金黄色葡萄球菌肠毒素B精制免疫球蛋白对小鼠中毒模型的免疫救治效果研究  

Protection from Staphylococcal Enterotoxin B Intoxication in a Mouse Model by Purified Immunoglobulin

作　　者：韩慧 李敏 杨晓岚 段跃强[2] 谷宏婧 范铁炯 李鑫 杨鹏辉 刘媛 闫芳[5] 郑源强[1] 石艳春[1] 赵忠鹏[2] HAN Hui;LI Min;YANG Xiao-Lan;DUAN Yue-Qiang;GU Hong-Jing;FAN Tie-Jiong;LI Xin;YANG Peng-Hui;LIU Yuan;YAN Fang;ZHENG Yuan-Qiang;SHI Yan-Chun;ZHAO Zhong-Peng(Key Laboratory of Molecular Biology,Inner Mongolia Medical University,Huhehaote 010058;State Key Laboratory of Pathogen and Biosecurity,Beijing Institute of Microbiology and Epidemiology,Beijing 100071;Shanghai Serum Bio-technology Co.,LTD,Shanghai 201707;Fifth Medical Center of PLA General Hospital,Beijing 100039;College of Animal Science and Veterinary Medicine,Shanxi Agricultural University,Taigu 030800,China)

机构地区：[1]内蒙古自治区分子生物学重点实验室,内蒙古医科大学,内蒙古呼和浩特010058 [2]病原微生物生物安全国家重点实验室,军事科学院军事医学研究院微生物流行病研究所,北京100071 [3]上海赛伦生物技术股份有限公司,上海201707 [4]解放军总医院第五医学中心,北京100039 [5]山西农业大学动物科技学院,山西太谷030800

出　　处：《生物技术通讯》2019年第6期756-761,共6页Letters in Biotechnology

基　　金：国家重点研发计划（2017YFC1200900,2018ZX09J19106）;国家自然科学基金（81871252）

摘　　要：目的:研制金黄色葡萄球菌肠毒素B(SEB)精制免疫球蛋白,即马抗SEB免疫球蛋白F(ab′)2制品,为SEB中毒的救治提供候选药。方法:利用传统的发酵和纯化工艺制备天然SEB纯品,并建立小鼠实验动物模型;利用分子克隆和蛋白质纯化技术,克隆、表达、纯化重组SEB,加入适量弗氏佐剂充分乳化,免疫马匹,测定免疫马匹血清中和抗体效价后采血,分离血清,按照成熟的生产工艺制备治疗性抗体成品;对成品进行安全性检测及免疫救治效果评价。结果:纯化的天然SEB经SDS-PAGE和HPLC法测定纯度大于90%,对小鼠腹腔注射的半数致死量为0.5μg/kg;纯化的重组SEB表达量超过2 mg/mL,经SDS-PAGE和HPLC法测定纯度大于90%,对小鼠腹腔注射的半数致死量为50μg/kg;免疫马匹血清中和抗体效价达2000 U/mL以上时,采血并制备F(ab′)2成品,经SDS-PAGE和HPLC法测定纯度在80%以上,中和抗体效价超过2500 U/mL;安全性符合现行《中国药典》要求;对SEB中毒动物模型有很好的免疫救治效果。结论:建立了SEB精制免疫球蛋白制品生产工艺,制备的成品符合现行《中国药典》要求,为SEB中毒的救治提供了候选药。Objective:Until now,there have been no specific medicines to treat staphylococcal enterotoxin(SE)-poisonedpatients.Development of a safe and effective SE therapeutic preparation is urgently needed.Methods:To this end,we constructed SEB intoxication mouse model with purified natural SEB and lipopolysaccharide(LPS).Meanwhile,we prepared the pepsin-digested F(ab′)2 fragments of serum IgGs from the horses inoculated with Freund's-adjuvanted purified recombinant SEB antigens.The protective effects of the F(ab′)2 fragments against SEB were evaluated in a mouse model by observing death and weight loss.Results:The purity quotients of the natural and recombinant SEB were both more than 90%,which were assayed by SDS-PAGE and HPLC.Meanwhile,the half lethal dose(LD50)of the natural and recombinant SEB were 0.5 and 50μg/kg,respectively,which were assayed by Reed-Muench method.The purity quotients of the F(ab′)2 fragments against SEB were more than 80%,which were safe to the animal subjects and meet the requirements of Chinese Pharmacopoeia according to the reports of Joinn Laboratories(China)Co.Ltd.The in vivo assays suggested that the preparations could efficiently protect mice from a lethal dose of SEB,which can reach 2500 U/mL.Conclusion:This highly purified preparation should be a potential candidate for treating severe patients suffering from SEB.

关 键 词：金黄色葡萄球菌肠毒素B 免疫球蛋白 动物模型 

分 类 号：R392-33[医药卫生—免疫学]