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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。
Objective: To construct the eukaryotic expression plasmid pDsRed1-C3 / LOC51255 and to detect its expression and subcellular localization in human hepatocellular carcinoma cell line HepG2. Methods: The full length cDNA of LOC51255 was cloned from pET28b / LOC51255 recombinant plasmid by PCR. The fragment was subcloned into eukaryotic expression vector pDsRed1-C3. The constructed pDsRed1-C3 / LOC51255 eukaryotic expression plasmid was identified by double enzyme digestion and sequencing. The recombinant plasmids were transfected into HepG2 cells by lipofectamine 2000 and the expression of LOC51255 was observed by fluorescence microscopy. Positioning situation. Results: The recombinant plasmid pDsRed1-C3 / LOC51255 was successfully constructed by double enzyme digestion and DNA sequencing. Fluorescence microscopy confirmed that the recombinant plasmid was expressed in HepG2 cells. LOC51255 protein mainly localized in the cytoplasm of HepG2 cells. CONCLUSION: The eukaryotic expression plasmid pDsRed1-C3 / LOC51255 was successfully constructed and expressed in HepG2 cells. LOC51255 was also localized in the cytoplasm of HepG2 cells, which laid the foundation for further study on the function of human LOC51255 gene.