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

作　　者：丁金花[1] 李启峰 马旭升 肖红冉[1] 肖盛中 吴鹏[1] 盛金良[1] 陈创夫[1] DING Jin hua;LI Qi -feng;MA Xu -sheng;XIAO Hong -ran;XIAO Sheng -zhong;WU Peng;SHENG Jin -liang;CHEN Chuang -fu(College of Animal Science and Technology, Shihezi University, Shihezi , Xinjiang , 832000, China;College of Life Science, Shihezi University, Shihezi , Xinjiang , 832000, China;Tian Kang Biological Co.Ltd. , Urumqi , Xinjiang , 830001, China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]天康生物股份有限公司,新疆乌鲁木齐830001 [3]石河子大学生命科学学院,新疆石河子832000

出　　处：《动物医学进展》2018年第5期6-12,共7页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(U1303283)

摘　　要：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.

关 键 词：牛病毒性腹泻病毒E0 纳米抗体 生物淘选 噬菌体展示技术 

分 类 号：S852.653[农业科学—基础兽医学]