重组幽门螺杆菌热休克蛋白A的纯化研究  

Purification of recombinant helicobacter pylori hspA

作　　者：郭鹰[1] 邹全明[1] 朱永红[1] 吴超[1] 张卫军[1] 

机构地区：[1]第三军医大学医学检验系临床微生物学及免疫学教研室,重庆400038

出　　处：《免疫学杂志》2005年第4期285-287,291,共4页Immunological Journal

基　　金：国家"九五"重点科技攻关计划项目(96-901-01-54);"863"生物与现代农业技术领域生物工程技术主题课题(2001AA215161)资助

摘　　要：目的对重组幽门螺杆菌热休克蛋白A大肠杆菌工程菌表达的rHspA蛋白进行纯化研究。方法酵后的重组大肠杆菌进行高压破菌后,采用镍离子亲和柱层析和凝胶过滤柱层析相结合的方法分离纯化rHspA,使用SDS-PAGE和HPLC鉴定蛋白纯度,注射免疫家兔检测纯化后的rHspA的免疫学活性。结果所纯化获得的rHspA蛋白纯度>98%,总回收率为60%,并且保持了良好的免疫学活性。结论建立了从重组大肠杆菌中纯化高纯度rHspA的工艺,为Hp疫苗的进一步研究提供了材料。Objective To purify the recombinant helicobacter pylori HspA expressed in E.coli BL21(DE3). Methods The gene engineering E. coli of rHspA was broken by APV-1000, and then the supernatant was collected by centrifuge method. rHspA was captured by Chelating sepharose chromatography from the supernatant. After being concentrated and denaturalized, the captured sample was purified by Superdex 75 gel filter chromatography in denatured condition. The purified sample was desalted by sephadex G-25 and the immunogenicity of purified sample was detected by immunizing rabbits. Results After purified, the rHspA's purity was over 98%, the recovery rate of target protein was 60%. The purified rHspA has good immunogenicity demonstrated by rabbit immunization. Conclusion An effective method for purifying rHspA from the gene engineering E. coli is established, which is very important for the next researches in H. pylori engineered vaccines.

关 键 词：幽门螺杆菌 热休克蛋白 柱层析 

分 类 号：R392[医药卫生—免疫学]