F型肉毒毒素单克隆抗体的筛选及评价  

Preparing and Evaluation of Monoclonal Antibodies to Botulinum Toxin Type F

作　　者：马骏池 许永亮 张惟材 熊向华 张秀娟[2] MA Jun-chi;XU Yong-liang;ZHANG Wei-cai;XIONG Xiang-hua;ZHANG Xiu-juan(Institute of Biotechnology,Academy of Military Medical Sciences,Beijing 100071,China;College of Pharmacy,Harbin University of Commerce,Harbin 150076,China;School of Life Sciences,Liaoning University,Shenyang 110034,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所,北京100071 [2]哈尔滨商业大学药学院,黑龙江哈尔滨150076 [3]辽宁大学生命科学学院,辽宁沈阳1100361

出　　处：《药物生物技术》2022年第2期116-120,共5页Pharmaceutical Biotechnology

基　　金：国家科技重大专项(No.2018X09J18105-003)。

摘　　要：研究以筛选得到中和抗体为目的,通过抗体制备的常用手段——杂交瘤技术进行,以F型肉毒毒素受体结合结构域FHc端作为单克隆抗体的筛选抗原。利用现有抗原FHc蛋白辅助佐剂对动物进行免疫,免疫结束后无菌环境中取动物脾细胞,与预先培养的SP2/0细胞融合后HAT选择性培养基筛选得到阳性杂交瘤细胞株,大量培养后通过体内诱生法制备单抗,纯化后对所得鼠源抗体各指标进行评价,结果测得该单抗亚型为IgG1,κ型、经过Protein G柱纯化后抗体纯度可达到98%以上、与FHc端结构域亲和力可达到5.2×10^(-8)mol/L、轻重链基因调取后经Igblast tool分析得抗体可变区中轻链及重链CDR3区氨基酸序列为QHIRELTR、ARWLGDYAMDY,采用10倍半数致死量进行攻毒实验可得,该抗体中和效价在200 LD_(50)/mg,当抗体量达到50μg就可以实现完全保护。实验通过细胞融合的方式,在聚乙二醇的化学刺激下,筛得一株阳性杂交瘤细胞株,取细胞上清,以亲和力作为筛选指标,得到了一株针对F型肉毒毒素受体结合结构域FHc端的具有高亲和力和并同时具有中和效果的鼠源单克隆抗体,所得单抗为具有潜在临床应用价值的鼠源单克隆抗体,为后续治疗药物的开发即鼠源抗体人源化奠定了基础。The purpose of the research is to screen and obtain neutralizing antibodies.The F-type botulinum toxin receptor binding domain FHc end is used as the screening antigen of monoclonal antibodies through hybridoma technology,a common method of antibody preparation.Use the existing antigen FHc protein auxiliary adjuvant to immunize the animal.After the immunization,the animal spleen cells were collected in a sterile environment,and then fused with pre-cultured SP2/0 cells and then HAT selective medium is screened to obtain positive hybridoma cell lines.After culturing,the monoclonal antibody was prepared by in vivo induction method.After purification,the indicators of the obtained murine antibody were evaluated.The results showed that the monoclonal antibody was IgG1,κtype,and the purity of the antibody after purification by Protein G column could reach 98%or more.The affinity with the FHc terminal domain can reach 5.2×10^(-8)mol/L.Then the light and heavy chain genes are retrieval and analyzed by Igblast tool,the amino acid sequences of the light and heavy chain CDR3 regions in the antibody variable region are QHIRELTR,ARWLGDYAMDY.The neutralizing titer of the antibody is 200 LD_(50)/mg,and complete protection can be achieved when the antibody amount reaches 50μg through 10 LD_(50)challenge experiment.In the experiment,a positive hybridoma cell line was screened under the chemical stimulation of polyethylene glycol through the method of cell fusion,and the cell supernatant was taken,and the affinity was used as the screening index to obtain a cell line targeting the F-type botulinum toxin receptor.A murine monoclonal antibody with high affinity and neutralizing effect that binds to the FHc end of the domain was obtained,the monoclonal antibody is a murine monoclonal antibody with potential clinical application value.It is a mouse-derived human antibody and laid the foundation for the development of subsequent therapeutic drugs.

关 键 词：F型肉毒毒素 单克隆抗体 杂交瘤技术 细胞融合 抗体纯化 中和抗体 

分 类 号：R392.116[医药卫生—免疫学]