钩端螺旋体LigA基因原核表达及抗原性分析

Prokaryotic expression of leptospira Lig A gene and analysis of its antigenicity

目的:通过基因工程方法,获得表达量高、抗原性好的钩端螺旋体重组抗原,检测国内常见的15群15型钩端螺旋体阳性标准血清,初步验证其抗原性。方法:用PCR法扩增哥本哈根型钩体LigA基因,与表达载体pET-30a连接,获得LigA重组表达质粒,转化BL21(DE3)宿主菌,IPTG诱导表达。表达产物经亲和层析纯化后,采用Western blot和ELISA分析其抗原性。结果:获得在大肠杆菌中有高产量的重组蛋白,Western blot和ELISA结果显示重组抗原在检测国内常见的15群15型钩端螺旋体阳性标准血清方面具有良好的免疫活性。结论:可以通过原核表达哥本哈根型钩体LigA基因,重组蛋白可应用于钩体病血清抗体的检测,在钩体病诊断及疫苗研究中具有潜在的应用价值。

Objective：To evaluate the antigenicity of LigA protein which is obtained from Leptospira Copenhagen strain and expressed in E.coli by genetic engineering methods,and using LigA protein to detect 15 standard positive sera of domestic prevalent leptospira by WB and Elisa analysis.Methods：The LigA gene was amplified by PCR and constructed the recombinant expression plasmid with pET-30a,and then expressed in E.coli BL21（DE3）and induced with IPTG.The expression product was dissolved in urine solution and purified by affinity chromatography.We use Western-blotting and indirect ELISA to evaluate its antigenicity.Results：The recombinant protein was expressed with high yield in E.coli,meanwhile,the results obtained from Western-blotting and ELISA reveal that it has good antigenic activity.Conclusion：LigA sequence from Leptospira Copenhagen strain can be expressed in E.coli and maintains its antigenicity.It shows a potential role in the diagnosis of leptospirasis as well as the research on vaccine domain.