Screening Of Proteins Interacting With The Enterovirus713A Protein From T7-phage Display Library

Human Enterovirus71(EV71) is the main causative agent of hand, foot and mouth disease (HFMD). The viral genome RNA contains a single open reading frame coding for four structural proteins and seven non-structure proteins (2A-2C and3A-3D). The multiple functioning protein3A, which is essential for EV71replication, causes the intracellular membrane alterations, is involved in the formation of viral factory and inhibits anterograde traffic from the ER to the Golgi apparatus. The3A consists of two obvious domains. The C terminal hydrophobic domain is responsible for its direct membrane association. The N terminal of3A has several functional sub-domains, but host interacting proteins of which are barely known.This study tries to screen the host interacting protein of3A using T7phage display technology. The coding sequence of3A N terminal60aa obtained by PCR amplification and was inserted into pET28a(+) prokaryotic expression vector and transformed into Escherichia coli DH5a. The expression vector was transformed into E. coli BL21and the expression of recombinant His-3A-60aa protein was induced by IPTG and identified by SDS-PAGE electrophoresis and Western Blot. Then the His-3A-60aa protein was purified by Talon Resin Column. The purified protein was used as a selecting protein to bio-pan the T7select human lung cDNA library. After4rounds of bio-panning,19selected positive clones were sequencing and ribosomal protein L27a, ATP synthase FO subunit6, ferritin light chain were identified using homologous analysis, possibly to interact with3A protein These results will be useful for further studying the function mechanism of3A and pathogenic mechanism of EV71.