幽门螺杆菌粘附素基因保守区的克隆及免疫原性研究

Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori

目的 构建表达幽门螺杆菌 (Hp) 4种粘附素 (BabA2 、AlpA、AlpB和HopZ)保守区蛋白质的候选菌株 ,并研究其免疫原性。方法 利用PCR技术扩增粘附素保守区 (命名为CB)基因 ,将其定向插入pET 2 2b(+)载体 ,在BL2 1(DE3)大肠杆菌中表达 ;表达产物经亲和层析纯化和免疫印迹鉴定 ,ELISA法测定Hp感染者血清中抗CB抗体。结果 构建了 4种粘附素保守区的重组质粒 ,测序显示CB基因长 5 88bp ,编码 195个氨基酸。SDS PAGE和凝胶扫描分析 ,CB基因表达的蛋白质相对分子质量约为 2 2 5 0 0 ,其重组蛋白质表达量占菌体总蛋白质的 2 9%。经免疫印迹证实该重组蛋白质可以被Hp阳性患者血清所识别 ,ELISA法共检测了 5 5份血清 ,以快速尿素酶实验 (RUT)作为平行对照 ,两法的评价判断一致性程度的指标卡帕系数为 0 .76。结论 CB有可能成为一种有效的疫苗 ,用于幽门螺杆菌感染的预防和治疗。

Objective To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity. Methods The DNA of Hp was extracted. Primers were designed according to the C terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET 22b (+) and expressed in BL21 (DE3) strain of Escherichia coli . The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control. Results A recombinant plasmid pET 22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results. Conclusion CB has the potential to be used as a vaccine against Hp infection.