金黄色葡萄球菌肠毒素B 的中和性全人源抗体制备  

Preparation of neutralizing full human monoclonal antibodes against Staphylococcus enterotoxin B

作　　者：刘成华 刘玉 冯健男 沈倍奋 杨光(指导) LIU Chenghua;LIU Yu;FENG Jiannan;SHEN Beifen;YANG Guang(Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]军事医学研究院毒物药物研究所,北京100850

出　　处：《中国免疫学杂志》2022年第12期1480-1483,1493,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金面上项目(81871618)资助。

摘　　要：目的:制备靶向金黄色葡萄球菌外分泌蛋白肠毒素B(SEB)的中和性全人源单克隆抗体,为SEB诱发的中毒性休克综合征(SEBILS)提供新的治疗方法。方法:以金黄色葡萄球菌COL基因组为模板,利用PCR技术扩增seb基因,进一步构建原核表达载体pET28a-seb,并克隆至大肠杆菌BL21(DE3),经IPTG诱导表达目的蛋白,通过Ni2+柱亲和纯化获得SEB蛋白;利用SEB诱导的小鼠SEBILS模型检测SEB活性;进一步通过噬菌体展示技术筛选SEB结合的噬菌体克隆并进行序列测定,以测序正确的阳性克隆质粒为模板,扩增该抗体的可变区基因并构建重组表达载体转染至293E细胞,并利用Protein A树脂亲和纯化抗体;利用SPR检测其亲和力,通过SEBILS模型验证抗体活性。结果:获得了高纯度重组SEB蛋白,且具有很好的活性;筛选出两株全人源靶向SEB的单克隆抗体YG11-1和YG11-2,SPR结果表明YG11-1、YG11-2的亲和力相当(其KD值分别为16.59 nmol/L和21.35 nmol/L),且YG11-1和YG11-2均能有效抵抗SEB诱导的小鼠中毒性休克综合征,降低小鼠的死亡率,YG11-2(200μg/只)时保护率能够达到100%。结论:本研究获得了具有良好活性的重组SEB蛋白,并制备了抗SEB两株全人源单克隆抗体,为研制SEB中毒治疗制剂提供了新的选择。Objective:To obtain a specific neutralizing monoclonal antibody of Staphylococcus enterotoxin B for the new treatmeat of SEB induced lethal shock(SEBILS).Methods:seb gene was amplified by PCR using S.aureus COL genomic DNA as the template.The expression vector pET28a-seb was constructed and transformed into E.coli BL21(DE3).The recombinant SEB protein expression was induced by IPTG and purified with Ni2+affinity chromatography.The murine models for SEBILS was used to detect the activities of recombinant SEB protein.The specific binders of SEB were screened by phage display.The genes encoding VH and VL were amplified by PCR,respectively.The recombinant expression vectors containing VH or VL genes were constructed,respectively and co-transfected into 293E cells.Monoclonal antibodies from the culture supernatants were purified by the Protein A affinity resin.The affinity of antibody was detected by surface plasmon resonance(SPR)and the function of antibodies were determined in the SEBILS murine models.Results:Recombinant SEB protein was expressed and purified.Two full human monoclonal antibodies(named as YG11-1 and YG11-2)against SEB were obtained by phage display.The results of SPR showed that YG11-1 and YG11-2 specifically bond to SEB with high affinity(KD value was 16.59 nmol/L and 21.35 nmol/L,respectively).Furthermore,it was found that both YG11-1 and YG11-2 protected mice from lethal shock induce by SEB,and administration of YG11-2(200μg/mouse)completely rescued mice with SEBILS.Conclusion:Our findings present two novel human monoclonal antibodies that can specifically block the lethal shock induced by SEB,which provide a new treatment method for SEBILS.

关 键 词：金黄色葡萄球菌 原核表达 重组SEB 中和性全人源单克隆抗体 

分 类 号：R392[医药卫生—免疫学]