SARS病毒N蛋白特异性单链抗体的筛选

Isolation of the scFv to N protein of SARS virus from library

目的利用大容量人源性SARS单链抗体库筛选N蛋白的特异性单链抗体。方法利用RTPCR的方法克隆了SARS病毒的N蛋白基因,克隆入原核表达载体pBV220,在大肠杆菌DH5α中进行表达并经离子交换色谱纯化后得到了纯度达95%以上的N蛋白,然后以此蛋白包被25cm2组织培养瓶,对SARS单链抗体库进行先松弛,后严谨的筛选、富集,进行4轮筛选后对所筛的抗体进行ELISA鉴定。结果经过4轮的筛选、富集,得到了1株高亲和力的N蛋白单链抗体N18。结论在对SARS疾病的早期检测或筛查、SARS病毒的生活周期及发病机理的研究中N18将起到重要的作用。

Objective To isolate scFv antibody with high affinity and specificity of N protein of SARS-CoV from a large human fibrary of SARS. Methods DNA coding the N protein of SARS-CoV was obtained by RT-PCR and then cloned into pBV220. N protein was expressed in DH5α and purified by ion exchange chromatography. Panning was performed in 25cm^2 tissue flasks. In order to isolate antibody with high affnity without loosing the rare members, the panning procedure was under a loose condition in the first panning round and stricter condition in the later three rounds. The isolated clones were identified by phage ELISA and soluble scFv ELISA. Resuits One scFv with high affinity named N18 was obtained after four rounds of panning. Either in phage ELISA or soluble scFv ELISA, N18 could give intense positive signals. Conclusion N18 may take a place in scanning SARS patients in high risk population, studying the life cycle of SARS-CoV and mechanism of SARS or development of anti-SARS-CoV drugs.