Expression of coat protein of an ordinary strain of potato virus Y in Escherichia coli and production of polyclonal antibodies for diagnosis

Abstract

The coat protein gene (CP) of an ordinary strain of Potato virus Y (PVYO) was cloned into the expression vector, pET-28a(+). The insert was sequenced and analysis showed that the CP gene was in frame with intact N-terminal 6X histidine tags. An approximately 35 kDa recombinant fusion protein was observed in inclusion bodies of induced Escherichia coli BL21 cells. This fusion protein was purified and used as antigen to raise polyclonal antibodies in rabbits. In Western blot and dot blot immuno-binding assay (DIBA), both PVYO-CP IgG and PVYO IgG strongly reacted with the recombinant CP. The PVYO-CP IgG could detect PVYO in infected samples up to 1 : 3200 dilutions. A PVYO-CP ELISA kit was prepared and compared with conventional ELISA kit based on purified virus particles (PVYO ELISA kit). The PVYO-CP ELISA kit consistently detected the PVYO in DAS-ELISA of field samples and was as effective as PVYO ELISA kit. © 2013 Blackwell Verlag GmbH.