Expression in Escherichia coli of seven DNA fragments comprising the complete L1 and L2 open reading frames of human papillomavirus type 6b and localization of the 'common antigen' region.
Molecular cloning was used to express human papillomavirus type 6b (HPV-6b) antigens in Escherichia coli. Seven genomic DNA fragments of HPV-6b which together comprise the complete L1 and L2 open reading frames, known to code for capsid proteins, were cloned and expressed in E. coli as both beta-galactosidase and TrpE fusion proteins. Western blots of HPV-6b beta-galactosidase fusion proteins using 'genus-specific' antisera produced by immunization of rabbits with disrupted bovine papillomavirus type 1 (BPV-1) showed that polypeptides encoded by two DNA fragments from the mid portion of L1 of HPV-6b were cross-reactive. Only one of these two polypeptides reacted with antisera raised against disrupted HPV-1, directly demonstrating that this polypeptide contains the papillomavirus 'common antigen'. The cross-reactive region was confirmed by reversing antigen and antibody. Polyclonal antisera were raised against the seven HPV-6b beta-galactosidase fusion proteins and tested against BPV-1 virion proteins on Western blots. Only antiserum against the mid portion of L1 of HPV-6b reacted with the BPV-1 major capsid protein. HPV-6b fusion proteins were also used to test human sera for antibodies reactive in Western blots. Serum samples from 38 patients with documented HPV-6 infections and from 22 presumably uninfected controls were tested. Antibodies were not detected in any of the sera to any of the seven fusion proteins. HPV-6b beta-galactosidase fusion proteins are antigenic and can be used on Western blots to localize immunologically reactive sub-regions of proteins by reacting protein fragments with antisera from immunized animals. However, alternative methods will be required to detect anti-HPV antibodies in human sera.
Strike
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DG
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Bonnez
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Rose
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Reichman
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RC
D000328:Adult; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D015153:Blotting, Western; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics*; D003429:Cross Reactions; D004279:DNA, Viral / Q000235:genetics*; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology; D005786:Gene Expression Regulation; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D000914:Antibodies, Viral; D000942:Antigens, Bacterial; D000956:Antigens, Viral; D003062:Codon; D004279:DNA, Viral; D000939:Epitopes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase