牛病毒性腹泻病毒NS3蛋白纳米抗体的筛选及反应原性检测

本研究旨在获得高效特异性的牛病毒性腹泻病毒（BVDV）NS3（P80）非结构蛋白的纳米抗体。用BVDV灭活疫苗免疫羊驼，测得抗体效价后分离全血中的淋巴细胞。通过噬菌体展示技术构建羊驼重链抗体可变区噬菌体展示文库。经过连续3次吸附-洗脱-扩增的生物筛淘，从中挑选出与BVDV-NS3蛋白结合的噬菌体。对经菌液PCR、琼脂糖凝胶电泳鉴定到的单域抗体（VHH）克隆进行基因测序和同源性比对。用ELISA方法验证筛选出的纳米抗体的反应原性，找到与BVDV-NS3蛋白亲和力高的纳米抗体。结果表明，获得插入率为92.8%、库容为1.84×10¹⁴ CFU/mL的噬菌体展示文库。ELISA结果和氨基酸序列分析显示，成功得到1条与BVDV-NS3蛋白具有良好反应性且与VHH同源性较高的纳米抗体序列。本研究利用大肠杆菌成功表达BVDV-NS3抗原蛋白，建立BVDV纳米抗体噬菌体展示文库，筛选到针对BVDV重要抗原蛋白相应的纳米抗体且与VHH同源性较高。试验结果为牛病毒性腹泻/黏膜病的防控、诊断、治疗及纳米抗体药物的研制提供参考。

Screening and Reactogenicity Test of Bovine Viral Diarrhea Virus NS3 Protein Nanobody

The aim of this study was to obtain highly efficient and specific nanobodies of Bovine viral diarrhea virus (BVDV) NS3 (P80) non-structural proteins.The BVDV inactivated vaccine was used to immunize alpacas and lymphocytes in whole blood were isolated after antibody titer was measured.Phage display technique was used to construct phage display library of heavy chain antibody variable region of alpaca.The phage binding to BVDV-NS3 protein was selected through three successive adsorption-elution-amplification biological screening.Gene sequencing and homology comparison were performed for the cloning of positive nanobody (VHH) identified by PCR and agargel electrophoresis.ELISA was used to verify the reactogenicity of the selected nanobodies,and the nanobodies with high affinity to BVDV-NS3 protein were found.The results showed that the phage display library with insertion rate of 92.8% and storage capacity of 1.84×10¹⁴ CFU/mL was obtained.ELISA results and amino acid sequence analysis showed that a nanometer antibody sequence with good reaction to BVDV-NS3 protein and high homology to VHH was successfully obtained.In this study,Escherichia coli was used to successfully express BVDV-NS3 antigen protein,the phage display library of BVDV nanobody was established,the corresponding nanometer antibodies against the important antigen protein of BVDV were screened and had high homology with VHH.The results provided references for the prevention and control,diagnosis and treatment and the development of nanobody drugs of bovine viral diarrhea-mucosal disease.