幽门螺杆菌尿素酶B基因的克隆表达及活性测定

Cloning,expression and functional analysis of the UreB gene from Helicobacter pylori

为研究幽门螺杆菌(Hp)尿素酶B(UreB)的免疫学活性,采用PCR方法扩增Hp UreB基因,经双酶切构建表达载体pET28a-UreB,转化大肠杆菌BL21(DE3)感受态细胞,经过IPTG诱导进行表达,用纯化后的重组蛋白免疫BALB/c小鼠,ELISA检测血清抗体效价,并使用感染患者血清进行Western-blot鉴定。结果显示,扩增的Hp UreB基因序列与GenBank中的26 695标准株序列相比较核苷酸序列同源性达到96.73%,氨基酸序列同源性高达99.30%,SDS-PAGE分析发现表达的重组蛋白分子质量约为66ku,以可溶性形式存在。Western-blot结果显示重组UreB能被Hp感染患者血清特异性识别,同时,用纯化后的UreB蛋白免疫小鼠能诱导产生特异性体液免疫应答,表明UreB具有很好的免疫原性。试验证明表达出具有生物活性的UreB蛋白,为Hp快速检测方法的研究奠定了基础。

In order to study the immunological activities of urease B subunit（UreB） from Helicobacter pylori（Hp）,UreB gene of Hp was amplified by PCR,digested with restriction enzyme and cloned into the plasmid pET-28a. The recombinant expression vector pET28a-UreB was transformed into Escherichia coli BL21（DE3） strains,and recombinant protein UreB was expressed by IPTG induction. BALB/c mice were immunized with purified recombinant UreB protein, and antibody titer was measured by ELISA. The immu- noreactivity of recombinant protein was identified by Western-blot. The results indicated that the homology of nucleotide sequence of the cloned UreB gene was not less than 96.73% in comparison with 26 695 stan- dard strain sequences in GenBank,while the homology of its putative amino acid sequence was 99.30~. SDS-PAGE analysis showed that the molecular weight of recombinant protein was about 66 ku and presen- ted in the supernatant. The purified recombinant protein could react with serum from patients infected by Hp in Western-blot, specific humoral immune response could be induced by pureffied protein,which showed that the recombinant protein had immunogenicity. The results showed that the recombinant protein UreB was expressed successfully and possessed strong immunogenicity,which will be contributed to the develop-ment of rapid detection methods for detection of Hp infection.