家畜布鲁氏菌外膜蛋白OMP25基因的原核表达及其应用

Prokaryotic expression and application of outer membrane protein OMP25 of livestock Brucella

为了获得活性良好的布鲁氏菌基因工程抗原,对Brucella OMP25蛋白进行原核表达及初步应用,试验采用PCR方法从布鲁氏菌病S2疫苗株中扩增获得OMP25基因,构建了原核表达质粒p GEX-6P-1-OMP25,转化入大肠杆菌BL21中,经过IPTG诱导表达,应用Western-blot方法鉴定表达产物,并以纯化的OMP25重组蛋白为诊断用抗原,通过反应条件优化,初步建立Brucella抗体ELISA检测方法。又以羊布鲁氏菌普查检测血清120份为样本,采用试管凝集试验进行了比较分析。结果表明:成功构建了pGEX-6P-1-OMP25原核表达载体,并在BL21中得以诱导表达,经SDSPAGE和Western-blot分析,重组蛋白分子质量约为49 ku,优化试验确定抗原的最佳包被浓度为3.5μg/m L,血清最佳稀释度为1∶20。试管凝集试验血清样本的阳性率为83%,采用OMP25作为包被抗原的阳性检出率为68%,二者的符合率为85%。说明OMP25作为间接ELISA包被抗原用于羊布鲁氏菌血清学检测具有良好的反应性和特异性。

To obtain Brucella gene engineering antigen with good activity, and conduct prokaryotic expression of Brucella outer membrane protein （OMP） 25 protein and its preliminary application, the OMP25 gene was amplified from a vaccine strain $2 of brucellosis by PCR. The constructed recombinant plasmid pGEX -6P - 1 - OMP25 was transformed to Escherichia coli （E. coli） B1221 and induced with IPTG. The expressed products was identified by Western - blot using purified recombinant OMP25 protein as a diagnostic antigen. An indirect ELISA for detection of antibody against BruceUa was initially established through optimizing the reaction conditions. A tube agglutination test was used for a comparative analysis using 120 serum samples in general survey for the BruceUa infection as a specimen source. The results showed that the prokaryotic expression vector pGEX-6P- 1 - OMP25 was successfully constructed, which was induced and expressed in E. coli BE21. The molecular mass of recombinant protein was about 49 ku by SDS - PAGE and Western - blot analysis, and the optimal antigen coating concentration determined by an optimization test was 3.5 p,g/mL, and the optimal serum dilution was 1：20. Ihe positive rate of serum samples detected by the tube agglutination test was 83%, and the positive detection rate using the OMP25 as the coating antigen was 68%, and the coincidence rate both methods was 85%. The results indicate that the OMP25 protein used as the coating antigen in indirect ELISA test for the serological diagnosis of Brucella melitensis has good reactivity and specificity.