Expression of the recombinant coat protein of Potato virus X in Escherichia coli

Abstract

Objective Potato virus X (PVX) is a member of the genus Potexvirus in the family Alfaflexiviridae. This virus is one of the most common and widespread viruses infecting potato worldwide. Due to the wide distribution and economic damage of the virus in Iran, identification and detection of the virus is necessary using non-expensive methods. Production of polyclonal antibody through molecular approaches can be a useful method for detection of virus in the nature. Materials and methods In this research, an infected potato sample was collected from Zarand (Kerman province) and its PVX infection was confirmed by ELISA test. This sample was inoculated on test plant and the coat protein (CP) gene of this isolate was amplified in RT-PCR test with specific primers comprising BglII and NcoI restriction enzymes. Amplified product was cloned into expression prokaryotic vector (pQE60 plasmid). followed by transformation of the E. coli strain M15 competent cells. Results Subsequent to induction of CP expression, total protein was extracted and run onto 12.5% SDS-PAGE. An approximate 24 kDa band was observed on the gel corresponded to the PVX coat protein. Furthermore, expression of the PVX CP was confirmed by dot blot technique. Conclusions In this study, a recombinant antigen suitable for the detection of potato X virus was prepared which was well identified by antiserum produced by injection of complete virus particles into rabbits in dot blot analysis. The production of multiclonal antibodies by recombinant viral antigen will be an important step in accelerating and facilitating the serological identification process of plants infected with this virus.

Keywords

• viral coat protein
• e. coli
• recombinant protein expression
• potato virus x