驼源天然噬菌体纳米抗体文库的构建及抗CD22纳米抗体的筛选  

Construction of a camel-derived natural phage nanobody display library and screening of anti-CD22 nanobodies

作　　者：何婉钧 崔愷 张西倩 蒋丹 徐广贤 HE Wanjun;CUI Kai;ZHANG Xiqian;JIANG Dan;XU Guangxian(School of Medical Technology,Guangdong Medical University,Guangdong Medical University,Dongguan 523000,China;Precision Medicine Center affiliated to Dongguan First Hospital,Guangdong Medical University,Guangdong Medical University,Dongguan 523000,China;Guangdong Provincial Key Laboratory of Medical of Medical Immunology and Molecular Diagnostics,Guangdong Medical University,Dongguan 523000,China)

机构地区：[1]广东医科大学医学技术学院,广东东莞523000 [2]广东医科大学附属东莞第一医院精准医学中心,广东东莞523000 [3]广东医科大学广东省医学免疫与分子诊断重点实验室,广东东莞523000

出　　处：《细胞与分子免疫学杂志》2025年第3期254-261,共8页Chinese Journal of Cellular and Molecular Immunology

基　　金：宁夏回族自治区重点研发计划(2022CMG03123,2022CMG02041);广东省基础与应用基础研究基金(2023A1515140148);广东医科大学附属东莞第一医院高层次人才科研资助计划项目(GCC2023004);广东医科大学博士科研启动基金(4SG23190G)。

摘　　要：目的筛选出抗CD22特异性纳米抗体,为其作为免疫治疗制剂提供基础。方法构建天然噬菌体纳米抗体展示文库,分析其多样性。以生物素化CD22抗原为靶点,进行3轮生物素链霉亲和素液相筛选法,初步通过ELISA和基因测序鉴定抗CD22纳米抗体序列。结果经测定,构建的噬菌体纳米抗体展示文库容量为3.89×10~9 CFU/mL,插入有效片段高于85%。基于此文库,初步筛选出7条抗CD22纳米抗体,其氨基酸序列比对结果显示,整体相似性为70.34%,均为亲水性蛋白。蛋白-蛋白复合物对接预测结果表明,5条纳米抗体序列的模拟蛋白都可以配对连接到CD22,主要作用力为疏水相互作用和氢键。结论本研究为靶向CD22的嵌合抗原受体T细胞的研究提供基础,成功构建天然噬菌体纳米抗体展示文库,获得5条抗CD22特异性的纳米抗体。Objective To screen the anti-CD22-specific nanobodies to provide a basis for immunotherapy agents.Methods The naive phage nanobody library was constructed and its diversity was analyzed.Three rounds of biotinylated streptavidin liquid phase screening were performed by using biotinylated CD22 antigen as the target,and the sequence of nanobodies against CD22 were identified by ELISA and gene sequencing.Results The capacity of the constructed naive phage nanobody library was 3.89×109 CFU/mL,and the insertion of effective fragments was higher than 85%.Based on this library,seven anti-human CD22 nanobodies were screened,and the amino acid sequence comparison results showed that the overall similarity was 70.34%,and all of them were hydrophilic proteins.The results of protein-protein complex docking prediction showed that the mimetic proteins of the five nanobody sequences could be paired and linked to CD22,and the main forces were hydrophobic interaction and hydrogen bonding.Conclusion This study provided a basis for the study of chimeric antigen receptor T cells targeting CD22,successfully constructed the natural phage nanobody library and obtaining five anti-CD22-specific nanobodies.

关 键 词：CD22 纳米抗体 噬菌体展示文库 

分 类 号：R392[医药卫生—免疫学] R730.51[医药卫生—基础医学] S852.43[农业科学—基础兽医学]