羊种布鲁菌omp25的原核表达与免疫原性检测

Expression and immunogenicity of the recombinant protein omp25 of Brucella melitensis strain

目的在大肠杆菌中表达羊种布鲁菌omp25基因并鉴定重组蛋白的免疫原性。方法从羊种布鲁菌中用聚合酶链反应技术(PCR)扩增得到布鲁菌omp25基因片段,并将目的基因插入原核表达载体PET-32a中,构建重组质粒PET-32a/omp25转入大肠杆菌E.coli Rosetta中并进行诱导表达,用SDS-PAGE和western-blot检测表达蛋白。结果成功构建了重组质粒PET-32a/omp25,并在大肠杆菌Rosetta中获得了重组蛋白,重组蛋白与布鲁菌阳性血清发生特异性反应。结论重组质粒PET-32a/omp25可以在大肠杆菌Rosetta中成功表达,并且重组蛋白可与布鲁菌阳性血清发生特异性反应,说明该重组蛋白有良好的免疫原性,该研究为疫苗的研制及布鲁菌病的检测打下良好的基础。

To express omp25 gene of Brucella in E.coli Rosetta and detect the immunogenicity of the recombinant protein,the target fragment of omp25 gene was amplified by PCR from the B.melitensis M306 strain and was cloned into expression vector PET-32a.After transforming into competent E.coli Rosetta and inducing by IPTG,the recombinant protein was expressed.As detected by SDS-PAGE and western-blot,this recombinant protein has immunogenicity.It was confirmed DNA sequencing and restriction enzymes cleavage that the recombinant plasmid PET-32a/omp25 was constructed successfully.Furthermore,the result of SDS-PAGE and western-blot assay showed that PET-32a/omp25 was expressed successfully in E.coli Rosetta and the recombinant protein could react with B.melitensis positive serum.This study provides a good foundation for the diagnosis of brucellosis and the preparation of new types of vaccines of Brucella.