Li Yanning, Li Guangqi, Wang Hongxia, Jiang Dan, Chu Yuankui, Zhang Aijun, Xu Guangxian
Department of Medical Laboratory, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China.
Department of Medical Laboratory, School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004; Department of Medical Laboratory, School of Medical Technology, Guangdong Medical University, Dongguan 523000, China. *Corresponding author, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2021 Jan;37(1):67-72.
Objective To screen the sequence of nanobodies against human CD20, and obtain anti-CD20-human IgG Fc nanobodies with high affinity and specificity. Methods Based on the naive phage display library, 4 rounds of liquid affinity screening were performed using biotinylated CD20 antigen as the target, and positive clones were identified by ELISA. Prokaryotic expression vector CD20-IgG Fc/pCZN1 was constructed and transformed into E.coli Arctic Express, and the expression of the recombinant protein was induced by IPTG at low temperature and purified by Ni column. The purified product was identified by ELISA and Western blot analysis. Results The specific CD20 nanobody showed good repeatability and hydrophilicity. The purity of anti-CD20-human IgG Fc nanobodies was higher than 85%. ELISA indicated that anti-CD20-human IgG Fc nanobodies had high affinity with CD20 antigen, and Western blot analysis demonstrated they could specifically recognize CD20 antigen. Conclusion The sequence of anti-CD20 nanobody was successfully obtained using the naive phage nanobody library. The purified anti-CD20-human IgG Fc nanobody has high affinity and specificity.
目的 筛选抗人CD20纳米抗体序列，获得具有高亲和力和特异性的抗CD20-人IgG Fc纳米抗体。方法 以原始噬菌体展示文库为基础，采用生物素化的CD20抗原作为靶标进行4轮液相亲和筛选，通过ELISA鉴定阳性克隆。构建原核表达载体CD20-IgG Fc/pCZN1并转化至大肠杆菌Arctic Express中，用IPTG在低温下诱导重组蛋白表达，经镍柱纯化。纯化产物通过ELISA和Western blot分析进行鉴定。结果 特异性CD20纳米抗体表现出良好的重复性和亲水性。抗CD20-人IgG Fc纳米抗体的纯度高于85%。ELISA表明抗CD20-人IgG Fc纳米抗体与CD20抗原具有高亲和力，Western blot分析证明它们能特异性识别CD20抗原。结论 利用原始噬菌体纳米抗体文库成功获得抗CD20纳米抗体序列。纯化的抗CD20-人IgG Fc纳米抗体具有高亲和力和特异性。
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