高亲和力单克隆抗体CL15识别的表位  

The conformational epitope recognized by high affinity monoclonal anti-plasmin antibody from patients with antiphospholipid syndrome

作　　者：陈晓翔[1] 张宇杲[2] 杨程德[1] 顾越英[1] 吕良敬[1] 叶萍[1] 王元[1] 陈顺乐[1] 

机构地区：[1]上海交通大学医学院附属仁济医院风湿免疫科上海市风湿病研究所风湿病临床医学中心,上海200001 [2]复旦大学基因与分子实验室,上海200433

出　　处：《现代免疫学》2006年第4期297-301,共5页Current Immunology

基　　金：国家自然科学基金资助项目(30371332);上海市科委基础研究重点项目(03JC14039);教育部归国基金资助;上海市曙光学者计划项目;上海市教委重点学科基金资助项目(T0203)

摘　　要：鉴定抗磷脂综合征患者来源的抗纤溶酶单抗CL15所识别的结构域，并运用生物信息学和计算机辅助设计对CL15进行表位分析。使用分子克隆的方法克隆并纯化了纤溶酶的结构域，ELISA方法检测CL15对天然纤溶酶和微小纤溶酶的结合能力。根据CL15的抗体的互补区（CDR）的已知序列，在PBD蛋白库中进行同源比较，获得了同源性较高的单抗IMFA的Fab段的晶状结构，用计算机软件把CL15的CDR区序列优化进入IMFA后，根据结构域的空间结构与CL15进行对接。结果，高亲和力结合纤溶酶的单抗CL15识别纤溶酶的结构域，通过对CL15的晶体结构的同源模建，计算机模拟与微小纤溶酶结合，CL15在纤溶酶的酶活性区的606～789中的部分氨基酸序列有明显的相互作用，存在表位为空问构象，能将纤溶酶的丝氨酸活化中心屏蔽。CL15能识别微小纤溶酶，抗原表位在纤溶酶的丝氨酸的活性中心区域。CL15能阻断丝氨酸蛋白酶的活性中心的关键氨基酸，降低纤溶酶的活性，可能是最终导致抗磷脂综合征病人血栓形成的原因之一。To identify tbe domain recognized by the high affinity monoclonal anti-plasmin antibody CL15 of from patients with antiphospholipid syndrome patient and to predict the potential epitope of CL15 by bio-informatics, the plasmin enzyme domain （micro-plasminogen, uPLG）, was cloned and purify and the binding ability of CL15 to native plasmin and uPLG; were analyzed by ELISA. The information of the conformational determination regions（CDR）of monoclonal antibody CL15 and the crytal structure of model monoclonal antibody IMFA were collected in reference and protein data hank （PBD, http：//www, rcsb. org/pdb/） . The heavy and light chains sequences CDR were blasted in PBD, then the CDR sequences were optimized into an crystal structure of an Fab I MFA. According to the conformational structure of theuPLG, in-silicon calculated the interaction between CL15 and uPLG. It was demonstrated that the high affinity anti-plasmin monoclonal antibody CL15 could identify the enzyme activity center of plasmin. The recombined fusion protein uPLG was recognized by 471.15, and the epitope of uPLG was conformational determination covered the amino acid sequence from 606 to 789, binding to one key amino acid of activity center of plasmin. It is concluded that monoclonal antibody CL15 can bind to the protease domain of plasmin and interact with a key a mino acid in activity center, which may inhibit the degration of fibrinolysis and cause thrombosis in antiphospholipid syndrome.

关 键 词：表位 生物信息学 纤溶酶 抗磷脂综合征 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]