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

作　　者：臧金凤 陈妍[1] 吴锦艳[1] 王光祥[1] 尹双辉[1] 栾志舫 曹小安[1] 尚佑军[1] 张志东[1] 刘湘涛[1] 张超 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室/农业部畜禽病毒学重点开放实验室,兰州730046 [2]河北农业大学动物医学院,河北保定071001 [3]石家庄市兽用生物制品供应站,石家庄050000

出　　处：《黑龙江畜牧兽医》2016年第7期29-32,37,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家现代农业产业技术体系项目(NYCYTX-39);内蒙古科技重大专项"巴美肉羊产业化技术研究与集成应用"

摘　　要：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.

关 键 词：布鲁氏菌 OMP25蛋白 克隆 原核表达 间接ELISA 

分 类 号：Q344.13[生物学—遗传学]