重组布鲁菌外膜蛋白Omp19的制备方法及免疫原性研究

Preparation Method and Immunogenicity of Recombinant Brucella Outer Membrane Protein Omp19

[目的]探索重组布鲁菌Omp19蛋白的制备方法,评价其在实验动物中的免疫原性,为中试工艺放大奠定基础。[方法]利用摇瓶培养对重组Omp19蛋白进行原核诱导表达,对培养基类型、诱导剂浓度、诱导温度、诱导时间、诱导时的菌体密度等条件进行筛选;利用阳离子交换层析、疏水层析和阴离子交换层析对重组Omp19蛋白进行纯化制备;利用SDS-PAGE和分子排阻高效液相色谱法(SEC-HPLC)对重组Omp19蛋白的纯度进行鉴定;利用动物实验评价制备的重组Omp19蛋白的免疫原性。[结果]摇瓶培养试验表明,重组布鲁菌Omp19蛋白较优的表达条件为:使用LB培养基,在菌密度OD600 nm值为0.6~1.0时加入0.5 mmol/L的IPTG,于28℃诱导5 h;经3步纯化后,获得了高纯度的重组Omp19蛋白;免疫原性研究表明,经方法优化后制备的重组Omp19蛋白联合佐剂可以较好地刺激BALB/c小鼠产生特异性抗体。[结论]优化了重组布鲁菌外膜蛋白Omp19的制备方法,评价了其在小鼠中的免疫原性,为重组布鲁菌疫苗的研发提供了实验基础。

[Objective]To explore the optimal preparation method of recombinant Brucella outer membrane protein Omp19,to evaluate its immunogenicity in experimental animals,and to lay the foundation for the follow-up pilot-scale study.[Method]The recombinant Omp19 protein was expressed by prokaryotic induction using the shake flask culture method,and the effects of different culture mediums,IPTG concentrations,induction temperatures,induction durations,and bacterial densities on protein expression were evaluated and optimized.The recombinant Omp19 protein was expressed and purified by cation exchange chromatography,hydrophobic chromatography,and anion exchange chromatography methods.The purity of the recombinant Omp19 protein was characterized using SDS-PAGE and SEC-HPLC methods.The immunogenicity of the recombinant Omp19 protein was evaluated by animal experiment.[Result]Shake flask culture test showed that the better expression conditions of recombinant Brucella Omp19 protein were as followed:using the LB medium culture and inducing at OD600 nm of 0.6~1.0 of bacterial densities with 0.5 mmol/L IPTG at 28℃for 5 h.The highly pure recombinant Omp19 protein was obtained after the three steps of purification.Immunogenicity studies revealed that the recombinant Omp19 protein could effectively stimulate the production of specific antibodies when formulated with adjuvants in BALB/c mice.[Conclusion]This research optimized the preparation method of recombinant Brucella Omp19,evaluated its immunogenicity in mice,and provided the experimental basis for recombinant Brucella vaccine development.