幽门螺杆菌UreB抗原表位预测及其有效表位噬菌体展示的筛选

Prediction of epitopes in urease subunit B of Helicobacter pylori and screening by phage display

目的 筛选幽门螺杆菌（Hp）尿素酶B亚单位（UreB）分子中的抗原表位，为研制多抗原肽（MAP）疫苗奠定基础。方法 采用生物信息学技术对UreB的T细胞和B细胞表位进行预测和分析。构建ureB基因原核表达系统，Ni-NTA亲和层析法提纯目的重组表达产物rUreB，常规皮内免疫法制备兔抗血清。选择UreB主要T细胞和B细胞联合表位肽并构建其噬菌体展示系统，PEG／NaCl沉淀法提纯重组噬菌体，SDS-PAGE鉴定目的重组PⅢ蛋白（rPⅢ）。分别以商品化抗跏全菌IsG和rUreB抗血清为一抗，采用Western blot对上述表位肽进行鉴定和筛选。结果 与GenBank中相关序列比较，所克隆的ureB基因核苷酸和氨基酸相似性分别为96％～99．5％和96％～100％。rUreB表达量约为细菌总蛋白的52％，提纯后仅见单一蛋白条带。预测的4个主要表位肽UreB230、UreB322、UreB479和UreB527在M13噬菌体中获得成功表达，采用不同一抗的Westernblot均显示相似的阳性结果，但UreB322和UreB527反应强度明显高于UreB230和UreB479。结论 本研究成功地构建ureB基因高效原核表达系统和UreB主要T细胞和B细胞联合表位肽噬菌体展示系统。所采用的生物信息学技术可有效地预测抗原表位。UreB322和UreB527是UreB有效抗原表位，可作为幽门螺杆菌MAP疫苗的候选表位。

Objective To screen the antigenic epitopes in urease subunit B（UreB） of Helicobacterpylori for laying the foundation for further development of multiple antigenic peptide （MAP） vaccine. Methods The epitopes of T and B cells in UreB molecule were predicted and analyzed using bioinformatic technique. A prokaryotic expression system was constructed. Ni-NTA chromatography was applied to extract the target recombinant ex- pression product rUreB and routine intracutaneously immunization was performed to prepare rabbit anti-rUreB serum. The major T and B combined epitope peptides of UreB were selected to construct their phage display systems. PEG/NaCl precipitation and SDS-PAGE were used to extract the recombinant phage and identify the target recombinant Pm （rPm） protein, respectively. By using commercial anti-whole H. pylori IgG or rUreB antiserum as the first antibody , Western blot assays were established to identify as well as to screen the epitope peptides . Results In comparison with the corresponding sequences in GenBank, the similarities of nucleotide and putative amino acid sequences of the cloned ureB gene were 96%-99.5% and 96%-100%, respectively. The output of rUreB was approximately 52% of the total bacterial proteins and the purified rUreB showed a single band in SDS-PAGE gel. The four major predicated epitope peptides UreB230, UreB322, UreB479 and UreB527 were successfully expressed in their recombinant M13 phages. Western blot assays using the different first antibodies displayed similar positive results and the reactive intensities in the assays of both UreB322 and UreB527 were remarkably stronger than those of UreB230 and UreB479. Conclusion A prokaryotic expression system of ureB gene with high efficieney and phage display system of the major UreB T and B combined epitope peptides are successfully constructed in this study. The applied bioinformatic techniques can be used to efficiently predict antigenic epitopes. UreB322 and UreB527 are confirmed to be the efficient antigenic epitopes of UreB that can be used as candidate epitopes for de- veloping MAP vaccine of H. pylori.