Molecular genetic studies on citrus psorosis virus

Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV) the type species of the genus ophiovirus, is the putative causal agent of psorosis disease. In this study CPs V was detected from naturally citrus cv. Navel orange trees showing psorosis virus like symptoms. CPsV was detected serologically using monoclonal antibodies (MAbs) specific to CPsV by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and then the molecular detection by reverse transcription-polymerase chain reaction (RT -PCR) confirmed the serological results. The full length of cp gene fragment was amplified and cloned into pGEM- T easy vector and the new created plasmid that named pRl (pGEM-T + CPsV cp gene) was transferred to E. coli strain JMIO9. A large scale production of the recombinant DNA plasmid was prepared for sequencing that determined the insert by 1323 bp. DNA sequence results and deduced amino acids were used for alignment in the GENBANK database and results confirmed that the determined sequence belonging to CPsV-cp gene. The cp gene fragment was sub-cloned into p, T-30a(+) expression vector to create new plasmid that named pR2 (p, T-30a(+) + CPsV cp gene) which transformed in E .coli strain BL21. Expression of the cp gene induced by IPTG and the presence of the target protein confirmed by rapid screening and time course analysis, then the affinity chromatography technique was used to purify the expressed protein (CPsV-CP). Then the specificity of the purified expressed protein was determined via DAS-ELISA using the MAbs specific to the Italian isolate of CPsV. Some properties of the purified expressed protein were determined using the DNA star software package (Expert Sequence Analysis Software, USA). Then the purified expressed protein (CPsV-CP) was used in injection of two white mice for production of antiserum and the raiesd polyclonal antibodies (P Abs) were evaluated via western blotting analysis and triple antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA).