title: Expression of Mycobacterium tuberculosis ferric uptake regulator A gene in Escherichia coli and generation of monoclonal antibodies to FurA.
abstract: Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.
authors.affiliation: Department of Otorhinolaryngology, General Hospital of Second Artillery, Beijing, P.R. China.
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.forename: Li-Mei
authors.initials: LM
authors.lastname: Wang
authors.forename: Yin-Lan
authors.initials: YL
authors.lastname: Bai
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.forename: Chang-Hong
authors.initials: CH
authors.lastname: Shi
authors.forename: Hai
authors.initials: H
authors.lastname: Zhang
authors.forename: Ying
authors.initials: Y
authors.lastname: Xue
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000378:metabolism*; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D007501:Iron / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D012995:Solubility
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron