title: High-level expression of Mycobacterium tuberculosis protein EspB in E. coli and preparation of novel anti-EspB monoclonal antibodies.
abstract: The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis. The ESX-1 secreted protein EspB is cleaved close to its C-terminus during secretion and is necessary for inhibiting phagosome maturation. In this study, the EspB gene of M. tuberculosis H37Rv was cloned into the expression vector of pET21a(+) and was effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as a soluble form in cell lysate and was first purified by a column packed with Ni-NTA resin and then a column packed with DEAE-Sepharose Fast Flow matrix. Using the purified protein to immunize BALB/c mice, five monoclonal antibodies were produced. As shown by ELISA and immunoblotting, the five respective antibodies could recognize the EspB protein. These monoclonal antibodies will provide powerful reagents for further investigation of EspB protein functions.
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dongdaije, Fengtai, Beijing, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.forename: Shuling
authors.initials: S
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: Yin
authors.forename: Shuang
authors.initials: S
authors.lastname: Ji
authors.forename: Dayong
authors.initials: D
authors.lastname: Dong
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Fu
authors.forename: Junjie
authors.initials: J
authors.lastname: Xu
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000378:metabolism*; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000378:metabolism; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D037521:Virulence Factors / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D037521:Virulence Factors