ABSTRACT
Objective To investigate the prokaryotic expression and immunoreactivity of BspE,a type Ⅳ secretion protein of Brucella,and the effect of recombinant protein BspE on cytokines.Methods According to the BspE gene of Brucella M5-90 published in GenBank,the gene fragments were synthesized by a company and then ligated into PUC57 vector for sequencing.The sequenced gene was cloned into a prokaryotic expression vector pET-28α and transformed.Induced expression was performed in E.coli DE3 competent cells.The obtained target protein was purified by a Ni-NTA affinity column,and its reactogenicity was analyzed by Western blotting.Mouse RAW264.7 cells were treated with 25 g/L BspE recombinant protein for 12,24,48 h,and the control group was treated with the same amount of BSA instead of BspE,and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β level.Results The recombinant expresed plasmid of pET-28α-BspE was successfully obtained.The results of Western blotting showed a single band with a relative molecular mass of about 30.1 × 103,and the recombinant protein BspE had good reactogenicity,and IL-1β levels (ng/L)were significantly elevated by the recombinant protein BspE (12 h:43.27 ± 2.13 vs 30.24 ± 1.66,24 h:57.78 ± 3.44 vs 41.22 ± 1.22,48 h:72.52 ± 3.04 vs 46.77 ± 2.75,t =8.38,7.86,10.89,P ＜ 0.05).Conclusions BspE recombinant protein has better immunoreactivity and can increase the expression level of IL-1β in mouse macrophages.This study provides a scientific basis for the role of effector proteins in the pathogenesis of Brucella.
ABSTRACT
Objective:To investigate the effects of TGF-β1 on T lymphocytes of BALB/c mice infected with Echinococcus granulosus( E.granulosus ) in vitro.Methods: The inhibitor group:the spleen cells of BALB/c mouse were co-cultured with E.granulosus and SB525334.The control group:the spleen cells of BALB/c mouse were co-cultured with E.granulosus and PBS.The blank group:the spleen cells of BALB/c mouse were co-cultured with RPMI-1640 medium and SB525334.The lymphocytes were collected at 48 h post-infection.The T lymphocyte subsets, the number of CD4+CD25+T cells, the number of NK cells, and the expression of NKG2D receptor were detected by flow cytometry.The NK cell activity was determined with the lactate dehydrogenase leakage assay(LDH).Results:The inhibition the TGF-β1 receptors resulted in the increase of in the number of CD4+T cells,the decrease in the number of CD8+T cells,the increase of in the ratio CD4+/CD8+T cells,the decrease of in the number of CD4+CD25+T cells,the increase in the expression of the NKG2D receptors,the increase in the lysis rate of Yac-1 cells by NK cells,and a positive cor-relation between the expression of activity receptor NKG2D and killing activity of NK, which were mediated by E.granulosus.Conclusion: The inhibition of TGF-β1 receptors can enhance the immune response of T lymphocytes against E.granulosus infection in vitro.
ABSTRACT
Objective:To understand the expression levels of Tim-3,a new proinflammatory factor in the early stages of Echinococcus granulosus infection in mice.Methods: BALB/c mice were infected with E.granulosus.Peritoneal macrophages and spleen cells were collected at 1,5,9 and 13 days post-infection.At different time points ,the levels of Tim-3 in peritoneal macrophages and spleen CD3+lymphocyte subsets were detected by FCM , and the relative expression of TLR 4 mRNA was detected by qRT-PCR.Results:There was no significant difference in the expression levels of Tim-3 of CD3+spleen lymphocyte subsets between E.granulosus group and control group (P>0.05).The expression levels of Tim-3 of spleen macrophages (9,13 days) and peritoneal macrophage (5,9,13 days) were much higher in E.granulosus infected group than those in control group with statistical significance (P<0.05).The numbers of macrophages were no change.Compared with control groups,the relative expression of TLR4 mRNA at 1 day post-infection was statistically higher in E.granulosus infected group ( P<0.05 ).Conclusion:During early stage of E.granulosus infection in mice,the levels of Tim-3 expression are upregulated,while the expression of TLR4 are downregulated,which may inhibit the function of macrophages resulting in host-immunity-defensive-system inhibition and immune tolerance of E.granulosus to host.