Résumé
Objective To investigate the role of cHSP60 in the pathogenesis of murine cervicitis.Methods Fifty female C3H/HeN mice were randomly and equally classified into 5 groups, including the control group receiving no treatment and 4 groups receiving intravaginal inoculation of cHSP60 (cHSP60 group),live elementary bodies of Chlamydia trachomatis mouse pneumonitis (MoPn group), inactive elementary bodies of MoPn (inactive MoPn group) and growth medium (medium group), respectively. Five days after the inoculation,cervical tissue was resected from these mice and subjected to pathological examination. Results There were varying degrees of inflammatory reaction characterized by neutrophil infiltration, necrosis and shedding of mucosal cells in the cervices of mice in cGSP60 and MoPn groups. No statistical difference was observed in the incidence of cervicitis (90% vs. 80%, P ＞ 0.05), neutrophile count [76.00 (25.0 - 80.0) vs. 25.00 (8.75 -32.5), P＞ 0.05] or inflammation score [12.5 (11.5 - 14.25) vs. 9.00 (8.00 - 11.5), P ＞ 0.05] between the cHSP60 and MoPn group. The inflammatory reaction was weak with decreased incidence of cervicitis (40%),inflammation score [0.00 (0.00- 12.50)] and neutrophile count [0.00 (0.00- 15.50)] in inactive MoPn group compared with the cHSP60 and MoPn groups (all P ＜ 0.05). A small number of neutrophils migrated into the superficial layer of cervical mucosa in only 2 mice in the medium group. Conclusion cHSP60 may be a primary pathogenic factor in chlamydial genital tract infection.
Résumé
Objective To clone and express Chlamydia trachomatis (Ct) heat shock protein 60 (hsp60) gene. Methods The hsp60 gene fragment was amplified from Ct chromosomal DNA by PCR. After purification and digestion with enzymes Sal I and Not I , the hsp60 gene fragment was inserted into the compatible site of prokaryotic expression vector pET-28a. The constructed recombinant plasmid was identified by PCR, restriction enzyme cleavage and sequencing, then, it was transfected into an expression strain Escherichia coli BL21 (DE3). The expression of fusion protein was induced by isopropy-β-D- thiogalactoside (IPTG) in the host bacteria, and the expressed product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western-blot. Results PCR and restriction enzymes cleavage analysis confirmed that the hsp60 gene was successfully cloned into the recombinant plasmid. DNA sequencing showed that the sequence of cloned gene was fully consistent with the published sequence in Genebank. As revealed by SDS-PAGE, the size of expressed fusion protein approximated 60 kilodaltons, and Western-blot confirmed the expressed product to be the expected protein. The final concentration of fusion protein was 17.85 mg/L with a purity of more than 90%. Conclusions A recombinant expression plasmid pET-28a-hsp60 is successfully constructed in this study, and soluble hsp60 protein is expressed by the recombinant plasmid-transfected E. coli.