We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.
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机译:我们之前报道过一种特异于幽门螺杆菌脲酶的小鼠单克隆抗体(MAb),称为L2,强烈抑制其酶促活性。在这里,为了深入了解该抗体如何影响脲酶活性,确定了该抗体识别的表位。通过筛选覆盖两个亚基(UreA和UreB)整个序列的一组重叠的合成肽,我们鉴定了一段UreB衍生的19个氨基酸(aa)残基(UB-33; aa 321至339,CHHLDKSIKEDVQFADSRI),被L2抗体特异性识别。从任一端对19-mer肽的进一步连续氨基酸删除,使我们能够确定最小表位为L2反应性的8个氨基酸残基(F8; SIKEDVQF)。该表位似乎恰好位于短序列上,该短序列在尿素酶的活性位点上形成了一个瓣,表明L2抗体的结合在空间上抑制了尿素(尿素酶的底物)的进入。最后,用19-mer肽或8-mer最小表位免疫兔子会产生抗尿酶抗体,该抗体能够抑制酶活性。由于脲酶对于幽门螺杆菌的毒力至关重要,因此诱导抗体产生以抑制其酶活性的抗原肽可能作为预防和治疗幽门螺杆菌感染的疫苗可能是有用的工具。
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