抗寨卡病毒非结构蛋白1单克隆抗体的制备与鉴定

目的制备抗寨卡病毒非结构蛋白1的小鼠杂交瘤单克隆抗体,并对筛选获得的单抗进行抗体亚型鉴定以及免疫结合特性鉴定。方法将真核表达纯化获得的寨卡病毒非结构蛋白1按50μg/只的量免疫3只SPF级BABL/c小鼠,经过3次皮下多点免疫及1次腹腔加强免疫后测定小鼠血清抗体效价,取抗体滴度高的免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合,使用有限稀释法进行亚克隆,采用间接ELISA筛选分泌高滴度单抗的杂交瘤细胞株,使用小鼠单克隆抗体分型试剂盒对筛选的单抗进行亚型鉴定,分别使用免疫印迹与间接免疫荧光法检测单抗与寨卡病毒非结构蛋白1以及寨卡病毒的免疫结合特性。结果寨卡病毒非结构蛋白1免疫刺激小鼠产生高滴度的抗体,ELISA滴度为1∶64000。通过筛选获得2株能高效分泌抗寨卡病毒非结构蛋白1单抗的杂交瘤细胞株(命名为1C9-F12,4B2-F3),分泌的单抗的亚型均为重链γ1和轻链Kappa;Western blot及IFA法检测2株单抗均能与寨卡病毒非结构蛋白1及寨卡病毒发生特异性结合。结论成功筛选到2个抗寨卡病毒非结构蛋白1的小鼠单克隆抗体,该两个单抗均具有寨卡病毒非结构蛋白1及寨卡病毒结合特性,为建立检测寨卡病毒感染的ELISA方法及治疗性抗体研究奠定了基础。

Preparation and characterization of monoclonal antibodies against Zika virus nonstructural protein 1

Objectives To prepare monoclonal antibodies(mAbs)against Zika virus nonstructural protein 1(ZIKV NS1)and to characterize the isotope type and immune binding properties of those mAbs.Methods Recombinant ZIKV NS1 protein was obtained from a eukaryotic expression system and purified.It was then used to immunize 3 SPF BABL/c mice at a dose of 50μg per mouse.After 3 rounds of multipoint subcutaneous immunization and 1 round of intraperitoneal booster immunization,the serum antibody titers of the mice were measured.The spleen cells of the mouse with the highest antibody titer were subjected to cell fusion with mouse myeloma SP2/0 cells.After subcloning using the limiting dilution method,indirect ELISA was used to screen the cell lines that secret high titers of anti-ZIKV NS1 mAbs.The obtained mAbs were further characterized in terms of isotope typing using an SBA clonotyping system/HRP kit.The binding specificity of the mAbs with the recombinant ZIKV NS1 and the ZIKV virion was respectively determined using Western blotting and an indirect fluorescence assay(IFA).Results After immunization with the recombinant ZIKV NS1 protein,high titers of antibody were stimulated in the mice,the highest of which was 1∶64,000.Two hybridoma cell lines(1 C9-F12 and 4 B2-F3)secreting anti-ZIKV NS1 mAbs with a high level of efficiency were obtained.Both antibodies had aγ1 heavy chain and a kappa light chain.Western blotting indicated that the two antibodies were able to specifically bind with the recombinant ZIKV NS1,producing a band at 43 ku.IFA indicated that both antibodies were able to bind with the ZIKV virion with a high of specificity in contrast to the negative and positive controls.Conclusion Two mAbs against ZIKV NS1 that are highly capable of binding with the recombinant ZIKV NS1 and the ZIKV virion were obtained.This finding provides the basis for establishment of an immune assay to diagnose a ZIKV infection and research on anti-ZIKV antibodies.