人突触囊泡蛋白2C单克隆抗体的制备及鉴定

摘要

目的:制备人突触囊泡蛋白2C(SV2C)单克隆抗体.方法:纯化SV2C/L4蛋白,免疫BALB/c小鼠并制备杂交瘤细胞,筛选针对SV2C/L4的特异性抗体,鉴定亚型及测定效价;选取效价最高的抗体进行大量制备,并经SDS-PAGE、Western印迹、间接ELISA和动物实验对抗体纯度、特异性、亲和力及中和活性进行检测.结果:得到纯度大于95％的SV2C/L4蛋白,经2轮筛选共获得13株鼠单抗,抗体重链大多为IgG1、IgG2a型,轻链均为κ型;选取效价最高的30号抗体进行大量制备,纯化后抗体的纯度大于99％,亲和力为6.7 nmol/L,中和活性为100 LD50/mg.结论:筛得1株高亲和力、具有中和活性的特异性SV2C鼠单抗,为肉毒中毒的治疗提供了新的选择,也为阐明BoNT/A受体间的关系奠定了基础.%Objective:To prepare and identify monoclonal antibodies(mAbs) against synaptic vesicle glycoprotein 2C(SV2C).Methods:BALB/c mice were immunized with the purified protein SV2C/L4 and hybridomas were produced by fusing immune splenic B cells with Sp2/0 myeloma cells.The positive hybridoma cells were screened by indirect ELISA.An antibody with the highest titer was selected and large-scale prepared.Its characteristics was identified by SDS-PAGE,Western blotting,ELISA and Bioassay.Results:The SV2C/L4 protein was purified up to 95％ and used as antigen to immunize mice.Thirteen specific mAbs were screened from the hybridoma cells.The isotypes of heavy chains were mostly IgG1 and IgG2a subclass with kappa chains.An antibody numbered as 30 was selected with the highest titer and large-scale prepared by mouse ascites.The purity was up to 99％,the affinity was 3.5 nmol/L and the neutralizing activity was 100 LD5dmg.Conclusion:A specific anti-SV2C antibody was successfully prepared,which is significant for further investigation on the relationship between the receptor SV2C and botulinum neurotoxin A(BoNT/A).