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Establishment of a Bacterial Expression System and Immunoassay Platform for the Major Capsid Protein of HcRNAV, a Dinoflagellate-Infecting RNA Virus.
Microbes Environ. 2012 Dec 7;27(4):483-9
Authors: Wada K, Kimura K, Hasegawa A, Fukuyama K, Nagasaki K
Abstract
HcRNAV is a small icosahedral virus that infects the shellfish-killing marine dinoflagellate Heterocapsa circularisquama, which harbors a dicistronic linear single-stranded RNA (ssRNA) genome ca. 4.4 kb in length. Its major capsid protein (MCP) gene sequence is not expressed by various strains of Escherichia coli, possibly because of a codon usage problem. To solve this problem, a chemically modified (i.e., de novo synthesized) gene was designed and cloned into the pCold-GST expression vector, and transformed into E. coli strain C41 (DE3), in which codon usage was universally optimized to efficiently express the polypeptide having the viral MCP amino acid sequence. The bacterially expressed protein, which was purified after a procedure involving denaturation and refolding, successfully formed virus-like particles that significantly resembled native HcRNAV particles. The purified, denatured protein was used as an antigen to immunize rabbits, and the resulting antiserum was shown to be strongly reactive to not only the bacterially expressed recombinant protein, but also to native HcRNAV MCP by Western blotting and dot immunoassays, respectively. These results indicate that an antiserum recognizing native HcRNAV MCP was successfully obtained using bacterially expressed HcRNAV MCP as the antigen.
PMID: 23047150 [PubMed - in process]
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