目(No.20050159023)

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。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 ceils.

关 键 词：Microchidia2 转染 蛋白表达 胃癌 

分 类 号：R735.2[医药卫生—肿瘤]