口蹄疫病毒O型和A型交互反应性单克隆抗体的筛选与鉴定

不同血清型口蹄疫病毒(FMDV)之间不能产生交叉免疫保护,但是血清学的交叉反应确实存在。本研究旨在建立一种筛选型间交互反应性抗体的方法,以期为解析FMDV血清型间保守的抗原结构奠定基础。本方法主要依据单个B细胞抗体技术,首先采用两种不同的荧光染料FluoProbes 647H和Pacific Blue分别标记O型与A型FMDV 146S灭活抗原,然后利用流式细胞分选技术,从免疫牛外周血单个核细胞(PBMCs)中分选出结合两种抗原的特异性B细胞,通过单个B细胞抗体基因测定,获得Ig G抗体重链与轻链可变区编码序列,并将可变区基因插入含有牛IgG抗体恒定区的pcDNA3.4载体中,构建IgG抗体的重链与轻链表达质粒。将重链与轻链表达质粒共转染中国仓鼠卵巢悬浮细胞(CHO-S)进行完整抗体表达。结果显示,通过该方法获得了5株FMDV特异性单克隆抗体,其中4株(H64、R82、I16、R29)经间接免疫荧光(IFA)检测都可特异性识别O和A型FMDV;ELISA结果显示,H64、R82、I16与O、A型抗原均具有较好的亲合力;Western-blot结果显示,I16可特异性结合O、A型FMDV结构蛋白VP2中的线性表位,说明在衣壳蛋白VP2上存在血清型间保守的抗原表位,通过本研究进一步加深了对FMDV抗原结构的认识。

Screen and identification of cross-reactive monoclonal antibodies against foot-and-mouth disease virus serotype O and A

Although no cross protection was observed between different serotype of foot-and-mouth disease virus(FMDV),the serological cross reactivity was indeed observed between different serotypes.This study aimed to develop the method to screen the serologically cross-reactive monoclonal antibody(mAb) against different serotype FMDV,which would provide useful tools for exploration of the conserve antigenic structure among serotypes of FMDV.Single B cell antibody technique was used in this study for this purpose.Firstly,146 S antigens of serotype O and A were labeled with two different fluorescent dyes of Fluo Probes 647 and Pacific Blue dye,respectively.Single antigen-specific B cell was sorted from the PBMCs of immunized cattle by flow cytometry,then the genes of IgG variable region of heavy and light chains(VH and VL) were amplified by single B cell gene detection method.Amplified VH and VL genes were cloned and inserted into the pc DNA3.4 vector containing the constant region genes of heavy and light chains of the bovine Ig G to construct antibody expression vectors.These recombinant vectors were co-transfected into CHO-S cells for antibody expression.The results showed that five FMDV-specific m Abs were obtained by this method.Among them,four m Abs(H64,R82,I16,R29) can specifically bind to both FMDV type O and A antigens by IFA;Three mAbs(H64,R82 and I16) showed good affinity with type O and A antigens by indirect ELISA;And only one mAb(I16)can specifically bind to capsid protein VP2 of FMDV type O and A by Western-blot,indicating a conserved antigenic epitope exist on the capsid protein VP2.This study further deepen the understanding on the antigenic structure of FMDV capsid.