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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 the SARS-CoV Spike receptor binding domain (RBD) and screen for highly potential neutralizing human antibodies, we screened for an antibody library using prokaryotic expressed and purified SARS-CoVSRBD. Lymphocytes were obtained from healthy human blood from a number of SARS-infected healthy individuals. All the antibody genes were amplified by PCR and inserted into Pcomb3x vector to construct an anti-SARS virus phage antibody library. The phage display technique was used to screen human SARS-CoVSRBD-binding antibodies and the positive clones were identified by ELISA and Western blot. The competitive ELISA was used to detect the binding of human antibodies to the binding of SARS-CoVSRBD to its receptor ACE2. As a result, we constructed a panel of immunoblotted Fab phage antibody with a capacity of 6.2 × 107, and the recombination rate was 75%. Nine human Fab antibodies against SARS-CoVSRBD were screened out by ELISA and Western blot. One of them blocked the binding of SARS-CoVSRBD to its receptor ACE2. The results of this experiment show that the availability of human anti-SARS-CoVSRBD genetically engineered antibodies will provide a new way to prevent, treat and diagnose SARS diseases on the one hand, and at the same time suggest that the SARS-CoV subunit vaccine can be used in SARS diseases Prevention.