Zhao Aizhi, Qin Weijun, Han Yueheng, Wen Weihong, Zhang Wenhong, Lian Zhonghui, Chen Gang, Zhang Zhuoli, Peng Jianqiang, Wang He, Guo Yinglu
Vector Gene Technology Company Ltd., Beijing 100176, People's Republic of China.
Microbes Infect. 2007 Jul;9(8):1026-33. doi: 10.1016/j.micinf.2007.04.008. Epub 2007 Apr 21.
To develop reagents for early diagnosis and therapeutic drugs against SARS-associated coronavirus (SARS-CoV), a large (3 x 10(9)) immunized human antibody library was constructed from peripheral blood mononuclear cells from six SARS convalescent patients. A single chain variable fragment antibody (N18) with high affinity against N protein of SARS-CoV was isolated. Sequence analysis revealed that the VL gene was composed of VL3h (V lambda subgroup) and JL2 regions and the VH gene was composed of VH1-69 (VH1 subgroup), D2-15, D3-22 and JH6 regions. Soluble N18 antibody was expressed in Escherichia coli HB2151, purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blot. The potential application for early diagnosis was evaluated using N protein capture ELISA in which N18 antibody demonstrated high sensitive activity in detecting N protein of SARS-CoV. Finally, the potential usefulness of the N18 antibody in prophylaxis, vaccine design and therapy of SARS is discussed.
为开发针对严重急性呼吸综合征相关冠状病毒（SARS-CoV）的早期诊断试剂和治疗药物，从6名SARS康复患者的外周血单个核细胞构建了一个大型（3×10⁹）免疫人抗体文库。分离出一种对SARS-CoV的N蛋白具有高亲和力的单链可变片段抗体（N18）。序列分析表明，VL基因由VL3h（Vλ亚群）和JL2区域组成，VH基因由VH1-69（VH1亚群）、D2-15、D3-22和JH6区域组成。可溶性N18抗体在大肠杆菌HB2151中表达，通过镍-亚氨基三乙酸亲和层析纯化，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质免疫印迹法进行验证。使用N蛋白捕获酶联免疫吸附测定法评估其在早期诊断中的潜在应用，其中N18抗体在检测SARS-CoV的N蛋白时表现出高灵敏度活性。最后，讨论了N18抗体在SARS预防、疫苗设计和治疗中的潜在用途。
