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目的:构建pcDNA3.1(-)/XAPC7真核表达载体并检测其在人肝癌细胞系SMMC-7721中的表达。方法:采用PCR法从pET28b/XAPC7重组质粒中克隆得到XAPC7cDNA全长序列,将之与pMD18-T载体连接、测序后将该片段亚克隆到真核表达载体pcDNA3.1(-)中。构建好的pcDNA3.1(-)/XAPC7真核表达质粒经酶切鉴定后,采用脂质体法将该重组质粒转染人肝癌细胞系SMMC-7721,经G418筛选,得到阳性克隆细胞株,再应用半定量RT-PCR技术检测转染前后该细胞株XAPC7基因的mRNA表达水平。结果:pcDNA3.1(-)/XAPC7经酶切鉴定及DNA测序证实,目的基因XAPC7的序列完全正确,真核表达载体构建成功;经RT-PCR检测,重组质粒转染株的XAPC7基因mRNA表达水平高于对照组,证实XAPC7基因已经稳定转染到SMMC-7721细胞中并得到表达。结论:成功地建立了人基因XAPC7的稳定转染细胞株,为进一步研究XAPC7的功能奠定了实验基础。
Objective: To construct eukaryotic expression vector pcDNA3.1 (-) / XAPC7 and to detect its expression in human hepatocellular carcinoma cell line SMMC-7721. Methods: The full length of XAPC7 cDNA was cloned by PCR from pET28b / XAPC7 recombinant plasmid and ligated with pMD18-T vector. After sequencing, the fragment was subcloned into eukaryotic expression vector pcDNA3.1 (-). The constructed pcDNA3.1 (-) / XAPC7 eukaryotic expression plasmid was identified by restriction enzyme digestion, and the recombinant plasmid was transfected into human hepatocellular carcinoma cell line SMMC-7721 by lipofectamine. After screening by G418, the positive cloned cell line was obtained, Semi-quantitative RT-PCR was used to detect the mRNA expression level of XAPC7 gene before and after transfection. Results: The recombinant plasmid pcDNA3.1 (-) / XAPC7 was confirmed by restriction enzyme digestion and DNA sequencing. The sequence of XAPC7 gene was completely correct and the eukaryotic expression vector was constructed successfully. The expression of XAPC7 mRNA was detected by RT-PCR Higher than the control group, confirmed that XAPC7 gene has been stably transfected into SMMC-7721 cells and expressed. Conclusion: Stable transfected cell line of human gene XAPC7 was successfully established, which laid the experimental foundation for further study on the function of XAPC7.