| Fascioliasis is a zoonotic parasitic disease caused by liver flukes of the genus Fasciola,such as Fasciola gigantica,Fasciola hepatica or its intermediate form.Fasciola gigantica is distributed in tropical and subtropical regions,and F.hepatica is mainly located in temperate regions,which causes huge economic losses to livestock industry and seriously threatens to public health.Calcium-binding protein(CaBp)belongs to the worm calcium-binding protein family and has calcium binding sites.It plays roles in various biological processes by binding to different signaling molecules and participates in regulating cell survival,proliferation,adhesion,migration,angiogenesis and many other characteristic biological processes of tumors.Leucine aminopeptidases(LAPs)are metalloproteinases which are widely found in prokaryotic and eukaryotic cells,which cleave N-terminal residues from proteins and peptides,and have cytosolic enzymes,interaction,protein turnover,tumor growth and tissue invasion,metabolism and other immune functions.In this study,the combination of morphological and molecular biological techniques was used to confirm the identity of liver flukes representing F.gigantica.According to the gene sequence of F.hepatica CaBp2(Fh CaBp2),FgCaBp2 gene was amplified,and analyzed using bioinformatics tools.Recombinant FgCaBp2(r FgCaBp2)and recombinant Fg LAP(r Fg LAP)proteins were expressed and purified,and soluble expression of the two proteins was achieved.The two recombinant proteins were identified to react with goat-anti F.gigantica sera and polyclonal antibodies raised in rabbits by Western-blot,and specific bands appeared at about 23 ku and 63 ku,respectively,indicating that these two proteins are potential diagnostic antigens.In this study,the effects of r FgCaBp2 and r Fg LAP on the functions of goat peripheral blood mononuclear cells(PBMCs)were examined,including specific binding ability to goat PBMCs,secretion of cytokines,total NO production,cell proliferation,cell migration and apoptosis and effects on monocyte phagocytosis.Indirect immunofluorescence assay(IFA)demonstrated that the two proteins have good binding ability to goat PBMCs;r FgCaBp2 significantly inhibited the secretion of IFN-?,IL-4 and IL-10;however,it had significant promoting effects at high concentration.It could significantly promote the secretion of TGF-? at low concentration.r Fg LAP significantly promoted the secretion of TGF-?,significantly inhibited the secretion of IL-4,IL-10 and IFN-? at low concentration.A low concentration of r FgCaBp2 significantly promoted the production of NO,but high concentration of r FgCaBp2 inhibited the production of NO.The low concentration of r Fg LAP had a weak promoting effect,while r Fg LAP at high concentration significantly promoted the production of NO.Both proteins significantly promoted the proliferation of goat PBMCs and both proteins significantly promoted the migration of goat PBMCs in the migration chamber.r FgCaBp2 inhibited apoptosis,while r Fg LAP had weak inhibiting effect on apoptosis.Both proteins interacted with goat monocytes to promote the phagocytic capacity of monocytes.Taken together,the present study obtained the recombinant proteins of r FgCaBp2 and r Fg LAP.The two proteins were recognized by positive anti-F.gigantica sera,indicating that they have strong immunological activity.The functional effects of the two proteins on goat PBMCs indicate that they have immunomodulatory effects on host immune responses.It is speculated that these two proteins may play an important role in anti-host immunity of F.gigantica.These results provided foundation for further identification and evaluation of diagnostic antigens,for revealing the interaction between excretory-secretory(ES)products of F.gigantica and the host,and for elucidating the immune evasion mechanism of F.gigantica. |
PDF Full Text Request