عنوان مقاله [English]
Background and Objectives
Beet curly top Iran virus (BCTIV) and Beet curly top virus (BCTV) are the causal agents of beet curly top disease in Iran. It has been reported that both viruses are distributed in different geographical regions and host plants in Iran. Development of a simple, fast and low cost serological method such as ELISA for detection of these viruses, is a necessary demand. To do serological tests such as ELISA, an antibody is needed. Production of polyclonal antibodies against geminiviruses is a hard candidate because of low concentration and localization of their particles in the phloem tissue of their hosts. Therefore, expression and production of geminiviral recombinant proteins in bacterial system is an alternative method to obtain polyclonal antibodies.
Material and Methods
Specific forward and reverse primers containing BamHI/HindIII digestion sites at the 5′ end of each primer, respectively, were designed to amplify the full-length fragment of BCTIV V1 open reading frame encoding the viral coat protein (CP). The amplified 751 bp fragment was cloned into the pTZ57R/T cloning vector. V1 gene was released from pTZ57R/T by enzymatic digestion and cloned into the expression vector pQE30. The recombinant pQE30-BCTIV V1 was transformed into Escherichia coli strain M15. The expression of protein was induced by adding 1 mM of isopropylthio-d-galactoside (IPTG) to the transformed bacterial cells and grown at 37 ºC in liquid LB medium. Expression of recombinant protein was analyzed by SDS-PAGE electrophoresis and Western blot hybridization.
Analysis of the expressed proteins by SDS-PAGE electrophoresis, revealed a protein band with a position corresponding to molecular weight of approximately 30 kDa that was consistent with the predicted molecular weight of BCTIV CP.Time course of induction by IPTG influenced on level of the new recombinant protein expression, thus four hours induction had the highest level of expression. Western blot analysis confirmed the identity of expressed protein via using anti-His antibody to detect expressed recombinant His-BCTIV CP.
One of the most popular bacterial protein expression system is IPTG inducible system wherein IPTG regulate level of new recombinant protein expression. Our results by SDS-PAGE electrophoresis and Western blot analysis revealed a high level of expression of the fusion protein in IPTG-induced bacterial cells. However, a negligible rate of expression of recombinant protein was also observed in non-induced bacterial cells. This suggests low appearance and activity of responsible promotor even without inducing protein expression with IPTG. Expression of conserved plant viral proteins in bacterial systems following purification of the resulted recombinant proteins would facilitate production of specific polyclonal antibodies.