Expression of human papillomavirus type 16 E6 protein by recombinant baculovirus and use for detection of anti-E6 antibodies in human sera.
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.
Stacey
S N
SN
Cancer Research Campaign Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K.
Bartholomew
J S
JS
Ghosh
A
A
Stern
P L
PL
Mackett
M
M
Arrand
J R
JR
D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002277:Carcinoma / Q000382:microbiology; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics*; D011233:Precipitin Tests; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012097:Repressor Proteins; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D002583:Uterine Cervical Neoplasms / Q000382:microbiology
D000914:Antibodies, Viral; D000956:Antigens, Viral; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins