Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library.
Lpp20, an outer membrane protein of Helicobacter pylori (H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli (E. coli) as a recombinant fusion protein with glutathione-S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114-117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.
Li
Yan
Y
School of Biotechnology, Southern Medical University, Guangzhou, Guangdong Province, 510515, PR China.
Ning
Yun-shan
YS
Wang
Yun-dan
YD
Hong
Yan-hua
YH
Luo
Jun
J
Dong
Wen-qi
WQ
Li
Ming
M
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D007074:Immunoglobulin G / Q000276:immunology; D008074:Lipoproteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D014611:Vaccination
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D007074:Immunoglobulin G; D008074:Lipoproteins; C089630:Lpp20 protein, Helicobacter pylori; D019151:Peptide Library; D011993:Recombinant Fusion Proteins