重组人幽门螺杆菌尿素酶B亚单位及其生物学性质

Recombinant Helicobacter pylori urease B subunit and its biological properties

目的基因重组人幽门螺杆菌(Hp)尿素酶B亚单位(UreB)并分析重组蛋白的生物学性质。方法采用PCR从我国临床分离的Hp菌株中克隆UreB基因,并通过DNA重组技术构建非融合表达UreB的重组质粒pET-11C-UreB,转化至大肠杆菌BL21(DE3),经IPTG诱导表达。结果重组UreB在大肠杆菌中表达,相对分子质量约62 000,表达率26%,N端15个氨基酸序列为MKKISRKEYVSMYGP,肽图及氨基酸组成成分分析结果与理论预测值吻合。以重组UreB免疫BalB/c小鼠,能产生抗UreB抗体。ELISA及Western blotting分析显示,重组UreB具有良好的免疫原性和反应性,表现出与天然Hp UreB相似的生物学功能。结论为重组UreB作为Hp亚单位疫苗成分奠定了免疫学基础。

Objective To perform genetic recombination of the urease B subunit (UreB) of Helocobacter pylori(Hp) and examine the biological properties of the recombinant protein. Methods The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain. Results The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62 000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP. The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp. Conclusion The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.