柯萨奇病毒A6型多克隆抗体的制备及鉴定

Preparation and identification of polyclonal antibody against Coxsackie virus A6

目的制备兔抗柯萨奇病毒A6型(Coxsackie virus A6,CVA6)多克隆抗体,并鉴定其特异性。方法将杆状病毒-昆虫细胞表达系统中共表达的CVA6病毒样颗粒(virus-like particle,VLP)纯化后免疫日本大耳白兔,制备其多克隆抗体;用CVA6-R69-XY-2016、CVA10-R97-XY-2016、CVA16-V217-XY-2016、EV71-V54-XY-2016及CVA6 Gdula毒株分别感染人恶性胚胎横纹肌瘤RD细胞,体外中和试验及ELISA法检测中和抗体和结合抗体效价,Western blot法及间接免疫荧光试验(indirect immunoinfluscent assay,IFA)检测抗体特异性。结果制备的兔抗CVA6 VLP多克隆抗体与CVA6-R69-XY-2016及CVA6 Gdula毒株的中和抗体效价均可达1024,多克隆抗体仅与这2种病毒的结构蛋白VP1、VP2及VP3产生特异性反应,仅在这2种病毒感染的RD细胞中产生明显的绿色荧光;经ELISA分析,其结合效价达1×10^7。结论成功制备了兔抗CVA6 VLP多克隆抗体,其特异性良好,可为CVA6血清型中和试验鉴别、病毒结构蛋白定性定量分析提供抗体试剂。

Objective To prepare and identify the rabbit polyclonal antibody against Coxsackie virus A6(CVA6).Methods The virus-like particles(VLPs)of CVA6 were co-expressed by using baculovirus-insect cell expression system and purified,with which Japanese white rabbits were immunized to prepare polyclonal antibody(PcAb).Human embryo malignant rhabdomyoma RD cells were infected with CVA6-R69-XY-2016,CVA10-R97-XY-2016,CVA16-V217-XY-2016,EV71-V54-XY-2016 and CVA6 Gdula strains respectively,and determined for neutralizing antibody titer by neutralization assay in vitro,for binding antibody titer by ELISA,and for antibody specificity by Western blot and indirect immunoinfluscent assay(IFA).Results The titers of prepared PcAb against CVA6 VLPs as well as the neutralizing antibodies against CVA6-R69-XY-2016 and CVA6 Gdula strains reached 1024.The PcAb showed only specific reactions with structural proteins VP1,VP2 and VP3 of CVA6-R69-XY-2016 and CVA6 Gdula strains,and green fluorescence was observed only in the RD cells infected with the two strains.ELISA showed a binding titer of 1×10^7 of the prepared PcAb.Conclusion Rabbit PcAb against CVA6 VLPs was successfully prepared,which showed high specificity and provided a reagent for identification of serotype of CVA6 by neutralization assay as well as qualitative and quantitative analyses of virus structural protein.