表达MERS-CoVS1蛋白重组狂犬病病毒rSRV9-MERSS1纯化方法的建立

Establishment of apurification method for recombinant rabies virus expressing MERS-CoVS1 protein rSRV9-MERSS1

建立一种可线性放大、应用于表达中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)S1蛋白重组狂犬病病毒rSRV9-MERSS1的规模化纯化方法。将收获的病毒去除细胞碎片、灭活后,分别通过3种方法(醋酸锌沉淀+Sepharose 4FF凝胶过滤层析、500 000中空纤维超滤柱+Sepharose 4FF凝胶过滤层析、500 000中空纤维超滤柱+CellufineTM Sulfate亲和层析)进行纯化,经比较筛选出一种温和、目的蛋白回收率高、纯度高的纯化方法。结果显示,500 000中空纤维超滤柱+Sepharose 4FF凝胶过滤层析回收的抗原含量最高,50 mL重组病毒原液经纯化后可得到1.82 mg蛋白,杂蛋白去除率达到97.02%,病毒纯度较高,电镜下可见到病毒囊膜刺突。结合成本等方面综合考虑,此方法优于其他2种纯化方法。本试验筛选获得了一种适用于rSRV9-MERSS1的纯化方法,为rSRV9-MERSS1规模化纯化工艺的建立与MERS新型疫苗的研制奠定了基础。

To establish a method that can be linearly amplified and applied to the purification of recombinant rabies virus expressing MERS-CoV S1 protein(rSRV9-MERSS1).After the harvested virus was removed cell debris and inactivated,three purification methods(zinc acetate precipitation+Sepharose 4 FF gel filtration chromatography,500 000 hollow fiber ultrafiltration column+Sepharose 4 FF gel filtration chromatography,500 000 hollow fiber ultrafiltration column+CellufineTM Sulfate affinity chromatography)were used to screen a mild,high recovery and high purity purification method.The results showed that 500 000 hollow fiber ultrafiltration column+Sepharose 4 FF gel filtration chromatography recovered the highest antigen content,50 mL recombinant virus stock can obtain 1.82 mg protein after purification,the removal rate of impurity protein reached97.02%,virus purity was higher,capsule spur of virus can be seen in electron microscope.Considering cost and other aspects,this method was superior to the other two purification methods.This study screened and obtained a suitable purification method for rSRV9-MERSS1,which laid a foundation for the establishment of large-scale purification process for rSRV9-MERSS1 and the development of MERS vaccine.