利用转基因小鼠筛选全人源炭疽致死因子中和抗体

王潇霖¹ 迟象阳¹ 刘炬¹ 刘威岑¹ 刘树玲¹ 邱顺芳¹ 温中华¹ 范鹏飞¹ 刘坤¹ 宋小红¹ 付玲¹ 张军¹ 于长明¹

(1.军事医学科学院生物工程研究所, 北京 100071)

【摘要】炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。

【关键词】 炭疽致死因子; 转基因小鼠; 流式分选; 记忆B细胞; 单细胞PCR; 全人源单克隆抗体;

【DOI】

【基金资助】 国家自然科学基金(No.81172980) National Natural Science Foundation of China(No.81172980) 国家高技术研究发展计划(863计划)(No.2014AA021407)资助 National High Technology Research and Development Program of China(863 Program)(No.2014AA021407)

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