关键词: 戊型肝炎病毒, HepG₂细胞, 全长扩增, 瞬时转染, 体外

Abstract: Objective To establish a HEV genome amplification and cloning method, and the full-length HEV RNA transient transfer cells in vitro. Methods Sera from five patients with serum anti-HEV IgG positive were obtained. After reverse transcription of HEV RNA with specific primers for its cDNA was finished, we designed general segmentation and overlapping PCR primers for further amplification step by step, and then we restructured amplified PCR product respectively,and finally obtained the total full-length HEV RNA cDNA fragments. The sequences were determined by cloning sequencing. The HEV full-length cDNAs were transcribed in vitro, extracted the transcription products of RNA （removal of DNA template） to obtain a high concentration of full-length HEV RNA products, transfected HEV RNA into HepG₂ cells by pulse current,and the secretion of HEV antigen and HEV RNA in supernatants were assayed 24 h,48 h and 96 h after transfection. Results We searched about 300 HEV genome sequences from GenBank and designed the segmentation of recombinant PCR method, which could quickly get over HEV genome sequence;the sequences were determined by T-A clone sequencing; Preparation of high purity of transcription HEV mRNA in vitro was success,and we evaluated the HEV phenotype effectively in vitro;HEV antigen in supernatants was detected positive before HEV RNA showed up; There was no obvious difference between HEV type I and type IV replication in vitro,and both of them peaked up at 48 h after transfection. Conclusion We successfully establish a full-length HEV RNA transient transferred HepG₂ cells in vitro,which might be useful for the subsequent study of HEV molecular virology and phenotypic resistance research.

Key words: Hepatitis E virus, HepG₂ cells, Full length amplification, Transient transfection, In vitro