抗GⅡ.3型诺如病毒阻断型单克隆抗体的制备和鉴定

The Preparation and Characterization of Blocking Monoclonal Antibodies Against GⅡ.3 Norovirus

诺如病毒(norovirus,NoV)是引起非细菌性急性肠胃炎的主要病原体之一。目前国内外还没有关于抗GⅡ.3NoV组织血型抗原(histo-blood group antigens,HBGAs)阻断型单克隆抗体(monoclonal antibody,mAb)的报道,本研究制备了抗GⅡ.3型NoV HBGA阻断型mAbs,并对这些抗体特性进行了初步的鉴定。采用纯化的GⅡ.3型(GenBank登录号:KY767665)NoV病毒样颗粒(virus-like particles,VLPs)免疫BALB/c小鼠。将GⅡ.3,GⅡ.4(GenBank登录号,KF306214),GⅡ.3S/GⅡ.6P,GⅡ.6S/GⅡ.3P,GⅡ.4-VP1/GⅡ.3-P2主要衣壳蛋白(VP1)抗原或嵌合抗原作为包被抗原采用间接ELISA方法分别对细胞克隆株进行筛选,采用体外HBGA-VLP结合阻断实验对筛选的mAbs进行阻断活性鉴定,利用基于涵盖GⅡ.3突环区(protruding domain,P区)的重叠多肽ELISA和western blot(WB)对阻断抗体结合位点进行特性分析。体外HBGA-VLPs阻断实验显示三株细胞分泌抗体具有阻断活性。挑选目标株制备腹水并对mAbs进行纯化。WB结果显示三株HBGA阻断型mAbs只识别非变性的GⅡ.3 VP1蛋白;基于GⅡ.3 P区重叠多肽的间接ELISA结果显示三株HBGA阻断型mAbs与被检测多肽无结合活性。本研究制备了具有HBGAs阻断活性的GⅡ.3 NoV特异性mAbs,全部只识别非变性的GⅡ.3 VP1蛋白,且结合位点位于GⅡ.3 VP1 P2区。抗GⅡ.3 NoV HBGAs阻断型mAbs的获得为后续研究GⅡ.3 NoV的进化、感染机制和HBGAs结合位点提供了原材料。

Norovirses(NoVs)are one of the leading causes of acute non-bacterial gastroenteritis worldwide.There are currently no reports about histo-blood group antigens(HBGAs)-blocking monoclonal antibodies(mAbs)against GⅡ.3 NoV and thus in this study we generated and characterized anti-GⅡ.3 HBGA-blocking mAbs.We immunized BALB/c mice with GⅡ.3 NoV(GenBank accession number,KY767665)virus-like particles(VLPs)and screened cell clones with indirect enzyme-linked immunosorbent assay using a combination of different antigens,including GⅡ.3,GⅡ.4(GenBank accession number,KF306214),chimeric GⅡ.3 S/GⅡ.6 P,chimeric GⅡ.6 S/GⅡ.3 P,and chimeric GⅡ.4-VP1/GⅡ.3-P2 VP1 proteins.The blocking mAbs were characterized by in vitro HBGA-VLP binding blockade assay,Western blot and indirect ELISA assay.mAbs secreted by three cell clones exhibited HBGA-blocking activity and those antibodies were massively produced by ascite induction method.Peptide-based indirect ELISA and WB indicated that the three blocking mAbs didn’t recognize peptides derived from the P domain and only bound non-denatured GⅡ.3 VP1 proteins.In this study,we generated and characterized three blocking mAbs against GⅡ.3 NoV for the first time and our results indicate that they bind the P2 domain in a conformation-dependent manner.These blocking mAbs will facilitate the clarification of evolutionary pattern,entry pathway and HBGA binding interface of GⅡ.3 NoV.