牛病毒性腹泻病毒纳米抗体文库构建与筛选

Construction and Screening of Nanobody Library Against BVDV

旨在获得牛病毒性腹泻病毒(BVDV)特异性纳米抗体。通过用BVDV-E0重组蛋白对羊驼进行免疫,分离血液中的淋巴细胞。利用噬菌体展示技术,构建噬菌体展示文库,经过连续3次生物淘筛获得与BVDV-E0蛋白结合的噬菌体,对所得VHH序列进行测序和基因比对。用ELISA筛选出抗BVDV-E0的高亲和力纳米抗体,并验证纳米抗体的亲和力和活性。结果成功构建了插入率为86%、库容量为1.3×1011 cfu的噬菌体表达文库,经过筛选获得5个BVDV-E0阳性单克隆,将这些基因克隆至原核表达体系,表达和纯化后获得了高纯度的BVDV-E0纳米抗体,经亲和力的鉴定获得2个不同的高亲和力的VHH基因,并且能结合人工抗原E0,还能够被竞争抗体所阻断。研究结果为用于组装检测BVDV试剂盒和研制BVDV疫苗奠定了基础。

The aim was to obtain the specific nanobodies against bovine viral diarrhea virus（BVDV）.Using BVDV-E0 recombinant protein to immunize the alpaca,the lymphocytes in the blood were separated.The phage display library was constructed by using phage display technology,and then the phages combined with BVDV-E0 protein were obtained through three successive bioscai sieves,the obtained VHH sequences were sequenced and alignmented.The high affinity nanobody against BVDV-E0 was screened by ELISA,and then the affinity and activity of the nannobody were verified.The results showed that phage expression library with a insertion rate 86%,the database 1.3×1011 cfu was successfully constructed.Five BVDV-E0 positive clones were obtained by screening,these genes were cloned into prokaryotic expression system,and the high purity BVDV-E0 nanobodies were obtained.Two different high affinity VHH genes were obtained by affinity identification.The high purity BVDV-E0 nanobodies could be combined with artificial antigen E0,and also be blocked by competitive antibodies.Thies study laid a foundation for further study on assembly of BVDV kit and development of BVDV vaccine.