牛病毒性腹泻病毒纳米抗体文库构建与筛选
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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.
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.
引文
[1]Moennig V,Lindberg H H A.BVD control in Europe:current status and perspectives[J].Animal Health Res Rev,2005,6(1):63-74.
[2]韩猛立,黄新,钟发刚,等.牛病毒性腹泻病流行现状与防制[J].中国奶牛,2010(3):35-38.
[3]Lang Q,Wang F,Yin L,et al.Specific probe selection from landscape phage display library and its application in enzymelinked immunosorbent assay of free prostate-specific antigen[J].Anal Chem,2014,86(5):2767-2774.
[4]贾莹.BVDV NS3表位串联及抗体间接ELISA方法的建立.东北农业大学[J].2011.
[5]项勋,段纲,李志敏,等.牛病毒性腹泻-粘膜病病毒(BVDV)的分子生物学研究进展[J].家畜生态学报,2004,25(4):202-204.
[6]范进江,薄新文,钟发刚,等.牛病毒性腹泻病毒基因组结构与蛋白功能研究进展[J].动物医学进展,2008,29(5):68-72.
[7]周陶然,詹守斌,翟飞,等.纳米抗体技术及其在疾病诊断和治疗中的应用[J].生物化学与生物物理进展,2016,43(10):936-945.
[8]Fridy P C,Li Y,Keegan S,et al.A robust pipeline for rapid production of versatile nanobody repertoires[J].Nat Meth,2014,11(12):1253-1260.
[9]Goethals L R,Bos T J,Baeyens L,et al.Camelid reporter gene imaging:ageneric method for in vivo cell tracking[J].Ejnmmi Res,2014,4(1):1-8.
[10]Maass D R,Sepulveda J,Pernthaner A,et al.Alpaca(Lama pacos)as a convenient source of recombinant camelid heavy chain antibodies(VHHs)[J].J Immunol Meth,2007,324:1-2.
[11]Rümenapf T,Unger G,Strauss J H,et al.Processing of the envelope glycoproteins of pestiviruses[J].J Virol,1993,67(6):3288-3294.
[12]蒋禄峰,赵月兰,李苏楠,等.牛病毒性腹泻病毒HB-DCZ株E0基因的克隆与抗原表位预测[J].中国兽医学报,2013,33(7):979-984.
[2]韩猛立,黄新,钟发刚,等.牛病毒性腹泻病流行现状与防制[J].中国奶牛,2010(3):35-38.
[3]Lang Q,Wang F,Yin L,et al.Specific probe selection from landscape phage display library and its application in enzymelinked immunosorbent assay of free prostate-specific antigen[J].Anal Chem,2014,86(5):2767-2774.
[4]贾莹.BVDV NS3表位串联及抗体间接ELISA方法的建立.东北农业大学[J].2011.
[5]项勋,段纲,李志敏,等.牛病毒性腹泻-粘膜病病毒(BVDV)的分子生物学研究进展[J].家畜生态学报,2004,25(4):202-204.
[6]范进江,薄新文,钟发刚,等.牛病毒性腹泻病毒基因组结构与蛋白功能研究进展[J].动物医学进展,2008,29(5):68-72.
[7]周陶然,詹守斌,翟飞,等.纳米抗体技术及其在疾病诊断和治疗中的应用[J].生物化学与生物物理进展,2016,43(10):936-945.
[8]Fridy P C,Li Y,Keegan S,et al.A robust pipeline for rapid production of versatile nanobody repertoires[J].Nat Meth,2014,11(12):1253-1260.
[9]Goethals L R,Bos T J,Baeyens L,et al.Camelid reporter gene imaging:ageneric method for in vivo cell tracking[J].Ejnmmi Res,2014,4(1):1-8.
[10]Maass D R,Sepulveda J,Pernthaner A,et al.Alpaca(Lama pacos)as a convenient source of recombinant camelid heavy chain antibodies(VHHs)[J].J Immunol Meth,2007,324:1-2.
[11]Rümenapf T,Unger G,Strauss J H,et al.Processing of the envelope glycoproteins of pestiviruses[J].J Virol,1993,67(6):3288-3294.
[12]蒋禄峰,赵月兰,李苏楠,等.牛病毒性腹泻病毒HB-DCZ株E0基因的克隆与抗原表位预测[J].中国兽医学报,2013,33(7):979-984.