【摘要】 目的:构建pDsRed1-C3/LOC51255真核表达质粒并检测其在人肝癌细胞株HePG2中的表达和亚细胞定位。方法:采用PCR法从pET28b/LOC51255重组质粒中克隆得到LOC51255cDNA全长序列,并将该片段亚克隆到真核表达载体pDsRed1-C3中。构建好的pDsRed1-C3/LOC51255真核表达质粒经双酶切和测序鉴定后,采用脂质体法将该重组质粒转染人肝癌细胞株HePG2,再用荧光显微镜直接观察LOC51255在其中的表达及定位情况。结果:经双酶切及DNA测序结果证实成功构建重组质粒pDsRed1-C3/LOC51255;荧光显微镜观察证实该重组质粒能在HePG2细胞中表达,且LOC51255蛋白主要定位于HePG2细胞的细胞质。结论:成功构建pDsRed1-C3/LOC51255真核表达质粒,并在HePG2细胞中得到表达,同时证实LOC51255定位于HePG2细胞的胞质,为进一步研究人类LOC51255基因的功能奠定了基础。更多还原

【Abstract】 AIM:To construct the recombinant eukaryotic expression vector pDsRed1-C3/LOC51255 and investigate the subcellular localization of LOC51255 protein in HePG2 cell line.METHODS:The cDNA of LOC51255 was amplified from the recombinant plasmid pET28b/LOC51255 by PCR.The aim gene fragment LOC51255 from pMD18-T/LOC51255 was subcloned into eukaryotic expression vector pDsRed1-C3.The recombinant plasmid pDsRed1-C3/LOC51255 was identificated by BamH I/Xho I double digestion and sequence analysis,and then transfected into HePG2 cell line by lipofectamine 2000.The expression of LOC51255 protein and its subcellular localization were detected by fluorescence microscope.RESULTS:The construction of the recombinant plasmid pDsRed1-C3/LOC51255 was proved by restriction enzyme digestion analysis and DNA sequencing.Its red fluorescent protein was mainly detected in the cytoplasm of HePG2 cell line.CONCLUSION:The eukaryotic expression plasmid pDsRed1-C3/LOC51255 has been successfully constructed and expressed in the HePG2 cell line.LOC51255 protein has been proved to be located in the cytoplasm of HePG2 cell line.Our research will help further studies on the function of human gene LOC51255.更多还原