Prelimary Studies On Genital Tract Inflammation And Immune Mechanisms In Mice Induced By PORF5Plasmid Protein Of Chlamydia Trachomatis

Objective: Chlamydia trachomatis (Ct) is an important pathogen that affects humanhealth seriously. Immuno-pathological damage caused by excessive inflammation isthe major pathogenic mechanism responsible for chlamydial diseases. The secretoryplasmid protein pORF5plays an important role in Ct persistent infection. The aim ofthis study was to investigate inflammatory response in urogenital tissue of BALB/cmice induced by pORF5plasmid protein and the levels of inflammatory cytokinesTNF-α, IL-1β and IL-6. The results of this study could provide some importantinformation for further exploring the pathogenesis mechanism and prevention theoryof the plasmid protein pORF5in Ct infection.Methods: After identified by PCR and restriction enzyme digestion, the recombinantplasmid pGEX-6p-1/pORF5was transformed into E.coli XL1-blue to express pORF5fusion protein induced by IPTG. The recombinant protein was purified withGlutathione Sepharose4B and digested with PreScission Protease to obtain the targetprotein without GST tag.The pORF5target protein was analyzed and identified bySDS-PAGE and Western-Blot. After condension by fast lyophilization, theconcentration was measured by BCA kit. The pORF5target protein was inoculated inposterior fornix of BALB/c mice on day1,3and6respectively, and the mice weresacrificed on day7. The pathomorphism change of oviduct was observed by using HEdyeing. The leukocytes of peripheral blood and vagina lavage liquid were countedwith blood cell counting plate and WBC sortings were observed in Wright’s staining.The levels of TNF-α, IL-1β and IL-6in the blood serum and the vagina’s lavage liquid were detected by ELISA. MTT was employed to study proliferation activity of spleenlymphocytes.Results:1The recombinant protein GST-pORF5was expressed succussfully with a relativemolecular weight about54kDa in solube form. After purification with GlutathioneSepharose4B, and digested with PreScission Protease,3μg/μL of the target proteinwithout GST tag was obtained.2Significant hydrosalpinx in the oviduct regions developed in BALB/c miceinoculated with pORF5protein, but there were almost normal oviducts in PBSControl Group and Normal Group. The genital tract tissues from pORF5-inoculatedmice exhibited pathology such as infiltration of inflammation cells includingneutrophil, monocyte macrophage, lymphocyte and so on, while the genital tracttissues from the other two groups did not show any obvious change. The leukocytecounting of peripheral blood in pORF5Group was more than that of the NormalGroup（P﹤0.05）and the PBS Group（P﹥0.05）. The percentage of the neutrophilfrom Group pORF5was higher than that of the Normal Group（P﹤0.05）and thePBS Group（P﹥0.05）. The percentage of the lymphocyte was lower than that ofthe Normal Group （P﹤0.05） and the PBS Group（P﹥0.05）. The percentage ofthe monocyte was higher than that of the Normal Group （P﹥0.05） and the PBSGroup（P﹥0.05）. The leukocyte counting of the BALB/c mice vaginal fluid fromGroup pORF5was more than that of the Normal Group（P﹤0.05） and the PBSGroup（P﹥0.05）.3The level of TNF-α in peripheralblood from Group pORF5was obviously higherthan that of the Normal Group and the PBS Grou（pP﹤0.01）. The level of IL-6wasobviously higher than that of the Normal Group （P﹤0.01）and the PBS Group（P﹤0.05）. The level of TNF-α in the BALB/c mice vaginal fluid from Group pORF5was more than that of the Normal Group and the PBS Group（P﹤0.01）. The levelof IL-1β was obviously higher than that of the Normal Group and the PBS Group（P﹤0.05）. The level of IL-6was obviously higher than that of the Normal Group and the PBS Group（P﹤0.01）. The level of TNF-α, IL-1β and IL-6in the BALB/cmice spleen cell from Group pORF5was more than that of the Normal Group andthe PBS Group（P﹤0.01）.4The levels of TNF-α, IL-1β and IL-6in the BALB/c mice spleen cell from GrouppORF5was more than that of the Nomal Group and the PBS Group（P﹤0.01）.Thelymphocyte proliferative activity in the BALB/c mice inoculated with pORF5protein was obviously higher than that of the Normal Group and the PBS Group（P﹤0.01）.Conclusion:1GST-pORF5fusion protein with a relative molecular weight about54kDa wasexpressed and purified successfully, and pORF5protein of about28kDa was alsoobtained.2pORF5plasmid protein could cause inflammatory pathological damage in theoviduct tissues of BALB/c mice.3pORF5plasmid protein increased the production of the inflammatory cytokinesTNF-α, IL-1β and IL-6in BALB/c mice.