口蹄疫病毒VP2蛋白特异性单克隆工程抗体的表达与活性鉴定

Expression and activity identification of a monoclonal antibody specific for VP2 protein of foot-and-mouth disease virus

为了在牛上应用单个B细胞抗体技术制备口蹄疫病毒(FMDV)特异性单克隆抗体,本研究利用生物素标记O型FMDV 146S抗原,从免疫牛外周血单个核细胞(PBMCs)中分选单个抗原特异性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,将可变区基因插入含有牛Ig G抗体恒定区的pcDNA3.4载体中,构建完整Ig G抗体的表达质粒。将表达质粒转染中国仓鼠卵巢悬浮细胞(CHO-S)进行抗体表达与纯化。经病毒中和试验(VNT)、间接免疫荧光试验(IFA)、酶联免疫吸附试验(ELISA)、Western-blot验证抗体的生物活性。结果显示,获得了4株FMDV特异性单克隆工程抗体,其中2株(A35、B57)可以中和O型FMDV;2株(A7、E32)IFA检测为阳性,其中E32可特异性结合O型和A型FMDV两种抗原,但没有病毒中和活性;ELISA结果显示,E32具有较好的亲合力;Western-blot结果显示,E32可特异性结合衣壳蛋白VP2,说明在衣壳蛋白VP2存在型间保守的抗原位点,为通用型FMDV抗原检测方法的研究提供了材料。

To produce FMDV-specific m Abs by single B cell antibody technique in cattle,in this study,using biotin-labeled type O FMDV 146S antigen as a bait,single antigen-specific B cell was sorted from the PBMCs of immunized cattle by flow cytometry,then the Ig G variable region genes of the isolated single B cell were cloned and inserted into the pcDNA3.4 vector containing the constant region genes of the bovine Ig G to construct antibody expression vectors. These constructed expression vectors were transfected into CHO-S for antibody production and purification. The biological activity of the expressed cattle-derived antibodies was verified by VNT,IFA,ELISA,and Western-blot. Finally,four FMDV-specific antibodies were obtained. Of which,two strains(A35,B57) neutralized O-type FMDV and the other two strains(A7,E32) were non-neutralizing,but obviously bound with FMDV tested by IFA.The E32 strain showed better affinity by ELISA test and could bind both the O-type and A-type FMDV. The Western-blot results showed that E32 could specifically bind to the capsid protein VP2 of FMDV,which suggested the existence of a serotype-independent linear conserved antigenic site on the VP2. In conclusion,the cattle-derived m Ab E32 strain was a potential tool for the study of the universal FMDV antigen detection method.