Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

摘要

Background: Cucumber mosaic virus (CMV) has isometric particles with a diameter of about 28 - 29 nm. Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost. Objectives: To this end, our aim was to express the CMV CP gene in E. coli to be used as the antigen for antibody production in the future. Materials and Methods: Coat Protein (CP) gene cDNA from an isolate (B13) of Cucumber Mosaic Virus (CMV) was subcloned from pTZ57RCMVCP to pET21a expression vector and transformed to E. coli strain Rosetta. Expression of CMV CP was successful and confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), wherein a ~30- kDa protein band was revealed. Induction by Isopropyl-Thiogalactoside (IPTG) at final concentrations of 0.5 to 2 mM appeared to produce similar results as to the amount of the expressed protein, which was judged by intensity of the band on SDS-PAGE. Results: The identity of the expressed protein was confirmed by immunoassays such as western blot, Dot-Immunobinding Assay (DIBA) and Enzyme-Linked Immunosorbent Assay (ELISA) by the use of anti-CMV antibody. Conclusions: This is the first report of expression of CMV CP gene in Iran, which is important for the preparation of anti-CMV antibody and paving the way for the use of the virus coat protein as a nanomaterial.