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目的构建人泛素偶联酶UBE2C/UbcH10荧光蛋白真核表达载体,并转染肝癌细胞,筛选建立稳定转染细胞株。方法从构建好的pMD19-T/UbcH10载体中PCR扩增UbcH10基因,PCR产物双酶切后克隆入pEGFP-N1真核表达载体中,构建真核表达载体pEGFP-N1/UbcH10,进行双酶切和测序鉴定。将构建好的真核表达载体pEGFP-N1/UbcH10和空载体pEGFP-N1利用脂质体Lip2000TM分别转染进入肝癌细胞SMMC7721中,建立肝癌细胞SMMC7721的G418细胞死亡曲线,选择G418的筛选浓度,经G418结合荧光显微镜观察筛选稳定转染细胞。将筛选获得的肝癌细胞进行有限稀释法单克隆化,收集单个细胞克隆扩大培养备用。结果经双酶切和测序鉴定,带有荧光蛋白标记的真核表达载体pEGFP-N1/UbcH10构建成功,经脂质体介导真核表达载体pEGFP-N1/UbcH10和空载体pEGFP-N1成功转入肝癌细胞SMMC7721中,经800μg/mL G418筛选并进行单克隆化获得稳定转染的细胞株SMMC7721-pEGFP-N1和SMMC7721-pEGFP-N1/UbcH10,阳性转化率达到94%以上。结论人泛素偶联酶UBE2C/UbcH10稳定高表达肝癌细胞株的建立,为该蛋白在肝癌发生发展过程中的具体作用机制的研究奠定了基础。
Objective To construct eukaryotic expression vector of human ubiquitin-conjugating enzyme UBE2C / UbcH10 and transfect it into hepatoma cells and screen the stable transfected cell lines. Methods The UbcH10 gene was amplified by PCR from the constructed pMD19-T / UbcH10 vector and cloned into pEGFP-N1 eukaryotic expression vector. The eukaryotic expression vector pEGFP-N1 / UbcH10 was constructed and double digested And sequencing identification. The constructed eukaryotic expression vector pEGFP-N1 / UbcH10 and empty vector pEGFP-N1 were transfected into hepatoma cell SMMC7721 respectively by liposome Lip2000TM to establish G418 cell death curve of hepatoma SMMC7721 cells, G418 combined with fluorescence microscopy screening stable transfected cells. The hepatoma cells screened by the screening were subjected to a limiting dilution method to be monoclonal, and a single cell clone was collected and expanded for further use. Results After double enzyme digestion and sequencing, the eukaryotic expression vector pEGFP-N1 / UbcH10 with fluorescent protein was successfully constructed. The recombinant plasmid pEGFP-N1 / UbcH10 and pEGFP-N1 The transfected SMMC7721 cells were stably transfected with SMMC7721-pEGFP-N1 and SMMC7721-pEGFP-N1 / UbcH10 by screening with 800μg / mL G418 and monoclonal antibody. The positive rate was over 94%. Conclusion The establishment of stable and highly expressed human hepatoma cell line UBE2C / UbcH10 by human ubiquitin-conjugate enzyme (UBE2C / UbcH10) laid a foundation for the study of the specific mechanism of this protein in the development and progression of hepatocellular carcinoma.