[Construction of a large human scFv library against SARS virus].
OBJECTIVE
To construct a large human scFv library against SARS virus by using in vivo recombination.


METHODS
Total RNA was isolated from the lymphocytes of 6 patients recovered from SARS. mRNA was isolated and reverse transcribed into cDNA. The V(H) and V(L) fragments were amplified from the cDNA and then assembled into scFv genes. The scFv genes were amplified and ligated into phagemid pDAN5. The primary library was constructed by transforming the recombinant phagemid into E.coli TG1. The secondary library was generated by in vivo recombination in E.coli BS1365 following the infection of BS1365 by primary library phages.


RESULTS
A primary library of 3x10(9) and a second library of 3x10(11) were constructed.


CONCLUSIONS
A large human scFv library against SARS virus with good diversity was constructed, which may be used for screening antibodies to SARS virus antigens.
Department of Immunology, Peking Union Medical University, Beijing 100005, China. zhaoaizhi@163.ccom
Ai-zhi
AZ
Zhao
Jian-qiang
JQ
Peng
Hui
H
Cao
Yu
Y
Kang
Lian-xian
LX
Cui
Xiao-bing
XB
Wu
Wei
W
He
D000914:Antibodies, Viral / Q000235:genetics* / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048348:Reverse Transcription; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D014169:Transformation, Bacterial
D000914:Antibodies, Viral; D018076:DNA, Complementary; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger