IBRV特异性噬菌体单域抗体库的构建、筛选及鉴定

Construction,screening and identification of bovine Infectious rhinobronchitis virus specific phage display single domain antibody

为了开发有效的用于治疗和诊断牛传染性鼻气管炎(infectious bovine rhinotracheitis,IBR)的抗体,试验用牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)免疫双峰驼,构建IBRV特异性噬菌体单域抗体库,并以IBRV gD蛋白为筛选抗原,对其进行筛选,对筛选得到的单域抗体进行诱导表达、纯化及鉴定。结果表明:构建得到的IBRV特异性噬菌体单域抗体库的库容为7×10^7;经3轮富集和筛选,phage-ELISA筛选出14个与gD蛋白结合的阳性克隆,并成功对阳性值较高的重组单域抗体IB68进行表达和纯化;经ELISA和Western-blot鉴定,重组单域抗体IB68可与gD蛋白产生抗原抗体结合反应,具有较高的亲和力。说明制备的IBRV特异性噬菌体单域抗体库具有较高的库容和多样性,且筛选得到的单域抗体具有较高的抗原抗体结合活性和免疫学反应性。

In order to develop an effective antibody for the treatment and diagnosis of Infectious bovine rhinotracheitis virus(IBRV),and constructed a single domain antibody library and screened against IBRV gD protein,and the positive clones were expressed,purified and identified.The results showed that the constructed IBRV-specific phage single domain antibody library was 7×10^7.After three rounds of enrichment and screening,phage-ELISA screened 14 positive clones bound to gD protein,and successfully expressed and purified IB68 clones with higher positive values.ELISA and Western-blot results confirmed IB68 could specific bind to gD protein with high affinity.These results indicated that we constructed a single domain antibody library with high capacity and diversity,and the single domain antibody screened from the library has a high binding activity and reactivity to IBRV.