摘要
目的:构建人Polo样激酶2(hPlk2)真核表达载体并证实该融合蛋白在细胞内的表达及定位。方法:提取工具细胞Hela的mRNA,反转录为cDNA。PCR扩增hPlk2基因cDNA全长,并将其亚克隆至pEGFP-C1表达载体中。然后将构建的重组质粒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检测。最后利用激光扫描共聚焦显微镜观察pEGFP-hPlk2在COS-7细胞内的定位。结果:hPlk2基因cDNA全长克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小为2 058 bp,并测序成功。Western blot检测到了pEGFP-hPlk2融合蛋白表达,分子量约为105 kDa。pEGFP-hPlk2在COS-7细胞中主要定位于细胞质和核周。结论:成功构建了hPlk2基因cDNA全长的真核表达载体,pEGFP-hPlk2蛋白在COS-7细胞中主要定位于细胞质和核周。
Objective：To construct the expression plasmid of human Polo-like kinase 2（hPlk2） gene and identify the expression and localization of the fusion protein.Methods：Total mRNA was extracted from Hela cells,and cDNA was synthesized by reverse transcription.The hPlk2 coding sequence was amplified by polymerase chain reaction（PCR） and subcloned into pEGFP-C1 vector.After the target region was identified by restriction 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-hPlk2 in COS-7 cells was observed with laser scanning confocal microscopy.Results：hPlk2 was constructed into the expressing vector pEGFP-C1 successfully.The length of the fragment identified by restriction enzyme digestion was 2058bp.The expression of pEGFP-hPlk2 fusion protein with a molecular weight of 105kDa was detected by Western blot.The pEGFP-hPlk2 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells.Conclusion：The recombinant plasmid of hPlk2 gene was successfully cloned into eukaryotic expressing vector,and the pEGFP-hPlk2 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells.
出处
《东南大学学报（医学版）》
CAS
2012年第2期162-165,共4页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(30800415
81070688)
