层析技术制备马抗中东呼吸综合征冠状病毒F(ab’)_2

Preparation of equine neutralizing F(ab')_2 against Middle East respiratory syndrom coronavirus by chromatography

目的采用层析技术制备高纯度的马抗中东呼吸综合征冠状病毒(Middle East respiratory syndrom coronavirus,MERS-CoV)F(ab’)_2。方法以MERS-CoV病毒样颗粒(virus-like particles,VLPs)对马匹进行多次免疫所制备的高免血清为原料,采用亲和层析技术提取血清IgG,经胃蛋白酶酶切后,通过阴离子交换层析、疏水层析、凝胶过滤层析分别对F(ab’)_2进行纯化;以MERS假病毒检测所制备的IgG与F(ab’)_2中和活性。结果凝胶过滤层析实现了F(ab’)_2的高纯度分离,最终制备的F(ab’)_2收率约70%,纯度为93%以上,具有良好的中和活性,其半数抑制浓度(IC_(50))为5.13μg/ml。结论制备了高纯度马抗MERS-CoV免疫球蛋白F(ab’)_2,为人用抗MERS-CoV免疫球蛋白F(ab’)_2生产工艺的建立奠定了基础。

Objective To prepare highly purified equine neutralizing F（ab＇）2 against Middle East respiratory syndrome coronavirus（MERS-CoV） by chromatography. Methods Hyperimmuned equine serum was prepared by immunization with virus-like particles（VLPs）of MERS-CoV for several times, from which IgG was extracted by affinity chromatography and digested with pepsin digestion. The obtained F（ab＇）2 was purified by anion ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The neutralizing activities of prepared IgG and F（ab＇）2 were determined by pseudo-MERS virus. Results The F（ab＇）2 with high purity was obtained by gel filtration chromatography,of which the recovery was about 70% and the purity was more than 93%. The final F（ab＇）2 showed high neutralizing activity, of which the IC（50） was 5. 13 μg/ml. Conclusion Highly purified equine neutralizing F（ab＇）2 against MERS-CoV was prepared, which laid a foundation of development of production procedure of the F（ab＇）2.