Immunostimulatory role of rBmHSP60 from filarial parasite Brugia malayi

摘要

Objective:To evaluate the immunostimulatory potential of crossreactive molecule heat shock protein 60(HSP60)of filarial parasite Brugia malayi and Leishmania donovani.Methods:HSP60 of Brugia malayi(BmHSP60)was amplified using gene-specific primer,cloned in p Tri Ex4 vector,expressed in BL21-DE3 cells,and recombinant HSP60(rHSP60)of~65 k Da was purified by affinity chromatography using Ni-NTA column.The recombinant protein was desalted by the dialysis membrane,and the presence of endotoxin level was determined by Limulus amebocyte lysate assay.The recombinant protein was tested for cell proliferation,nitric oxide release,expression of Th1 and Th2 cytokines,and transcription factors(STATs)in vitro using murine macrophage cell line(J774 A.1).Results:Higher cell proliferation indicated that BmHSP60 had immunostimulatory potential.rBmHSP60 exposure upregulated the expression of iNOS,STAT1,STAT4,Th1 cytokines(IFN-γ,TNF-α,IL-12),and nitric oxide release.In addition,no remarkable change was observed in the expression of IL-6,IL-10,and STAT3 in macrophage cell line J774 A.1.The ELISA analysis showed the levels of IFN-γ,TNF-α,and IL-12 were upregulated while IL-10 level was downregulated,revealing that BmHSP60 triggered a Th1 immune response.Conclusions:Our study demonstrates that rBmHSP60 has immunogenic properties which effectively enhances the Th1 type immune responses,and can be used as an immunoprophylactic agent against leishmaniasis.Furthermore,in vivo studies are in progress to determine the protective role of rBmHSP60 against Leishmania donovani infection in a mouse model.