人COX6B1在哺乳动物细胞株中转染表达与定位的初步研究

目的构建带FLAG标签的人野生型细胞色素氧化酶亚基COX6B1表达载体,转染至HEK 293T、COS7细胞观察COX6B1蛋白在细胞内表达与定位。方法以含人COX6B1全长cDNA序列的质粒为模板,通过上游带有FLAG标签的引物,PCR方法扩增出COX6B1全长序列,然后插入真核表达载体pCDNA3.1中构建pCDNA3.1-FLAG-COX6B1表达载体。将构建的质粒转染至HEK 293T细胞,提取总蛋白,进行Western blot检测;转染至COS7细胞,在荧光显微镜下观察其细胞定位。结果成功构建了pCD-NA3.1-FLAG-COX6B1表达载体,其在HEK 293T细胞中能有效地表达;在COS7细胞中呈斑点状分布,且主要在胞质内分布,细胞核中未见其明显表达。结论 COX6B1在HEK293T、COS7细胞中均有表达,且主要分布在胞质内,这为进一步了解COX6B1的功能提供了一定的基础。

Human COX6B1 was transfected for expression and location of the preliminary study in mammalian cell lines

Objective To investigate the expression and localization of the product of COX6B1 gene,human wildCOX6B1 with FLAG tag expression vector constructed and expressed in appropriate mammalian cell lines.Methods The full length cDNA fragment of COX6B1 from plasmid was used to amplify with PCR,by virtue of the upstream primer with FLAG tags,then the fragment was inserted into pCDNA3.1 vector to construct expression vector pCDNA3.1-FLAG-COX6B1.Its expression was detected in HEK 293T cells transfected with FLAG-tagged COX6B1 gene by Western blot.The location of COX6B1 protein defined in COS7 cells transfected with FLAG-tagged COX6B1 gene was observed by fluorescence microscope.Results The expression vector encoding COX6B1 protein could express efficiently in HEK 293T cell line,and the product of COX6B1 gene was found mainly localized in the cytoplasm,not in the nucleus obviously.Conclusion The expression vector encoding COX6B1 protein can express efficiently in two cell lines of HEK293T and COS 7 and which is mainly localized in the cytoplasm.The results provide the basis for further function studies of COX6B1 protein.