抗犬瘟热病毒VHH抗体噬菌体库的构建与筛选

doi:10.16431/j.cnki.1671-7236.2020.06.006

Abstract

Abstract: In order to construct a specific nanobody library of canine distemper virus (CDV) and obtain VHH antibody against CDV,alpaca was immunized with CDV.After four immunizations,peripheral blood lymphocytes were collected,total RNA was extracted and transcribed into cDNA,and the nanoantibody sequence was amplified by nested PCR.The target fragment was connected to the pComb3x phage display vector,and transferred to the TG1 host bacteria.40 clones were selected for liquid PCR verification.13 positive monoclonals were selected randomly for sequencing.The auxiliary phage was added to save the phage display antibody library.After three rounds of panning,the phages with high binding capacity to CDV were enriched.Pichia pastoris was used to express two phages with high binding capacity.After purification by Ni column,the binding capacity of phages was identified by ELISA.The results showed that the titer of alpaca serum reached 1:25 000 after four immunizations,and the capacity of phage display library reached 3.41×10⁹ PFU.After three rounds of panning,the specific antibody library was diluted 100 times,and the ELISA was still positive,indicating that the phage with specific binding to CDV significantly enriched.The results of ELISA showed that the reactivity of the two purified antibodies to CDV was significantly higher than that of the control group.These results suggested that two specific CDV binding VHH antibodies were successfully screened in this study,which laid a foundation for the application of VHH antibody in the diagnosis and treatment of canine distemper.

Key words: canine distemper virus (CDV); VHH antibody; M13 phage display; eukaryotic expression

CLC Number:

S855.3

WANG Zhaoyang, JIANG Yajun, LIU Xueting, LIN Weidong, XIN Ting, HOU Shaohua, GUO Xiaoyu, ZHU Hongfei, JIA Hong. Construction and Screening of Phage Library Against VHH Antibody of Canine Distemper Virus[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1685-1693.

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Construction and Screening of Phage Library Against VHH Antibody of Canine Distemper Virus

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