Lu Dong-shui, Mao Xu-hu, Zou Quan-ming, Wu Chao, Yang Jun, Zhang Wei-jun, Wang Fu-kun, Xie Qing-hua, Luo Ping
Teaching and Research Section of Clinical Microbiology and Immunology, Department of Medical Laboratory, Third Military Medical University, Chongqing 400038, China.
Di Yi Jun Yi Da Xue Xue Bao. 2003 Jun;23(6):549-52.
To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein.
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.
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.
The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.
进行幽门螺杆菌（Hp）脲酶B亚基（UreB）的基因重组，并检测重组蛋白的生物学特性。
通过PCR从中国受试者临床分离编码Hp UreB的基因片段，随后克隆到表达载体pET-11C-UreB中，以在大肠杆菌BL21（DE3）菌株中进行非融合蛋白表达。
重组UreB在大肠杆菌BL21中实现表达，相对分子质量约为62,000，表达率为26%，重组UreB的前15个氨基酸为MKKISREYVSMYGP。肽图分析和氨基酸组成分析结果与先前的理论预测一致，酶联免疫吸附测定和蛋白质印迹表明重组蛋白在BalB/c小鼠中具有强免疫原性和反应性，可被BalB/c小鼠抗Hp多克隆血清或感染Hp的人血清特异性识别。
本研究结果为将重组UreB作为抗Hp亚单位疫苗成分奠定了坚实的免疫学基础。
