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

摘要/Abstract

摘要： 为构建特异性犬瘟热病毒（CDV）的纳米抗体库，获得抗CDV的VHH抗体，本试验利用CDV免疫羊驼，四免后采集外周血淋巴细胞，提取总RNA反转录为cDNA，利用巢式PCR扩增纳米抗体序列。将目的片段连接至pComb3x噬菌体展示载体，并电转至TG1宿主菌，挑取40个克隆进行菌液PCR验证，随机挑选13个阳性单克隆进行测序，计算抗体库库容量，加入辅助噬菌粒拯救获得的噬菌体展示抗体库。经过3轮淘选，富集对CDV结合力高的噬菌体。利用毕赤酵母系统表达两株结合力高的噬菌体，经Ni柱纯化后，利用ELISA进行噬菌体结合力的鉴定。结果表明，四免后羊驼血清效价达1：25 000，达到建库要求，构建的噬菌体展示文库库容量达3.41×10⁹ PFU。经过3轮淘选，特异性抗体库经稀释100倍后，ELISA检测仍为阳性，表明特异性结合CDV的噬菌体得到明显的富集。ELISA结果表明，两株纯化的纳米抗体与CDV的反应性显著高于对照组。以上结果提示，本研究成功筛选出2株特异性结合CDV的VHH抗体，为VHH抗体在犬瘟热的诊断和治疗方面的应用奠定了基础。

关键词: 犬瘟热病毒(CDV); VHH抗体; M13噬菌体展示; 真核表达

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

中图分类号:

S855.3

王召阳, 蒋亚君, 刘雪婷, 林伟东, 鑫婷, 侯绍华, 郭晓宇, 朱鸿飞, 贾红. 抗犬瘟热病毒VHH抗体噬菌体库的构建与筛选[J]. 中国畜牧兽医, 2020, 47(6): 1685-1693.

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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