Ultrastructural changes of genital tract Chlamydia trachomatis after treatment with azithromycin in vitro / 中华皮肤科杂志

ObjectiveTo observe the ultrastructural changes of Chlamydia trachomatis after treatment with azithromycin.Methods The Chlamydia trachomatis laboratory strain (D/UW-3/Cx) was cultured in McCoy cells with or without the presence of azithromycin of 0.0667,0.1340,0.1900,0.2680 and 0.3330 mg/L for 48 hours.The ultrastructural changes of host cells andChlamydia trachomatis were observed by transmission electron microscopy.ResultsAfter 48-hour culture,vesicles increased in number both inside and outside of the inclusion bodies with the rise in azithromycin concentration; there were abnormally large reticulate bodies,some of which experienced abnormal division and even necrosis or breakdown; the number of elementary bodies was decreased,while their size was enlarged,with a more wrinkled outer membrane.No inclusionbodieswereseenwhentheconcentrationofazithromycinwas0.333mg/L. Conclusions Azithromycin can induce an increment in the outer membrane of Chlamydia trachomatis,formation of vesicles,abnormal enlargement or breakdown of reticulate bodies,and a decrease in elementary bodies.

Cloning and expression of Chlamydia trachomatis heat shock protein 60 gene / 中华皮肤科杂志

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.