精制马抗H5N1禽流感病毒免疫球蛋白疏水层析方法的优化

Optimization of Hydrophobic Chromatographic Method for Preparation of Purified Equine Immunoglobulin against Avian Influenza H5N1 Virus

目的对精制马抗H5N1禽流感病毒免疫球蛋白疏水层析方法进行优化。方法将胃蛋白酶直接加入制备好的高效价马抗H5N1禽流感病毒血清进行消化裂解,再加入饱和度为26%的硫酸铵溶液,盐析沉淀离心一次后,直接进行疏水层析进一步纯化F(ab′)2抗体。结果连续洗脱依次出现6个蛋白峰,50min左右,洗脱峰3开始出现,其中含大部分F(ab′)2抗体,此时硫酸铵摩尔浓度约为1.0～1.1mol/L。阶段梯度洗脱,当硫酸铵摩尔浓度降至1.0mol/L时,大量F(ab′)2片段被洗脱下来,纯度达90%以上。结论优化了精制马抗H5N1禽流感病毒免疫球蛋白的疏水层析方法,降低了硫酸铵用量,减少了离心次数,生产周期也大大缩短。

Objective To optimize the hydrophobic chromatographic method for preparation of purified equine immunoglobulin against avian influenza H5N1 virus.Methods The prepared high titer equine antiserum against avian influenza H5N1 virus was directly digested with pepsin,then added with 26% saturated ammonium sulfate for salting out.After the precipitate was removed by a single centrifugation,the supernatant was collected,from which F（ab＇）2 was purified by one-step hydrophobic chromatography.Results Six protein peaks were observed on the hydrophobic chromatographic profile after continuous gradient elution.The peak 3 containing a majority of F（ab＇）2 appeared about 50 min after elution when the concentration of ammonium sulfate was about 1.0 ～ 1.1 mol/L.However,a large quantity of F（ab＇）2 was eluted by phasic gradient elution when the concentration of ammonium sulfate decreased to 1.0 mol/L,which reached a purity of more than 90%.Conclusions The hydrophobic chromatographic method for preparation of purified equine immunoglobulin against avian influenza H5N1 virus was optimized,by which the consume of ammonium sulfate and the times of centrifugation decreased,while the production cycle was shortened significantly.