Treponema pallidum membrane protein Tp0971 induce macrophages to produce cytokines via TLR2/NF-κB pathway / 中国免疫学杂志

ABSTRACT

Objective:To investigate the production of cytokines from macrophages induced by Treponema pallidum membrane proteins Tp0971.Methods: The Tp0971 was amplified by PCR from a preparation of T.pallidum genomic DNA and then sub-cloned into the prokaryotic expression vector pET28a(+)to construct the recombinant plasmid pET28a(+)/Tp0971.The recombinant plasmids were transfected into E.coli Rosseta strain to express recombinant protein Tp0971 by IPTG induction.The expression products were purified by Ni-NTA affinity chromatography,and the concentration was determinated by BCA method.Detoxi-Gel was used to remove endotoxin contamination in during the protein preparation.After induced by PMA,cells were incubated with various concentrations of recombinant proteins Tp0971.The expression levels of IL-8,IL-1β and IL-6 were detected by ELISA.Cells were pretreated with anti-TLR2 antibody or TLR2 siRNA,or pyrrolidine dithiocarbamate,an inhibitor of NF-κB,for evaluation of the role of TLR2 and NF-κB in the production of cytokines by ELISA.Results: Tp0971 gene were amplified successfully by PCR,and the recombinant plasmids were confirmed by enzyme digestion and sequencing.SDS-PAGE results showed three recombinant proteins were expressed as the soluble with a relative molecular weight of 29 kD.0.5-10 μg/ml of Tp0971 could stimulate macrophages to produce IL-8,IL-1β and IL-6 dose-dependently.After cells were pretreated with siRNA or neutralizing antibody targeting TLR2 or the PDTC,the Tp0971 induced protein expression levels of proinflammatory cytokines were significantly reduced in macrophages.Conclusion: Tp0971 induces macrophages to produce proinflamatory cytokines via TLR2/NF-κB pathway.