Prokaryotic expression and monoclonal antibody preparation of Mycobacterium tuberculosis ferric uptake regulator B.
Ferric uptake regulator B (FurB) of Mycobacterium tuberculosis, which belongs to the Fur superfamily, is principally responsible for maintaining iron and zinc homeostasis in prokaryotes. This common feature of FurB and the role of FurB in iron and zinc metabolism contribute to research on the pathogenesis of mycobacteria. In this study, three novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurB protein as immunogen. The FurB gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pQE-80L and was effectively expressed in Escherichia coli DH5alpha. The expressed fusion protein existed as the insoluble form (inclusion bodies) in cell lysate and was purified on an Ni-NTA column. Using the fusion protein to immunize BALB/c mice, three monoclonal antibodies (DD12, BH1, and DH8) were produced. As shown by Western blot analysis and indirect immunofluorescence assay, the three respective antibodies could recognize the FurB protein. These results suggest that the antibodies against FurB may provide a powerful tool for investigating the function of FurB in the pathogenesis of tuberculosis.
Jiang
Hong
H
Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, Shaanxi, China.
Gao
Xue
X
Li
Yuan
Y
Xu
Zhi-Kai
ZK
Wang
Li-Mei
LM
Bai
Xue-Fan
XF
Yue
Ying
Y
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000276:immunology; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial