Chlamydia Trachomatis CT849 Protein Induces Expression Of Pro-inflammatory Cytokines In THP-1 Cells Via Activation Of JNK/ERK Signaling Pathways

Objective: To study the role of CT849,a protein of Chlamydia trachomatis,in the regulation of host cell inflammation and related mechanisms,providing reliable and accurate data support for further understanding of the pathogenicity of Ct.Method:Expression and identification of CT849 protein: CT849 whole gene sequence was seized in STD gene database to optimize codon synthesis to obtain CT849 gene sequence,construction of p GEX-4T-1/ct849 recombinant vector,sequencing and identification;The recombinant vector p GEX-4T-1/ ct849 was transformed into E.coli BL21(DE3)and induced to express.The concentration of CT849 protein was detected by purification,endotoxin removal and BCA assay;After using polymyxin B to remove CT849 protein endotoxin,the THP-1 cell 24 h was pretreated with PMA first,then the THP-1 cells were stimulated by the concentration of 0?g/ml?1.25?g/ml?2.5?g/ml?5?g/ml?10?g/ml,ELISA was used to detect the expression of TNF-??IL-1??IL-6 and IL-8 with the increase of CT849 protein concentration;The CT849 protein with a concentration of 10?g/ml was used to stimulate THP-1 cells in 0h?6h?12h?24h and 36 h,expression levels of proinflammatory cytokines of CT849 protein TNF-?,IL-1?,IL-6 and IL-8 in different time were detected by ELISA;THP-1 cells were pretreated with ERK inhibitor PD98059,JNK inhibitor SP600125 and p38 inhibitor SB202190 at a concentration of 30 ?mol/L for 2 h,and then THP-1 cells were stimulated with 10 ?g/ml CT849 for 24 h,the levels of ENK?JNK and p38 phosphorylation of CT849 protein were detected by ELISA and western blot.Result : 1.The prokaryotic expression recombinant vector p GEX-4T-1/ct849 was constructed,and the sequence of the ct849 sequence-optimized codons in the STD gene database was found to be 100% in conformity with the obtained target gene;The vector can express the target protein(44.3k Da)in E.coli BL21(DE3),and after purification,the purity can reach 90%.2.The CT849 protein at concentrations of 0?g/ml?1.25?g/ml?2.5?g/ml?5?g/ml?10?g/ml stimulated THP-1 cells pretreated with macrophages of PMA for 24 h,with the increase of CT849 concentration,the levels of TNF-??IL-1??IL-6 and IL-8 also increased to some extent.3.10?g/ml CT849 treated cells,IL-6?IL-8 or IL-1? expression was highest at 24 h and TNF-? expression was highest at 12 h.4.The cells were treated with CT849.After treatment,phosphorylation levels of ERK and JNK were significantly increased,but there was no change in the level of p38 phosphorylation.5.The ERK and JNK inhibitors decreased the levels of TNF-??IL-1??IL-6 and IL-8 induce by CT849.Conclusion: CT849 induces the production of TNF-??IL-1??IL-6 and IL-8 inflammatory cytokines in THP-1 cells through activation of JNK / MAPKs signaling pathways.