幽门螺杆菌尿素酶B亚单位基因克隆表达及临床应用  

Cloning and expression of urease B subunit (UreB) of Helicobacter pylori

作　　者：吴超[1] 王宁[1] 袁小澎[1] 鲁东水[1] 邹全明[1] 

机构地区：[1]第三军医大学临床微生物学及免疫学教研室,重庆400038

出　　处：《中华检验医学杂志》2001年第5期296-298,共3页Chinese Journal of Laboratory Medicine

基　　金：国家"九五"重点科技攻关课题 (96 90 1 0 1 5 4)

摘　　要：目的　获得重组表达的人幽门螺杆菌 (Hp)尿素酶B亚单位蛋白 (UreB) ,并将其运用于Hp感染的临床检测。方法　应用聚合酶链反应 (PCR)技术从临床分离的Hp菌株基因组中扩增出编码UreB的基因 (ureB) ,将其克隆于质粒PinPointTMXa Ⅲ上进行序列分析和蛋白表达 ,通过亲和层析得到纯化的重组UreB蛋白并用于 113例消化性溃疡患者Hp感染的酶联免疫吸附试验 (ELISA)法检测。结果　克隆的ureB基因核苷酸序列与GenBank公布的序列相比较 ,同源性为 96 .44 % ,推定氨基酸序列同源性为 99 6 5 %。纯化的重组UreB蛋白相对分子量约为 6 6 0 0 0 ,纯度为 90 %以上 ,并保持较好的免疫原性。应用纯化的重组UreB蛋白联合ELISA法检测 113例消化性溃疡患者Hp感染的灵敏度和特异性分别为 92 .0 %、98.5 %。结论　重组UreB蛋白作为抗原用于ELISA法检测Hp感染 ,保持了较高的灵敏度和特异性 ,可用于Hp感染的临床检测与诊断。Objective To obtain the recombinant urease B subunit (ureB) of Helicobacter pylori (Hp) and apply it in serological detection of Hp infected patients. Methods Urease B subunit gene was amplified from the complete genome of Helicobacter pylori by PCR, and cloned into the PinPoint TM Xa Ⅲ fusion expression vector, then was sequenced. The protein of urease B subunit was expressed in E.coli JM109 and purified with affinity chromatography. 113 serum samples of peptic ulcer patients were detected by ELISA combined with purified UreB protein. Results DNA sequence analysis showed the nucleic acid sequence homology of ureB gene was 96.44% and the putative amino acid sequence homology was 99.65%. The recombinant UreB protein was composed of 571 amino acid residues and kept original immunologic reaction with corresponding antibody, and its purity was over 90% after affinity chromatography. The results of ELISA associated with recombination UreB antigen showed the sensitivity and specificity was 92%, 98.5%. Conclusion The recombinant UreB protein will be of value for clinical serodiagnosis and epidemiological study of Helicobacter pylori.

关 键 词：尿毒酶B亚单位蛋白 基因表达 幽门螺杆菌 血清学检测 酶联免疫吸附试验 

分 类 号：R378[医药卫生—病原生物学]