原发性胆汁性肝硬化自身抗原M2蛋白的发酵与纯化

Fermentation and purification of primary biliary cirrhosis autoantigen M2 protein

目的利用补料分批培养技术大量发酵含有重组质粒pET-28/BPO的DH5-α大肠埃希菌,获得高效表达的M2蛋白,为诊断试剂盒的研发及原发性胆汁性肝硬化(PBC)发病机制研究作准备。方法发酵过程中,溶氧控制在30%以上,温度37℃,基础培养基培养2.5 h后补加甘油作为碳源的补料,4 h后加入IPTG至终浓度1 mmol/L继续培养,诱导表达6 h,收集菌体,溶解后按每克细菌加8μl 50 mmol/L蛋白酶抑制剂PMSF,破菌后用安玛西亚金属螯和层析系统纯化目的蛋白。结果发酵液最终菌体密度约15 g/L,OD600约8.0。表达的蛋白在上清中约占40%～50%,包涵体约占50%～60%,纯化得到的蛋白浓度约0.6 g/L。结论分批补料并以甘油作为碳源,以IPTG为诱导剂,可在大肠埃希菌中获得高效表达的M2蛋白。为后期深入研究PBC发病机制奠定了基础。

Objective To prepare recombinant M2 protein by fed-batch fermentation of E. coli DH5-cx/pET-28-BPO for clinical detection and primary biliary cirrhosis （PBC） mouse model. Methods Dissolved oxygen and temperature were kept at more than 30% and 37℃. Glycero was used as carbon source when dissolved oxygen decreased about 2.5 hours later. IPTG was added up to 1 mmol/L, and then the bacteria grew another 6 hours. To avoid the degradation of the protein, PMSF was involved. M2 was isolated from the supernant of bacteria by immobilized metal affinity chromatography （MAC）. Results The bacteria were cultivated at the final concentration of 15g/L or so. OD600 was about 8.0 at last. There was about 40% N 50% M2 protein in the supernant as soluble form, 50% N 60% as inclusion bodies. The concentration of the purified protein was 0.6g/L. Conclusion The high expression M2 protein was collected by fed-atch fermentation with IPTG as inducer, it could be used in the following research of PBC.