Nucleotide sequence and expression in E. coli of the complete P4 type VP4 from a G2 serotype human rotavirus.
The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 in E. colic has not been achieved. In this study we present successful expression in E. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.
Mahajan
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NP
Department of Microbiology and Cell Biology. Indian Institute of Science, Bangalore, India.
Rao
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CD
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D036022:Capsid Proteins; D002417:Cattle; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005814:Genes, Viral; D000882:Haplorhini; D006385:Hemagglutination Inhibition Tests; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012330:RNA, Double-Stranded / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012401:Rotavirus / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012703:Serotyping; D012756:Sheep; D013045:Species Specificity; D013552:Swine
D036022:Capsid Proteins; D017931:DNA Primers; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus