布鲁氏菌bp26基因的原核表达及BP26-间接ELISA方法的初步建立  

Expression of Brucella bp26 Gene and BP26 Protein Used as the Diagnostic Antigen in Indirect ELISA

作　　者：唐利燕[1] 陈创夫[1] 王远志[2] 张辉[1] 张红星[3] 

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]石河子大学医学院,石河子832002 [3]石河子大学生命科学院,石河子832003

出　　处：《石河子大学学报（自然科学版）》2009年第4期437-440,共4页Journal of Shihezi University(Natural Science)

基　　金：973计划前期研究专项(2007CB116309);国际科技合作项目(2006DFA33740);国家自然科学基金项目(30760187);国家自然科学基金项目(30800813)

摘　　要：为了表达并纯化布鲁氏菌BP26蛋白作为包被抗原,建立间接ELISA检测方法。方法:从布鲁氏菌疫苗株M5-90克隆bp26基因,在大肠杆菌E.coli BL21(DE3)中表达,纯化BP26蛋白,进行SDS-PAGE、Western-blot检测,将纯化的BP26蛋白包被ELISA板,建立检测布鲁氏菌病的间接ELISA方法,用该方法检测42份绵羊血清,结果与标准试管凝集试验(SAT)进行比较。结果显示:正确表达并纯化了BP26蛋白,布鲁氏菌标准试管凝集试验检测的阳性率为30.85%(13/42);间接ELISA方法检测的阳性率为35.71%(15/42),二者阳性符合率为86.67%(13/ 15)。结论表达、纯化的BP26蛋白能够与布鲁氏菌阳性血清特异性结合,建立的ELISA方法比SAT方法更敏感。为以后M5-90疫苗株bp26基因缺失苗的应用提供配套性血清学检测奠定基础。To investigate the value of the recombinant BP26 protein in the serological diagnosis of sheep brucellosis caused by Brucella melitensis, recombinant BP26 protein was produced in Escherichia Coli and purified for use in an indirect enzyme-linked immunosorbent assay （I-ELISA）. In the study, one target fragment bp26 gene was amplified via PCR from the vaccine strain M5-90 and expressed in E. Coli. The results were compared with standard tube agglutination test （STAT） with 42 serum samples. It was found that the positive rates of serum samples revealed by the STAT and iELISA were 30.85% （13/42） and 35.71% （ 13/42）, respectively. These results indicated that the BP26 protein could be expressed successfully in E. Coli and has a good immunity in SDS-PAGE and Western-blot. The recombinant BP26-I-ELISA indicated that the BP26 protein may be an interesting potential as diagnostic antigens for brucellosis. BP26 coated iELISA,which may be equipped with Brucella vaccine M5-90 △bp26 mutant strain.

关 键 词：布鲁氏菌 BP26蛋白 间接ELISA 标准试管凝集实验 

分 类 号：S852.61[农业科学—基础兽医学] Q786[农业科学—兽医学]