OVEREXPRESSION AND CHARACTERIZATION OF THE CDNA-ENCODING THE COAT PROTEIN OF CUCUMBER MOSAIC-VIRUS (STRAIN ABI) ISOLATED IN KOREA

We constructed a recombinant CMVCP expression vector termed pMALCMV in which cDNA fragment encoding CMVCP is ligated into pMAL-c2, an E. col i expression vector. Overexpression of pMALCMV containing the entire o pen reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E. coli TB1 cells, which resulte d in the production of a fusion protein of MBP-CMVCP (M-r 67.7 kDa) th at was immunoprecipitable with rabbit polyclonal antiserum specific fo r MBP, The CMVCP (M-r 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purifie d fusion protein with Factor Xa. The partial amino acid sequences of t he cleaved proteins were confirmed at the amino terminus by peptide se quencing. The CMVCP antiserum was also prepared by intraperitoneal inj ection of this purified CP into a BALB/c mouse, Immunoblot analysis sh owed that the purified CMVCP from the Factor Xa cleavage reaction was an authentic overexpression product of the cloned CMVCP. Using an RNA mobility shift assay, it was demonstrated that CMVCP can bind to its o wn RNA transcript in a concentration dependent manner. However the com plex formed between CMVCP and its RNA was abolished by the addition of a polyclonal antibody that had been raised against CMVCP, confirming that the overexpressed CMVCP specifically interacts with its own RNA. Thus, our results earn provide a basis for the development of a hybrid oma cell line expressing the monoclonal antibody for CMVCP and molecul ar cloning of their genes, which may lead to the creation of CMV-resis tant transgenic plants.