幽门螺杆菌临床分离株尿素酶基因B的原核表达、纯化及免疫活性  

Prokaryotic expression,purification and immunological activity of UreB of Helicobacter pylori isolated from clinical specimens

作　　者：周珍文 关锐梨 夏慧敏 邓秋连 谢永强 黄勇 廖灿 龚四堂 

机构地区：[1]广州市妇女儿童医疗中心,510120

出　　处：《实用医学杂志》2010年第20期3673-3675,共3页The Journal of Practical Medicine

基　　金：国家自然科学基金(编号:30801054);广东省自然科学基金(编号:8451012001001570);广州市医药卫生科技基金(编号:2008-YB-067);广州市妇女儿童医疗中心博士启动基金(编号:30307-3200818)

摘　　要：目的:在大肠杆菌中表达幽门螺杆菌临床分离株尿素酶B(UreB),纯化重组蛋白并分析其免疫活性。方法:将pGEX-4T-1-UreB重组菌BL21复舒,挑单菌落接种于含氨苄的LB培养基中,0.5mmoL异丙基硫代半乳糖苷(IPTG)诱导重组融合蛋白的表达,使用纯化的重组蛋白免疫小鼠,ELISA检测血清抗体效价,并使用感染患者血清进行Western-blotting鉴定重组蛋白反应原性。结果:SDS-PAGE示在约87kD相应分子量可见融合蛋白表达,产物主要在表达上清以可溶形式存在,纯化蛋白免疫小鼠能诱导产生特异性体液免疫应答,ELISA检测特异性IgG效价为1:51200,Westernblotting示重组蛋白能被幽门螺杆菌感染患者血清识别。结论:UreB在大肠杆菌中获得高效表达,表达产物具有较好的免疫原性及反应原性。Objective To express Helicobacter pylori urease B （UreB）in E.coli BL21,and purify recombinant fusion protein and analyze its immunological activities.Methods Recombinant strain pGEX-4T-1UreB /BL21 was resuscitated,and 0.5 mmoL isopropyl thiogalactoside （IPTG） was used to induce recombinant Ureb protein expression.Balb /c mice were immunized with purified recombinant Ureb protein and the polyclonal antibodies were obtained.Antibody titer was measured by ELISA assay.The immunoreactivity of recombinant protein was identified by Western-blotting assay.Results The expressed product was about 87 kDa in size,and the fusion protein mainly exists in the medium supernatant.Specific humoral immune response could be induced by purified protein,the specific IgG antibody titer was 1：51 200.Western-blotting assay showed that the expressed recombinant protein could be identified by serum from Helicobacter pylori infection patients.Conclusion Recombinant UreB protein was expressed in E.coli efficiently,and the expressed products showed good immunogenicity and immunoreactivity.

关 键 词：螺杆菌 幽门 尿素酶B 免疫活性 

分 类 号：R378[医药卫生—病原生物学]