Isolation and identification of an scFv antibody against nucleocapsid protein of SARS-CoV.
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.
Zhao
Aizhi
A
Vector Gene Technology Company Ltd., Beijing 100176, People's Republic of China. zhaoaizhi@gmail.com
Qin
Weijun
W
Han
Yueheng
Y
Wen
Weihong
W
Zhang
Wenhong
W
Lian
Zhonghui
Z
Chen
Gang
G
Zhang
Zhuoli
Z
Peng
Jianqiang
J
Wang
He
H
Guo
Yinglu
Y
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D000086462:Coronavirus Nucleocapsid Proteins; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000276:immunology*; D019151:Peptide Library; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D017422:Sequence Analysis, DNA
D000914:Antibodies, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D007128:Immunoglobulin Fragments; D019590:Nucleocapsid Proteins; D019151:Peptide Library