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目的构建戊型肝炎病毒(Hepatitis E virus,HEV)RdRp基因真核表达质粒,并检测其在PLC/PRF/5、A549和HepG2 3种细胞中的表达,为后续RdRp的研究提供实验依据。方法利用RT-nPCR法从阳性HEV粪便中扩增RdRp基因片段,插入pcDNA3.0真核表达载体中,并在其3′端插入EGFP报告基因,构建融合表达质粒pcDNA3.0-RdRp-EGFP,脂质体法转染PLC/PRF/5、A549和HepG2细胞,荧光显微镜观察报告基因的表达,RT-nPCR检测RdRp基因mRNA的转录情况,Westernblot检测RdRp蛋白的表达。结果重组表达质粒经酶切鉴定和测序证实构建正确;RdRp基因和蛋白在A549和HepG2细胞中可高效、特异性表达。结论成功构建了RdRp基因真核表达质粒,并在A549和HepG2培养细胞中大量表达,为进一步研究RdRp在HEV复制过程中的功能奠定了基础。
Objective To construct eukaryotic expression plasmid of RdRp gene of Hepatitis E virus (HEV) and to detect the expression of RdRp gene in PLC / PRF / 5, A549 and HepG2 cells. Methods RdRp gene fragment was amplified from feces of positive HEV by RT-nPCR and inserted into pcDNA3.0 eukaryotic expression vector. The EGFP reporter gene was inserted into its 3 ’end to construct the fusion expression plasmid pcDNA3.0-RdRp-EGFP. The expression of RdRp gene mRNA was detected by fluorescence microscopy and RdRp protein was detected by RT-PCR. The expression of RdRp protein was detected by Western blot. Results The recombinant plasmid was confirmed by restriction enzyme digestion and sequencing. The RdRp gene and protein were efficiently and specifically expressed in A549 and HepG2 cells. Conclusion The eukaryotic expression plasmid RdRp was successfully constructed and expressed abundantly in A549 and HepG2 cells, which laid the foundation of further study on the function of RdRp in HEV replication.