医学分子生物学杂志2017，Vol.14Issue(6)：317-322,6.DOI:10.3870/j.issn.1672-8009.2017.06.002
戊型肝炎病毒ORF3及其截短突变体真核表达载体的构建及其在A549细胞中的表达
Construction of Eukaryotic Expression Vectors for Hepatitis E Virus ORF3 and Its Truncation Mutant Genes and Their Expression in A549 Cells
摘要
Abstract
Objective To construct the eukaryotic expression vectors for hepatitis E virus (HEV)ORF3 and its truncation mutant genes, and examine their expression in human lung cancer A549 cells.Methods HEV ORF3 and its truncation mutant genes were amplified from PUC-HEV by RT-PCR, and inserted into the eukaryotic expression vector pcDNA3.1 with a Flag reporter in the downstream.The recombinant plasmids pcDNA3.1-ORF3/Flag, pcDNA3.1-△D1-ORF3/Flag, pcDNA3.1-△D2-ORF3/Flag and pcDNA3.1-△P2-ORF3/Flag were transfected into A549 cells withthe mediation of Lipofectamine 2000 transfection reagent.The expression ofproteins encoded by ORF3 and its truncation mutants were detected by Western blotting.Results Restriction analysis and sequencing proved that the eukaryotic expression vectors pcDNA3.1-ORF3/Flag, pcDNA3.1-△D1-ORF3/Flag, pcDNA3.1-△D2-ORF3/Flag and pcDNA3.1-△P2-ORF3/Flag were construc-ted correctly.A549 cells were transfected with the vectors, and harvested after 24 h.The proteins encoded by ORF3 and its truncation mutants were detectable by Western blotting.Conclusion The eukaryotic expression vectors for ORF3 and its truncation mutants were successfully constructed and expressed in A549 cells, which laid a foundation to further study the function of HEV ORFs and its pathogenic mechanism.关键词
戊型肝炎病毒/ORF3/截短突变/A549肺癌细胞/基因表达Key words
hepatitis E Virus/ORF3/truncation mutant/A549 cells/gene expression分类
生物科学引用本文复制引用
郭映秋,李饴,段素琴,王俊斌,禹文海,杨凤梅,赵远,马进,和占龙..戊型肝炎病毒ORF3及其截短突变体真核表达载体的构建及其在A549细胞中的表达[J].医学分子生物学杂志,2017,14(6):317-322,6.基金项目
中国医学科学院医学创新工程(CIFMS, No.2016-12 M-2-001)This work was supported by a grant from the CAMS Innovation Fund for Medical Science(CIFMS, No.2016-12 M-2-001) （CIFMS, No.2016-12 M-2-001）
