论文部分内容阅读
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。
Anthrax is an infectious disease caused by Bacillus anthracis that poses a serious threat to human health. Anthrax toxin consists of three protein components: protective antigen (PA), lethal factor (LF) and edema factor (EF). PA and LF form lethal toxin (LT) and form edema toxin (ET) with EF. Since lethal toxins (LTs) play a major role in the injury and death of infected individuals, treatment with antibiotics alone in the late stages of anthrax infection is difficult to achieve and therapeutic neutralizing antibodies are the most effective anthrax therapeutics available. Currently obtained anthrax toxin antibodies are mostly anthrax PA antibodies, the United States FDA has approved Ruixi Baku (human PA monoclonal antibody) for the treatment of inhaled anthrax. Once Bacillus anthracis has been artificially altered or a PA neutralizing epitope has been mutated, antibodies against a single epitope of PA will likely fail, so antibodies to LF will be an effective complement to anthrax therapy. At present, most of the foreign LF antibodies are murine antibodies and chimeric antibodies. However, full-human antibodies can avoid the disadvantages of high immunogenicity of murine antibodies. In this study, first, human anti-LF transgenic mice were immunized with LF antigen, and antigen-specific memory B cells were sorted from mouse spleen lymphocytes by flow cytometry. Two monoclonal antibodies against LF were rapidly obtained by single-cell PCR 1D7 and 2B9. Expi 293F cells were transiently transfected to produce antibodies, and 1D7 and 2B9 showed good neutralizing activity in the cell model by the toxin neutralization assay (TNA), and showed better synergistic effect when used in combination with PA monoclonal antibody . In conclusion, this study utilized the advantages of transgenic mice, flow cytometry and single-cell PCR to rapidly screen whole-body LF antibodies and opened up new ideas and methods for the rapid screening of all-human monoclonal antibodies.