ABSTRACT
Objective To study the possible molecular mechanism of IL-10 in promoting Chlamydia muridarum infection in mice. Methods C57BL/6 wild-type and IL-10 gene knockout ( IL-10-/-) mice were infected with Chlamydia muridarum. Indirect immunofluorescence assay was used to detect the growth of Chlamydia muridarum in the intestinal and genital tracts. The severity of genital diseases was assessed by hydrosalpinx scoring. Expression of IFN-γand IL-2 in blood was measured by ELISA. Re-sults Compared with the wild-type group, Chlamydia clearance in the intestinal and genital tracts of IL-10-/- mice was significantly faster, and the expression of IFN-γ and IL-2 increased significantly. In addi-tion, wild-type mice showed more serious hydrosalpinx. Conclusions IL-10 delays Chlamydia trachomatis clearance and promotes Chlamydia infection through inhibiting the expression of IFN-γ and IL-2, which ag-gravates hydrosalpinx.
ABSTRACT
Objective To construct a prokaryotic expression plasmid for CPSIT_p7 gene from Chlamydophila psittaci ( Cps) 6BC strain and to evaluate immunogenicity of the recombinant protein His-CPSIT_p7 and detect its dynamic expression at mRNA and protein levels in HeLa cells during persistent Cps infection.Methods The fusion protein His-CPSIT_p7 was expressed in E.coli BL21 and purified by Ni-NTA affinity chromatography .BALB/c mice were immunized with the recombinant protein to prepare polyclonal antibody for evaluation of the immunogenicity of His-CPSIT_p7 by ELISA.Penicillin sodium was used to establish a model of Cps persistence infection .RT-PCR and Western blot assay were performed to de-tect the expression of CPSIT_p7 at mRNA and protein levels during Cps persistent infection .Results The fusion protein His-CPSIT_p7 was successfully expressed with the use of constructed recombinant expression plasmid pET30 a-CPSIT_p7 and purified .ELISA result showed that the specific antibody titer against CPSIT_p7 reached 1 ∶1 000 000 on the 40th days after immunization .The expression of CPSIT_p7 at mRNA and protein levels were increased in a time-dependent manner in Cps-infected HeLa cells .The peak of mRNA level was reached at the time point of 36 hours after infection , followed by a time-dependent decrease during Cps acute infection .However , the expression of CPSIT_p7 at mRNA and protein levels were not decreased until 60 hours after infection during Cps persistent infection .Conclusion His-CPSIT_p7 protein was suc-cessfully expressed in the prokaryotic expression system and purified , showing an advantage of good immuno-genicity.Highly expressed CPSIT_p7 at mRNA and protein levels were detected during Cps persistent infection.
ABSTRACT
Objective To study the intracellular localization and temporal expression of CPSIT_0271 in Chlamydia psittaci-infected cells; and to investigate the effects of recombinant GST-CPSIT_0271 protein on the expression of proinflammatory cytokines including IL-6, IL-1βand TNF-αby THP-1 cells.Methods The gene encoding CPSIT_0271 of Chlamydia psittaci was expressed as fusion protein ( GST-CPSIT_0271 ) in E.coli.The polyclonal antibody was prepared by immunizing BALB /c mice with the purified recombinant pro-tein.Antibody titer was determined by ELISA .Indirect immunofluorescence assay ( IFA) was performed to lo-cate the endogenous CPSIT_0271 protein in C.psittaci-infected cells .The expression characteristics of CPSIT_0271 protein were detected by Western blot in C.psittaci-infected HeLa cells at different time points .The levels of IL-6, IL-1βand TNF-αwere analyzed by ELISA after stimulating THP-1 cells with different concentrations of CPSIT_0271 protein.Results CPSIT_0271 protein was found to express in the chlamydia inclusion of C.psittaci-infected HeLa cells .The expression of CPSIT_0271 protein was detected firstly at 36 h and increased at 48 h after C.psittaci infection.The titer of anti-CPSIT_0271 specific antibody in GST-CPSIT_0271 immu-nized mice reached to 1 ∶16 000.GST-CPSIT_0271 protein increased the levels of IL-6, IL-1βand TNF-αin THP-1 cells in a dose-dependent manner in the range of 2 to 5 μg/ml.The levels of TNF-αand IL-1βreached their peaks at 24 h, and IL-6 level peaked at 48 h upon the stimulation by 5 μg/ml of GST-CPSIT_0271 pro-tein.Conclusion CPSIT_0271 expressed in inclusion bodies of Chlamydia psittaci in the infected cells , sug-gesting it might be a late expression gene .GST-CPSIT_0271 protein shows good immunogenicity and enhances the expressions of IL-6, IL-1βand TNF-αin THP-1 cells.