title: Prokaryotic expression and functional analysis of the Mb1514 gene in Mycobacterium bovis.
abstract: The ability of mycobacteria to grow and invade target tissues is the key component in the process of Mycobacterium bovis infection. Therefore, analysis of the proteins responsible for cell invasion will assist clinicians in combating bovine tuberculosis. The Mb1514 gene of M. bovis encodes a hypothetical invasion protein (designated here as MbINV protein), whose function has not yet been directly identified. In this study, the Mb1514 gene from M. bovis was cloned, and expressed in E. coli. The recombinant MbINV protein (a single band of approximately 28 kDa) was purified for biological analysis. Our data demonstrated that recombinant MbINV protein significantly inhibited the viability of RAW264.7 macrophages in a dose-dependent manner (P < 0.05), and induced cell necrosis, indicating that the protein is toxic. MbINV protein infection significantly enhanced the mRNA expression levels of TNF-α, IL-1β, and NOS2 (P < 0.01), suggesting that MbINV protein may be one of the virulence factors which directly interact with macrophages and modulate the host immune response to M. bovis. An invasion inhibition assay showed that MbINV-inhibited M. bovis invasion of RAW264.7 cells in a concentration-dependant manner, demonstrating it is an invasion protein.
authors.affiliation: National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Yin
authors.forename: Xiangmei
authors.initials: X
authors.lastname: Zhou
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Xue
authors.forename: Lihong
authors.initials: L
authors.lastname: Tian
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Lifeng
authors.initials: L
authors.lastname: Yang
authors.forename: Deming
authors.initials: D
authors.lastname: Zhao
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009163:Mycobacterium bovis / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D053583:Interleukin-1beta; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse