hCAP基因真核表达载体的构建及蛋白的表达和定位

Construction of eukaryotic plasmid of human hCAP gene and the expression and localization of fusion protein

目的:构建hCAP真核表达载体并检测融合蛋白在细胞内的表达及定位。方法:提取工具细胞CV-1的mRNA,反转录为cDNA。PCR扩增hCAP基因的cDNA全长,并将其克隆至pEGFP-C1表达载体中。进一步将构建的重组载体进行酶切和测序鉴定,并转染到工具细胞COS-7中,提取细胞蛋白进行Western blot。最后利用激光共聚焦显微镜观察pEGFP-hCAP在NIH3T3成纤维细胞内的定位。结果:hCAP基因cDNA全长克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段为3 879 bp,测序证实成功。Western blot检测GFP-hCAP融合蛋白表达,相对分子质量(Mr)约为169 000。pEG-FP-hCAP在NIH3T3细胞中主要定位于细胞周边。结论:成功构建了真核表达载体pEGFP-hCAP,融合蛋白在NIH3T3细胞中主要定位于细胞周边。

AIM： To construct the expression plasmid of human c-Cbl-associated protein（hCAP） gene and identify the expression and localization of fusion protein.METHODS： Total mRNA was extracted from CV-1 cells,and cDNA was formed by reverse transcription.The hCAP coding sequence was amplified by polymerase chain reaction（PCR） and cloned into pEGFP-C1 plasmid.After the hCAP gene was identified by enzyme digestion and sequencing,the plasmid was transfected into COS-7 cells.The expression of the recombinant plasmid in COS-7 cells was detected by Western blot assay.The localization of pEGFP-hCAP in NIH3T3 cells was observed with laser confocal microscopy.RESULTS： hCAP was successfully constructed into the pEGFP-C1 expressing vector.The length of the fragment identified by restriction enzyme digestion was 3 879 bp.The expression of pEGFP-hCAP fusion protein with a molecular weight of 169kDa was detected by Western blot.The pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.CONCLUSION： The recombinant plasmid of hCAP gene was successfully cloned into eukaryotic expressing vector,and the pEGFP-hCAP fusion protein was mostly localized at the cell periphery of NIH3T3 cells.