一种全长扩增克隆HEV方法和HEV体外瞬时转染体系的建立

Full-length hepatitis E virus amplification and cloning and establishment of transient transfer system in vitro

目的建立HEV全长基因组扩增克隆和全长HEV RNA体外瞬时转染细胞方法和初步应用。方法取5例抗HEV IgG阳性患者血清,采用特异性引物逆转录HEV RNA为cDNA,设计通用的分段且相互重叠的PCR引物分步扩增,然后再分别将PCR产物进行重组PCR扩增,最终获得全长的HEV RNA cDNA产物片段。测序确定其序列,采用T-A克隆全长HEV cDNA序列,将HEV全长cDNA序列进行体外转录,提取转录后产物的RNA(去除DNA模板),获得高水平的HEV RNA产物,采用脉冲电流转染HepG2细胞,转染后24 h、48 h和96 h观察细胞上清HEV抗原分泌和HEV RNA载量。结果自Gen Bank获得约300条HEV全长基因组序列,设计了分段重组PCR方法,快速获得了HEV基因组全长序列;T-A克隆HEV全长基因组测序分析其序列,体外转录制备高纯度的HEV mRNA,通过电转方式转入HepG2细胞,在较短时间内检测出HEV RNA,有效地评价了HEV的体外表型。HEV抗原要先于HEV RNA出现;体外实验显示Ⅰ型和Ⅳ型HEV复制能力无明显差别,都在转染后48 h达到峰值。结论建立的分段重组PCR法扩增HEV全长基因组,体外电转细胞系后分泌HEV RNA,为后续HEV分子病毒学和表型耐药研究提供了条件。

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 HepG： cells in vitro,which might be useful for the subsequent study of HEV molecular virology and phenotypic resistance research.