[Construction of Recombinant Full-length Hepatitis E Virus Fused with EGFP and Assessment of Infectivity].

Abstract

The purpose of this study was to construct recombinant full-length hepatitis E virus(HEV)fused with enhanced green fluorescent protein(EGFP),and assess its infectivity in A549 cells. Two fragments from the full-length HEV genome and the EGFP gene were amplified by PCR. The EGFP gene was inserted downstream of the HEV ORF2 and then cloned into the pGEM® -7Zf(+)vector containing the T7 and SP6RNA polymerase promoters, producing pGEM-HEV-EGFP. The construction of the pGEM-HEV-EGFP recombinant plasmid was confirmed by restriction enzyme digest and sequencing. The pGEM-HEV-EGFP recombinant plasmid was transfected into A549 cells to assess infectivity using Lipofectamine. EGFP expression was observed at 24hpost-transfection,and expression of the HEV ORF2 was detected by immunofluorescence, confirming the presence of the HEV ORF2 and EGFP fusion protein. Cytopathic effects were observed at day seven post-transfection. The infectivity of pGEM-HEV-EGFP was confirmed by the presence of fluorescence after three continuous passages. The recombinant pGEM-HEV-EGFP vector was successfully constructed and effectively infected A549 cells, which will facilitate future studies on the mechanisms of HEV infection and pathogenesis.