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

doi:10.16431/j.cnki.1671-7236.2021.01.034

Abstract

Abstract: 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.

Key words: Bovine viral diarrhea virus (BVDV); NS3; nanobody; phage display technology

CLC Number:

S852.65

YANG Yan, QIAN Tianhao, ZHANG Yanhong, ZHANG Zhe, HAO Xiujing, LI Yan, LYU Wenhua, LIU Zhao, SHENG Jinliang. Screening and Reactogenicity Test of Bovine Viral Diarrhea Virus NS3 Protein Nanobody[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 303-311.

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

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