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目的:构建pcDNA3.1(+)/EMC6真核表达载体,并将其在人肝L-02细胞株中过表达。方法:查询Pub Med上EMC6的基因序列,针对该序列设计引物,提取L-02细胞的总RNA、聚合酶链式反应(PCR)扩增目的片段,采用基因重组技术将EMC6的cDNA定向插入真核表达载体pcDNA3.1中,构建pcDNA3.1(+)/EMC6真核表达载体,经菌落PCR,测序验证构建的准确性。应用脂质体转染方法将重组载体转入L-02细胞中,RT-PCR、蛋白质印迹法检测EMC6基因的mRNA和蛋白表达水平。结果:PCR和基因测序结果均表明pcDNA3.1(+)/EMC6重组质粒构建成功,RT-PCR和蛋白质印迹检测结果证实EMC6在人肝L-02细胞中过表达。结论:成功构建人pcDNA3.1(+)/EMC6重组质粒,并在L-02细胞中过表达。
OBJECTIVE: To construct pcDNA3.1 (+) / EMC6 eukaryotic expression vector and overexpress it in human liver L-02 cell line. Methods: The gene sequence of EMC6 in Pub Med was searched. Primers were designed for this sequence. The total RNA of L-02 cells was extracted. The target fragment was amplified by polymerase chain reaction (PCR) The eukaryotic expression vector pcDNA3.1 (+) / EMC6 was constructed in the nuclear expression vector pcDNA3.1, and the accuracy of the construction was verified by colony PCR and sequencing. The recombinant vector was transfected into L-02 cells by lipofection method. The mRNA and protein expression of EMC6 gene was detected by RT-PCR and Western blot. Results: PCR and gene sequencing results showed that pcDNA3.1 (+) / EMC6 recombinant plasmid was successfully constructed. The results of RT-PCR and Western blot confirmed that EMC6 was overexpressed in human liver L-02 cells. Conclusion: The pcDNA3.1 (+) / EMC6 recombinant plasmid was successfully constructed and overexpressed in L-02 cells.