SARS-CoV刺突蛋白受体结合区的表达及高潜在中和性人源抗体的制备  

Expression of Receptor-binding Domain of SARS-CoV Spike Protein and Isolation of Highly Potent Neutralizing Antibody

作　　者：李郁[1,2] 姚西英[1,2] 杨勇[1,2] 陈江浩[1,2] 张思河[1,2] 宋斐[1,2] 包国强[1,2] 杨向民[1,2] 陈志南[1,2] 

机构地区：[1]第四军医大学细胞工程研究中心第四军医大学西京医院,西安710032 [2]第四军医大学细胞工程研究中心第四军医大学唐都医院,西安710032

出　　处：《科学技术与工程》2005年第14期939-943,共5页Science Technology and Engineering

基　　金：此课题受"973"项目资助(项目号2003CB514127)

摘　　要：为表达SARS-CoV刺突蛋白S(Spike)受体结合区(RBD)并从中筛选高潜在中和性人源抗体,我们用原核表达并纯化的SARS-CoVSRBD进行抗体库的筛选。从多个曾患SARS的健康人血中获得淋巴细胞,PCR扩增全部抗体基因,插入Pcomb3x载体中,构建抗SARS病毒人源噬菌体抗体库。利用噬菌体表面呈现技术,从中筛选结合SARS-CoVSRBD的人源抗体并用ELISA及Westernblot鉴定阳性克隆,竞争ELISA检测人源抗体阻断SARS-CoVSRBD与其受体ACE2的结合情况。结果为构建了库容量6.2×107的免疫Fab噬菌体抗体库,重组率为75%,从中筛选获得9株特异抗SARS-CoVSRBD的人源Fab抗体,ELISA及Westernblot检测均为阳性。其中有一株能阻断SARS-CoVSRBD与其受体ACE2的结合。本实验结果表明:人源抗SARS-CoVSRBD基因工程抗体的获得,一方面将对SARS疾病的特异性预防,治疗和诊断提供新的途径,同时也提示SARS-CoV亚单位疫苗可以用于SARS疾病的预防。To express receptor binding domain of SARS-CoV spike and isolate highly potent neutralizing human Fab antibody against it, SARS- CoV RBD was expressed in E.coli and purified for screening human phage antibody library. Lymphocytes were isolated from blood of people who ever were infected with SARS-CoV. Genes of antibody were amplified from them and inserted to vector Pcomb3x. Phage display technique was used to screen Fab antibody against SARS- CoV S RBD. Positive clones were identified by ELISA and Western blot. Competitive ELISA was used to examine their inhibition of SARS-CoV S RBD binding to ACE2. The results showed that the phage antibody library was successfully constructed ;capacity of library was 6.2×107 and recombinant ratio was 75%; nine human Fab antibodies were isolated from it and all antibodies could bind to SARS-CoV S RBD and one of them can block SARS-CoV S RBD binding to ACE2. It is concluded that Human Fab antibodies against SARS-CoV S RBD lay the foundation of effective prophylaxis , diagnosis and antiviral therapy, moreover, acquirement of highly potential neutralizing antibody implies the possibility of developing subunit vaccine to prevent SARS.

关 键 词：严重急性呼吸综合征(SARS) 刺突蛋白 RBD 噬菌体抗体 FAB 

分 类 号：R511[医药卫生—内科学]