人Plk3基因真核表达载体的构建及蛋白表达和定位

Construction of human Plk3 gene plasmid vector and its fusion protein expression and localization

目的构建hPlk3真核表达载体并证实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hPlk3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Westernblot检测其表达。最后利用激光共聚焦扫描显微镜观察pEGFP-hPlk3在COS-7细胞内的定位。结果hPlk3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1941bp,并测序成功。Westernblot检测到了GFP-hPlk3融合蛋白表达,分子量约为98kDa。pEGFP-hPlk3在COS-7细胞中主要定位于细胞质和核周。结论成功构建了hPlk3基因cDNA全长的真核表达载体,pEGFP-hPlk3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 3 （hPlk3） gene and identify the expression and localization of the fusion protein. Methods Total mRNA was extracted from Hela cells, and cDNA was formed by reverse transcription. The hPlk3 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 transfeeted into COS-7 cells. The expression of the recombinant plasmid in COS-7 cells was detected by Western blot, the localization of pEGFP-hPlk3 in COS-7 cells was observed with laser scanning confocal microscopy. Results hPlk3 was constructed into the expressing vector pEGFP-C1 successfully, the length of the fragment identified by restriction enzyme digestion was 1941bp. The expression of pEGFP-hPlk3 fusion protein with a molecular weight of 98kDa was detected by Western blot. The pEGFP-hPlk3 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells. Conclusion The recombinant plasmid of hPlk3 gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-hPlk3 fusion protein was mostly localized in the cytoplasm and perinucleus of COS-7 cells.