Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran.
School of Advanced Biomedical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
Appl Microbiol Biotechnol. 2018 Aug;102(16):6899-6913. doi: 10.1007/s00253-018-9068-4. Epub 2018 Jun 3.
Infection with Helicobacter pylori may result in the emergence of gastric adenocarcinoma. Among various toxins assisting pathogenesis of H. pylori, the vacuolating cytotoxin A (VacA) is one of the most potent toxins known as the major cause of the peptic ulcer and gastric adenocarcinoma. To isolate single-chain variable fragments (scFvs) against two conserved regions of VacA, we capitalized on the phage display technology and a solution-phase biopanning (SPB). Characterization of scFvs was carried out by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR). Bioinformatics analyses were also performed in order to characterize the structural and functional properties of the isolated scFvs and the interaction(s) between the isolated antibodies (Ab)-antigen (Ag). After four rounds of biopanning, the positive colonies detected by scFv ELISA were harvested to extract the plasmids and perform sequencing. Of several colonies, three colonies showed high affinity to the VacA1 and two colonies for the VacA2. Further complementary examinations (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, SPR, and flow cytometry) displayed the high affinity and specificity of the isolated scFvs to the VacA. Docking results revealed the interaction of the complementarity-determining regions (CDRs) with the VacA peptide. In conclusion, for the first time, we report on the isolation of several scFvs against conserved residues of VacA toxin with high affinity and specificity, which may be used as novel diagnostic/therapeutic tool in the H. pylori infection.
幽门螺杆菌感染可能导致胃腺癌的出现。在协助幽门螺杆菌发病的各种毒素中，空泡细胞毒素 A（VacA）是已知最有效的毒素之一，是消化性溃疡和胃腺癌的主要原因。为了分离针对 VacA 两个保守区域的单链可变片段（scFv），我们利用噬菌体展示技术和溶液相生物淘选（SPB）。通过酶联免疫吸附试验（ELISA）、免疫印迹和表面等离子体共振（SPR）对 scFv 进行了表征。还进行了生物信息学分析，以表征分离的 scFv 的结构和功能特性以及分离的抗体（Ab）-抗原（Ag）之间的相互作用。经过四轮生物淘选，用 scFv ELISA 检测到的阳性菌落被收获提取质粒并进行测序。从几个菌落中，有三个菌落对 VacA1 具有高亲和力，两个菌落对 VacA2 具有高亲和力。进一步的互补检查（例如，十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、western blot、SPR 和流式细胞术）显示了分离的 scFv 对 VacA 的高亲和力和特异性。对接结果显示 CDR 与 VacA 肽的相互作用。总之，我们首次报道了针对 VacA 毒素保守残基的几种具有高亲和力和特异性的 scFv 的分离，这可能作为一种新的诊断/治疗工具用于幽门螺杆菌感染。
