牛病毒性腹泻病毒NS5A蛋白纳米抗体的筛选与鉴定

Screening and Identification of Nanobodies against Bovine Viral Diarrhea Virus NS5A Protein

为防控牛病毒性腹泻的流行,淘选得到抗BVDV-NS5A的纳米抗体序列,并探究其结合活性。通过重组质粒pET30a-NS5A诱导表达并纯化蛋白,免疫羊驼,获得重组质粒pCANTAB 5E-VHH,构建了一个初始的抗BVDV-NS5A纳米抗体展示文库。利用3轮固相亲和筛选,对筛选后的单克隆抗体通过ELISA验证其特异性与结合力并测序、分析。构建出具有较好多样性的噬菌体展示文库,测得库容为5.3×10^(7) CFU/mL,插入率73.9%。通过评价和分析显示,得到20株不同氨基酸序列的纳米抗体,其中7株与NS5A蛋白具有较好反应性。针对抗BVDV-NS5A蛋白淘选纳米抗体,并证明得到的纳米抗体有较好的结合力。研究结果有望用于研发BVDV诊断试剂盒、抗BVDV的候选药物,同时也为后续BVDV的致病机制研究奠定了基础。

In order to prevent and control the epidemic of bovine viral diarrhea,the nanobody sequence against BVDV-NS5A was selected and its binding activity was explored.The protein was induced and purified by recombinant plasmid pET30a-NS5A,and alpaca was immunized to obtain recombinant plasmid pCANTAB 5E-VHH.An initial nanobody display library against BVDV-NS5A was constructed.Using three rounds of solid phase affinity and screening,the specificity and binding ability of the screened monoclonal antibodies were verified by ELISA,sequenced and analyzed.The phage display library with good diversity was constructed,and the measured library capacity was 5.3×10^(7) CFU/ml,insertion rate 73.9%.Evaluation and analysis showed that 20 nanobodies with different amino acid sequences were obtained,of which 7 had good reactivity with NS5A protein.Panning nanobodies against BVDV-NS5A protein for the first time,and it is proved that the nanobodies had good binding ability.The results of panning are expected to be used to develop BVDV diagnostic kit,candidate drugs against BVDV,and lay a foundation for the subsequent research on the pathogenesis of BVDV.