Abstract: Helicobacter pylori (Hp) is associated with the development of chronic gastritis, peptic ulcer and gastric cancer. Epitope vaccine based on the enzyme urease of Hp is a promising option for prophylactic and therapeutic vaccination against Hp infection. An epitope vaccine CTB-UA composed of cholera toxin B subunit (CTB) and an epitope peptide named U21 from urease A subunit was constructed by analyzing the coupling sequence and linker between CTB and U21 using bioinformatics software. The recombinant expression vector pETCUA containing the fusion gene CTB-UA and its recombinant strain were constructed by gene cloning technology. After protein expression and optimization, the recombinant protein CTB-UA was purified by Ni^2+-charged column chromatography and anion-exchange chromatography using DEAE sepharose FF, about 51 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of CTB-UA was 94.8%. The activity of epitope vaccine CTB-UA was investigated by intraperitoneal immunization experiments in BALB/c mice. Mice immunized with CTB-UA using aluminum hydroxide adjuvant could induce high level of antibodies specific for both CTB and U21 by ELISA. The epitope vaccine CTB-UA with a scientific and reasonable structure was expressed at a medium level in E. coli and had good immunological specificity. This study will provide much experimental evidences for the development of epitope vaccines against Hp for human use.

Key words: Helicobacter pylori, urease, epitope

摘要： 幽门螺旋杆菌(Hp)是慢性胃炎、消化性溃疡和胃癌的重要致病因子。研发基于Hp尿素酶的表位疫苗是一种很有前景的防治Hp感染的策略。本研究主要通过生物信息学软件对Hp尿素酶表位肽U21和霍乱毒素B亚基(CTB)的连接顺序和间隔序列加以分析,设计出一种由U21和CTB构成的Hp表位疫苗CTB-UA。通过基因克隆技术构建含有融合基因CTB-UA的重组表达载体pETCUA及其重组菌株;重组菌株经经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化融合蛋白CTB-UA,获得高纯度(94.8%)的融合蛋白CTB-UA。并进一步通过腹腔注射免疫BALB/c小鼠,鉴定Hp表位疫苗CTB-UA的免疫学活性,经间接ELISA鉴定小鼠能够产生针对CTB和表位肽U21的高滴度特异性抗体。结果证明,Hp表位疫苗CTB-UA具有科学合理的结构,能在大肠杆菌表达系统中获得较高水平的表达,且具有较高的免疫学特异性,为研发防治Hp感染的表位疫苗奠定一定的实验基础。

关键词: 幽门螺旋杆菌, 尿素酶, 表位