原核表达的PD-1与PD-L1融合蛋白相互作用分析

目的 分析经原核表达纯化的PD-1和PD-L1融合蛋白分子间相互作用力及其生物学活性.方法 PD-1蛋白经过预浓缩后,用氨耦联的原理固定于CM5芯片表面,用HBS-EP缓冲液梯度稀释PD-L1蛋白,以20μl/min流速注射到耦联PD-1的传感芯片通道上,结合3 min,解离15 min;观察两者结合情况并用BIA Evaluation软件4进行分析.结果 PD-1在缓冲液的pH值为4.5、浓度为40μg/ml的状态下能稳定耦联于CM5芯片表面,RU值约为3300;稀释度为200 mmol/ml的PD-L1与PD-1能较好地结合,RU值为150,亲和常数为K_D=3.5×10~(-6).结论 经原核表达纯化的PD-1与PD-L1融合蛋白具有较强的特异性亲和力及良好的生物学活性,为下一步研究的开展打下基础.

Analyze the bioactivity of PD-1 and PD-L1 recombination protein which expressed by prokaryotic system

Objective To analyze the interaction of PD-1 and PD-L1 recombination protein and to know their bioactivity and affinity.Methods Stick the PD-1 protein on the surface of CM5 isensor chip by the method of Ammine coupling after being preconsentrated.Dilute the PD-L1 protein step by step and reject it to the passage on CM5 sensor chip which had been stick bv PD-1.The time of combination is 3 minutes and of separation is 15 minutes,respectively.Observe the procession and analyze data by BIA Evaluation software 4.Results On the consistency of 40 μg/ml.pH 4.5,the PD-1 protein could couple steady on the surface of CM5senser chip,RU is 3300.On the density of 200 mmol/ml PD-L1 could combine with PD-1 specifically,RU=150,K_D=3.5 × 10~(-6).Conclusion The PD-1 and PD-L1 recombination protein which we expressed by prokaryotic system have good affinity and bioactivity.The results could provide basic condition for later studv.