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. coli expression vector. Overexpression of pMALCMV containing the entire open reading frame of CMV cDNA sequence and the maltose binding protein (MBP) leader gene was facilitated in E. coli TB1 cells, which resulted in the production of a fusion protein of MBP-CMVCP (Mr 67.7 kDa) that was immunoprecipitable with rabbit polyclonal antiserum specific for MBP. The CMVCP (Mr 24.5 kDa) was isolated through a preparative SDS polyacrylamide gel following digestion of the affinity ligand purified fusion protein with Factor Xa. The partial amino acid sequences of the cleaved proteins were confirmed at the amino terminus by peptide sequencing. The CMVCP antiserum was also prepared by intraperitoneal injection of this purified CP into a BALB/c mouse. Immunoblot analysis showed 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 own RNA transcript in a concentration dependent manner. However, the complex 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 can provide a basis for the development of a hybridoma cell line expressing the monoclonal antibody for CMVCP and molecular cloning of their genes, which may lead to the creation of CMV-resistant transgenic plants.
Kim
Y M
YM
Department of Biochemistry and Bioproducts Research Center, Yonsei University, Seoul, Korea.
Kim
J H
JH
Kim
K H
KH
Hwang
Y S
YS
Paik
Y K
YK
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D017799:Cucumovirus / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007723:Korea; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C461599:maltose transport system, E coli