Position And Related Properties Studies Of Predicted Effector Proteins Secreted Via Type Ⅲ Secretion System Of Chlamydophila Psittaci

Objective: Chlamydophila psittaci is a strictly intracellular parasite andmultihomed zoonotic pathogen, and modern medical is still lack of effective drug forprevention and treatment. Looking for new key effector proteins between Cps andhost cells, which will be target of drug design, has become a hot topic in the medical.Available information indicates that the effector proteins of T3SS are the key of theinteraction of Cps and host cell. This study researched the localization and expressionphase of Cps T3SS effector protein, and their ability to induce THP-1to produce IL-6,IL-1β, TNF-α.Methods: Cps T3SS effector protein were prokaryotic expressed and purified. Thenthe GST-fusion proteins were used to immunize BALB/c mice, so polyclonalantibodies were obtained, which were detected the positioning of the predicted proteinin infected cells by indirect immunofluorescence analysis （IFA）. The proteins wereextracted after8h,18h,24h,36h,48h respectively in Hela cells infected with Cps, theprepared polyclonal antibodies were as first antibody to observe the expression phaseof proteins by Western Blot （WB）. GST-CPSIT₀271protein after being treated with100μg/ml polymyxin B and filtration sterilizated were used to stimulate THP-1cells,the cells were collected after6h,12h,24h,36h,48h respectively to detect the levels ofIL-6, TNF-α and IL-1β by using ELISA kits.Results:1.10Cps T3SS effect protein-coding genes: CPSIT₀139, CPSIT₀271,CPSIT₀363, CPSIT₀499, CPSIT₀700, CPSIT₀788, CPSIT₀803,CPSIT₀959, CPSIT₀965, CPSIT₀991were cloned; the inserted fragments ofconstructed recombinant plasmids were identified as the purpose genes by sequencing, because the results were exactly the same with the Genbank’s byBLAST; the recombinant bacteria were expressed6soluble proteins:CPSIT₀139, CPSIT₀271, CPSIT₀363, CPSIT₀700, CPSIT₀788,CPSIT₀959respectively by induction of IPTG, whose molecular weight weresimilar with the predicted ones; the specific antibody titer was1:16000forCPSIT₀139and CPSIT₀271,1:32000for CPSIT₀700and CPSIT₀788, and1:64000for CPSIT₀363and CPSIT₀959respectively;2. The location of predicted proteins were similar with that of Cps inclusion bodiesby IFA;3. WB results showed CPSIT₀139, CPSIT₀271, CPSIT₀363, CPSIT₀700,CPSIT₀788, CPSIT₀959were detected the strip after infection for24h,36h,18h,24h,36h,18h respectively;4. Different concentrations of GST-CPSIT₀271protein can significantly inducedproduction of IL-6, TNF-α and IL-1β in THP-1cells, when the concentration ofGST-CPSIT₀271protein was2μg/mL⁵μg/mL, CKs level showed a significantdose-dependent; when5μg/mL of GST-CPSIT₀271protein stimulated THP-1cells for24h, the levels of TNF-α and IL-1β reached their peaks, then graduallydecreased, the amount of IL-6arrived at48h.Conclusion:1. CPSIT₀139, CPSIT₀271, CPSIT₀363, CPSIT₀700, CPSIT₀788,CPSIT₀959recombinant proteins showed excellent immunongenicity and couldinduce the humoral responses efficiently; CPSIT₀139, CPSIT₀271,CPSIT₀363, CPSIT₀700, CPSIT₀788, CPSIT₀959were positioned ininclusion bodies;2. CPSIT₀139, CPSIT₀271, CPSIT₀700and CPSIT₀788may be late expressedgenes, CPSIT₀363and CPSIT₀959may be middle expressed genes;3. GST-CPSIT₀271protein can promote THP-1cells to secrete inflammatory cytokines IL-6, TNF-α and IL-1β.