Treponema Pallidum Adhesion Proteins Tp0155, Tp0483 Induce Macrophage Product Inflammatory Cytokine Via Activation Of NF-κB

Objective: This study was designated to investigate the roll of Treponema pallidum adhesion proteins Tp0155 and Tp0483 in the macrophage production of(CKs) IL-6,IL-1βand TNF-α;and take use of the key factor inhibitors of signal path contribute to study the production of(CKs), meanwhile we studied weather these signal path related to the production of(CKs)by the stimulated of Tp0155 and Tp0483.Methods: The genes sequence of Tp0155 and Tp0483 were obtained from Genbank, and they were amplified from T. pallidum Nichols strain complete genome by polymerase chain reactions, then sucloned into the prokaryotic expression vector pET28a (+) to construct the recombinant plasmid pET28a (+) / Tp0155and pET28a (+) / Tp0483. After PCR,restriction enzyme digestion, DNA sequencing, the recombinant plasmids were transfected into E.coli Rosseta strain to express their proteins by IPTG induction. Analysis and identify the protein by SDS-PAGE and Western blot; 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 PMA induced, THP-1 cells were incubated with various concentrations of Tp0155 and Tp0483 recombinant proteins, IL-1β,IL-6 and TNF-αproduction was detected by the quantitative ELISA technique.collet the sum protein of the disposed protein,analysis the phosphorylation of IκB. Pretreat THP-1 cell by 25μmol/L PDTC 30min, then stimulate macrophage by 5μg/mL Tp0155 and Tp0483 recombinant proteins for 24 h(IL-6 and IL-1β) and 48h(TNF-α)separately, the production was detected by the quantitative ELISA technique.Results: Tp0155 and Tp0483 genes 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 or inclusion bodies with a relative molecular weight of 46kDa(Tp0155) and 47kDa(Tp0483) in bacterial cells in form of solubility and inclusion body, their purity reached to 95% after Ni-NTA affinity purification; TAL showd the endotoxin level was less than 0.04EU/mL after endotoxin removal gel processed. After PMA induced, THP-1 cells were dose dependence stiulated by 0.5～5μg/mL Tp0155 and 0.5～7μg/mL Tp04883 recombinant proteins could product TNF-α,IL-1βand IL-6. Tp0155 and Tp0483 climbed to the peak value at 7μg/mLand 5μg/mL separately, after the peak value,the quantity of CKs will reduced with the growth of recombinant proteins concentration. Tp0155 and Tp0483 recombinant proteins stiulated THP-1 cells product TNF-α,IL-1βand IL-6 timedependently. The quantity of CKs grows with the time. IL-6,IL-1βand TNF-αclimb to the peak value after 48h and 24h separately. The result of western blot show that Tp0155 and Tp0483 are able to induce degradation of IκB, when pretreat PDTC, Tp0155 and Tp0483 induced IL-6 production to 53.0%,49.0% and decrease IL-1βproduction to 52.0%,54.0%, TNF-αproduction to 57%,58%; The difference between treated and control groups had statistical significance.Conclusion:1 Tp0155 and Tp0483could induce macrophages production of IL-6,IL-1βand TNF-αby dose and time dependence.2 Tp0155 and Tp0483 recombinant proteins colud active NF-κB by inducing degradation of IκB.3 The production of IL-6, IL-1βand TNF-αmay relatated to activation of NF-κB...