Abstracto

Expression of Cucumber mosaic cucumovirus coat protein in Escherichia coli and production of specific polyclonal antiserum

Ali. M.El-Borollosy, Sabah M.Hassan

Cucumber mosaic cucumovirus (CMV) was isolated from naturally infected cucumber plants serologically depending on indirect enzyme-linked immunosorbant assay (I-ELISA) and biologically by mechanical inoculated on Chenopodium amaranticolor as a local lesions host and maintained on Nicotianatabacum cv. (White Burley). Infected tobacco plant tissue used to perform immunocapture reverse transcriptase polymerase chain reaction (ICRT- PCR) to isolate the viral coat protein gene (cp). The agarose gel analysis of the PCR product indicated a single band with 657 bplength, which is the expected size for the CMV cp. The isolated CMV cp gene has been ligated into the PinPoint™ Xa-1 T-Vector depending on the T-A cloning principle. The new plasmids were transformed into competent Escherichia coli cells, further the gene integration success and orientation were screened by isolation of plasmids from transformed bacteria and treatment with BglII and BamHI restriction enzymes. It was found that total plasmid length was 3331 bp without insert, while fragment separated after restriction digestion was approximately 660 bp. The PCR product of the minipreparation indicated a single band with 657bp which is the expected size for the CMV cp gene.After induction of CMV coat protein (CP) expression using Isopropyl â-D-1-thiogalactopyranoside (IPTG), transformed bacteria lysate analyzed using SDS-PAGE revealed a band with amolecularweight of 25KDa (the expected of CMVCPmolecularweight). Western blotting analysis was carried out and confirmed that all the bands expected for viral CP react positively with CMV antiserum. The induced viral CP was purified from E. coli transformed cells using regenerated SoftLink™ Soft Release Avidin Resin yielding about 11 mg per 1 liter culture. Polyclonal antiserum produced in a New Zealand white rabbit by injecting of the produced fusion protein and Immunoglobulins G (IgGs) were purified from the collected antiserum. IgGs titration was performed using I-ELISA, the highest titers were 1:512 & 1:256 for antisera produced using fusion protein and purified virus, respectively. It was clearly observed that purified antiserum of E. coli viral fusion protein was more reactive and specific as it did not have any cross reactions with healthy tobacco plant sap.