针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

Toxin-neutralizing monoclonal antibodies to the different domains of anthrax protective antigen

炭疽保护性抗原(PA)是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。

Anthrax toxin from Bacillus anthracis is a three-component toxin consisting of lethal factor （ LF）, edema factor （EF）, and protective antigen （PA）. PA binds to target ceils and transports LF or EF into the cell cytosol where they carry out their enzymatic functions. PA can induce protective immunity to the infection of the bacterium and is the major component in the only anthrax vaccine approved by FDA of USA. Mouse hybridoma clones specifically secreting anti-PA monoclonal antibodies （MAbs） were generated by cell fusion technique and their ability to neutralize anthrax lethal toxin activities was screened in vitro on a toxin-sensitive cell line. Nine toxin-neutralizing MAbs obtained were then characterized for the domains of PA they recognize, and the epitope regions they bind were analyzed by competitive binding ELISA. It was found that these MAbs bind four potential neutralizing epitope regions in three different domains of PA. Four MAbs bind to two non-overlapping epitope regions in domain 4 of PA and may prevent the binding of PA to its cell receptor. Four MAbs bind to domain 2, a domain involved in membrane insertion. One MAb binds to domain 3, a region involved in the oligomerization of PA. The results provided supporting evidence that PA has several neutralizing epitopes, and offered potential immunotherapeutic agents for the treatment of anthrax.