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聚乙二醇(PEG)修饰是延长蛋白药物体内半衰期和降低免疫原性的有效途径。经过单因素实验,探索了PEG对LL-37与干扰素(IFN)-α2a融合蛋白修饰的最佳工艺条件,分别为:PEG分子量为5 000、融合蛋白浓度为0.6mg/mL、蛋白与PEG5000的物质的量之比为1∶10、反应温度为4℃、反应时间为4h、pH为9.0。经正交实验表明,3个主要因素对蛋白修饰率的影响顺序为融合蛋白浓度>蛋白mPEG5000-SS的物质量之比>pH,最佳工艺条件为融合蛋白浓度为0.6mg/mL、蛋白与mPEG5000-SS的物质的量之比为1∶10、pH为8.8。在最佳修饰条件下,三次平行实验的平均蛋白修饰率为86.98%。在最佳修饰条件下,蛋白的修饰率可稳定达到85%以上,LL-37与IFN-α2a融合蛋白的干扰素活性保持率为58%以上,抗菌活性的保持率可达到97%以上。
Polyethylene glycol (PEG) modification is an effective way to prolong the half-life and reduce the immunogenicity of protein drugs in vivo. After single-factor experiments, the optimal conditions for PEG-modified LL-37 and IFN-α2a fusion protein were explored: PEG molecular weight of 5000, fusion protein concentration of 0.6mg / mL, PEG5000 The ratio of the amount of the substance is 1:10, the reaction temperature is 4 ° C, the reaction time is 4h and the pH is 9.0. The orthogonal experiment showed that the order of the three main factors affecting the protein modification rate was the ratio of the mass of the fusion protein> protein mPEG5000-SS> pH. The optimal conditions were the concentration of fusion protein 0.6mg / mL, The amount of substance of mPEG5000-SS was 1:10 and the pH was 8.8. Under the best modification conditions, the average rate of protein modification in three parallel experiments was 86.98%. Under the best modification conditions, the modification rate of the protein can be more than 85%. The IFN activity of LL-37 and IFN-α2a fusion protein is above 58% and the retention rate of antibacterial activity can reach over 97%.