Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.

2.50
Hdl Handle:
http://hdl.handle.net/10541/108903
Title:
Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.
Authors:
Stacey, Simon N; Bartholomew, Jennifer S; Ghosh, Anna K; Stern, Peter L; Mackett, Mike; Arrand, John R
Abstract:
Existing assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.
Affiliation:
Cancer Research Campaign Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K.
Citation:
Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera. 1992, 73 ( Pt 9):2337-45 J. Gen. Virol.
Journal:
Journal of General Virology
Issue Date:
Sep-1992
URI:
http://hdl.handle.net/10541/108903
DOI:
10.1099/0022-1317-73-9-2337
PubMed ID:
1328490
Type:
Article
Language:
en
ISSN:
0022-1317
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorStacey, Simon Nen
dc.contributor.authorBartholomew, Jennifer Sen
dc.contributor.authorGhosh, Anna Ken
dc.contributor.authorStern, Peter Len
dc.contributor.authorMackett, Mikeen
dc.contributor.authorArrand, John Ren
dc.date.accessioned2010-08-03T10:03:31Z-
dc.date.available2010-08-03T10:03:31Z-
dc.date.issued1992-09-
dc.identifier.citationExpression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera. 1992, 73 ( Pt 9):2337-45 J. Gen. Virol.en
dc.identifier.issn0022-1317-
dc.identifier.pmid1328490-
dc.identifier.doi10.1099/0022-1317-73-9-2337-
dc.identifier.urihttp://hdl.handle.net/10541/108903-
dc.description.abstractExisting assays to detect antibodies to human papillomavirus type 16 (HPV-16) proteins in sera from cervical carcinoma patients rely primarily on bacterially produced recombinant proteins or synthetic peptides for use as target antigens. These methods have had limited success in the detection of antibodies against the E6 protein. To produce more authentic E6 protein for use in serological assays, we have employed a recombinant baculovirus vector to synthesize the protein in insect cells. Cells infected with the vector containing E6 gene sequences expressed a stable protein doublet comprising 18.5K and 19.1K bands. This protein reacted in Western blots with an antiserum raised against a purified E6 fusion protein produced in Escherichia coli. This antiserum, and several others raised against E. coli-derived E6 fusion proteins, were unable to recognize the baculovirus E6 protein in radioimmunoprecipitation assays (RIPAs). However, serum from a cervical carcinoma patient readily immunoprecipitated the baculovirus E6 protein, suggesting that the baculovirus-derived protein represented a realistic antigenic target. A RIPA was developed for the detection of anti-E6 protein antibodies in human sera. The assay was tested on a selected group of sera from carcinoma patients and controls, in comparison with a Western blotting method using bacterial fusion proteins. The baculovirus E6 protein-based RIPA showed a marked increase in detection rate over the Western blotting method. These findings suggest that serum antibodies to HPV-16 E6 protein may be more prevalent than has previously been shown.en
dc.language.isoenen
dc.subjectTumour Virus Infectionsen
dc.subjectUterine Cervical Canceren
dc.subject.meshAntibodies, Viral-
dc.subject.meshAntigens, Viral-
dc.subject.meshBaculoviridae-
dc.subject.meshBase Sequence-
dc.subject.meshCarcinoma-
dc.subject.meshFemale-
dc.subject.meshHumans-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshOncogene Proteins, Viral-
dc.subject.meshPapillomaviridae-
dc.subject.meshPrecipitin Tests-
dc.subject.meshRadioimmunoassay-
dc.subject.meshRecombinant Proteins-
dc.subject.meshRepressor Proteins-
dc.subject.meshTumor Virus Infections-
dc.subject.meshUterine Cervical Neoplasms-
dc.titleExpression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.en
dc.typeArticleen
dc.contributor.departmentCancer Research Campaign Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K.en
dc.identifier.journalJournal of General Virologyen
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