流产型布鲁菌Omp10、Omp25蛋白的表达纯化与鉴定

获得高纯度具有生物学活性的重组布鲁菌Omp10、Omp25融合蛋白,并进行抗原性的分析。将用PCR扩增出的布鲁菌Omp10、Omp25基因片段分别克隆到原核表达载体pET-32α中,构建pET-32α-Omp10/Omp25原核表达质粒。将其转入大肠杆菌BL21（DE3）PlysS中,用IPTG诱导表达,经HisTrap HP亲和层析柱分离纯化,分别用Western-blot和间接ELISA检测产物的抗原性。基因测序及酶切鉴定证明pET-32α-Omp10/Omp25原核表达载体构建成功。SDS-PAGE表明,Omp10、Omp25融合蛋白均以包涵体的形式在大肠杆菌中高效表达。经过包涵体的变性、复性及亲和层析纯化,成功获得了大小分别为34 000和44 000的融合蛋白,与预测的相对蛋白分子质量一致。Western和间接ELISA试验证明纯化的Omp10、Omp25融合蛋白能被免疫的牛布鲁菌阳性血清所识别。结果表明,成功获得了布鲁菌Omp10、Omp25融合蛋白,且均具有一定的免疫原性,通过血清学反应证实,Omp10、Omp25蛋白为布鲁菌病临床诊断试剂盒的研制奠定了基础。

Expression and purification of Omp10 and Omp25 recombinant proteins of Brucella abortus and their antigenicity research

The research aims at obtaining Brucella abortus’s Omp10 and Omp25 recombinant proteins with high purity and biologic activity and analyzing their antigenicity.A polymerase chain reaction(PCR) method was used to amplify the Omp10 and Omp25 gene regions,which were ligated with pET-32α prokaryotic expression vector into pET-32α-Omp10/Omp25 recombinant plasmids.The recombinant plasmids were transformed into Escherichia coli BL21(DE3) PlysS whose expression was induced by IPTG and then separated and purified through HisTrap HP affinity column.The methods of Western-blot and indirect ELISA were used to detect the products’ antigenicity.The results of gene sequencing and restriction enzyme digestion identification both proved that the pET-32α-Omp10/Omp25 vectors were successfully constructed.The SDS-PAGE electrophoresis showed that the Omp10 and Omp25 recombinant proteins were both highly expressed in the form of inclusion bodies in E.coli.After the denaturation,renaturation and affinity chromatography of inclusion bodies,the recombinant proteins in size of 34 000 and 44 000 were successfully obtained,which correspond to the expected protein molecular weight.The detection of Western-blot and indirect ELISA proved that purified Omp10 and Omp25 recombinant proteins could be identified by the Brucella positive serum from immunized cattle.The research showed that Omp10 and Omp25 recombinant proteins of Brucella abortus were successfully obtained and they both had certain immunogenicity.Serological reaction was also confirmed that the Omp10 and Omp25 recombinant proteins laid the foundation for the development of Brucellosis clinical diagnostic reagent kits.