hLMO4基因重组质粒的构建及蛋白表达和定位  

Construction of Fusion Plasmid of Human LMO4 Gene and Its Recombinant Protein Expression and localization

作　　者：李彦姝[1] 王春玉[1] 苏楠[1] 张红艳[1] 李丹妮[2] 李丰[1] 

机构地区：[1]中国医科大学细胞生物教研室细胞生物学卫生部重点实验室,辽宁沈阳110001 [2]中国医科大学,94期临床医学辽宁沈阳110001

出　　处：《解剖科学进展》2009年第1期85-87,92,共4页Progress of Anatomical Sciences

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

摘　　要：目的构建hLMO4真核表达载体并证实融合蛋白在细胞内表达及定位。方法提取人乳腺癌MCF-7细胞的mRNA,反转录为cDNA。PCR扩增hLMO4全长编码基因,亚克隆至pCDNA3.1-myc-hisA表达载体中。将构建的重组质粒测序并转染到胃癌细胞BGC823中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察pEGFP-LMO4在乳腺癌MCF-7细胞内定位。结果hLMO4全长基因序列克隆到了真核表达载体pCDNA3.1-myc-hisA中,酶切鉴定片段为500bp。Western blot检测到了融合蛋白表达,分子量约为23KD。pEGFP-LMO4在乳腺癌MCF-7细胞内定位以细胞核为主,在细胞浆内少量表达。结论成功的构建了hLMO4全长基因真核表达载体,pEGFP-LMO4蛋白定位于乳腺癌细胞质和核内。Objective To construct the expression plasmid of human LIM domain Only 4（hLMO4） gene and identify its recombinant protein expression and localization. Methods Total RNA was extracted from human breast cancer MCF-7 cells. The hLMO4 coding sequence was amplified by polymerase chain reaction（PCR） method and subcloned into pCDNA3.1-myc-his A vector. After the target region was sequenced, the plasmid was transfected into BGC 823 cell lines. The expression of the recombinant plasmid in BGC 823 cells was proved by Western blot. The localization of pEGFP-LMO4 in breast cancer cell MCF-7 was observed by using laser scanning confocal microscopy. Results hLMO4 had been constructed into expressing vector pCDNA3.1-myc-his A successfully. The length of the fragment was 500bp, identified by restriction enzymes digestion. The expression of myc-LMO4 fusion protein was detected by Western blot, with a molecular weight 23KD. The pEGFP-LMO4 protein was localized more in the nucleus, less in the cytoplasm. Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector, the pEGFP-LMO4 fusion protein was expressed in the nucleus and cytoplasm.

关 键 词：hLMO4 乳腺癌 WESTERN BLOT 

分 类 号：R737.9[医药卫生—肿瘤]