Screening and identification of mimotope of gastric cancer associated antigen MGb1-Ag

AIM: Using a monoclonal antibody against gastric cancer antigen named MGbl to screen a phage-displayed random peptide library fused with coat protein plII in order to get some information on mimotopes.lV^37BODS: Through affinity enrichment and EUSA screening,positive clones of phages were amplified. 10 phage clones were selected after three rounds of biopanning and the ability of specific binding of the positive phage clones to MGb1-Ab were detected by ELISA assay (DNA sequencing was performed and the amino acid sequences were deduced)By blocking test, specificity of the mimic phage epitopes was identified.RESULTS: There were approximately 200 times of enrichment about the titer of bound phages after three rounds of biopanning procedures. DNA of 10 phage clones after the third biopanning was assayed and the result showed that the positive clones had a specific binding activity to MGbl-Ab and a weak ability of binding to control mAb or to mouse IgG. DNA sequencing of 10 phage clones was performed and the amino acid sequences were deduced.According to the homology of the amino acid sequences of the displayed peptides, most of the phage clones had motifs of H(x)Q or L(x)S. And these 10 phage clones could also partly inhibit the binding of MGbl-Ab to gastric cancer cell KATO-Ⅲ. The percentage of blocking was from (21.0±1.6) %to (39.0±2.7) %.CONCLUSION: Motifs of H(x)Q and L(x)S selected and identified show a high homology in the mimic epitopes of gastric cancer associated antigen. There may be one or more clones which can act as candidates of tumor vaccines.

Helicobacter pylori specific immune response induced by conservative flagellin linear B-cell epitope

AIM:To testify the immunogenicity of a conservative B-cell linear epitope of Helicobacter pylori ( H pylori) flagellin A. METHODS: Different programs were used to analyze the secondary structure, molecular hydropathy, and surface accessibility of Hpyloriflagellin A. Linear B-cell epitopes were estimated based on the structural and physiochemical information. Analysis of residue divergence was proposed to screen a conservative linear epitope. The 29-peptide (Pep29mer) synthesized by chemical method, including the predicted conservative B-cell epitope and a known K^2d compatible T-cell epitope, was used to immunize mice, and then H pylori-specific antibodies were detected by ELISA. RESULTS: Based on the analyses of divergent amino acid residues, structural and physiochemical characteristics, it was strongly suggested that the short fragment NDSDGR was the core of a conservative linear epitope in flagellin A. Animals immunized by Pep29mer acquired efficient immune response. In detail, serum Hpylori-specific IgA and IgGl increased significantly in immunized group, while IgG2a only had an insignificant change. Hpylori-specific IgA in gastrointestinal flushing fluid also increased significantly. CONCLUSION: The conservative short fragment NDSDGR is the core of a linear B-cell epitope of flagellin A.