Antibody responses to HPV6b E polyproteins and production of monoclonal antibodies.
A range of fusion constructs (expressed in Escherichia coli) were produced that contained two or more HPV6b E proteins, producing a single continuous amino acid sequence corresponding to the sequences of the individual E proteins. The constructs also included a C-terminal hexahistidine tag fused in-frame to aid purification. The fusion proteins (polyproteins) were semipurified by Ni(++) metal affinity chromatography under denaturing conditions. Immunization of BALB/c mice with these polyproteins resulted in the production of specific E protein antibodies. The draining lymph nodes from these mice were used to produce monoclonal antibodies (MAbs). The specificity of the polyclonal and MAbs was confirmed by immunoblotting and by screening for reaction with a series of synthetic peptides of E proteins. HPV E polyproteins were found to be immunogenic and immunization with the polyproteins resulted in specific antibody responses to the component E proteins.
Pietrzykowski
E
E
Limited, Research & Development, 45 Poplar Road, Parkville, Victoria 3052, Australia. Elxbieta_Pietrzykowski@csl.com.au
Margetts
M B
MB
MacFarlan
R I
RI
Webb
E A
EA
Cox
J C
JC
Edwards
S J
SJ
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006825:Hybridomas; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008198:Lymph Nodes; D051379:Mice; D008807:Mice, Inbred BALB C; D009532:Nickel / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000276:immunology*; D027383:Papillomaviridae / Q000737:chemistry; D020815:Polyproteins / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D000596:Amino Acids; D000911:Antibodies, Monoclonal; C085208:E7 protein, Human papillomavirus 6b; D009856:Oncogene Proteins, Viral; D020815:Polyproteins; D011993:Recombinant Fusion Proteins; D009532:Nickel