The Effects Of Six Recombinant Enzymes In Haemonchus Contortus On Peripheral Blood Mononuclear Cells Of Goats In Vitro

Haemonchus contortus belongs to Trichostrongylidae Haemonchus nematodes, which seriously endangers cattle and other ruminants. The parasites mainly live in the abomasums of the hosts, occassionaly in intestines, taking nutrition by sucking the blood from hosts. Nowadays, there has been significant progress in immunology research of Haemonchosis, but still no commercial vaccine appears. So discover new antigens and further research parasite antigens are particularly important for vaccine development. H. contortus glutathione peroxidase (HC29), glyceraldehyde-3-phosphate dehydrogenase(GAPDH), glutamate dehydrogenase(GDH), enolase (ENO), triosephosphate isomerase (TPI) and aldolase(ENO) were recombinant expressed in this assay, and further study these six recombinant enzymes in vitro binding of goat peripheral blood mononuclear cells; effect on PBMCs proliferation and cytokine expression in PBMCs; macrophage phagocytosis and secretion of NO.IThe expression of six recombinant enzymes from Haemonchus contortus and the assay of recombinant enzymes binding to PBMCs from goatsThe plasmids were transformed into BL21,and then induced by IPTG after digestion with restrict enzymes. Blood samples were collected from jugular vein of no H. contortus infected goats.PBMCs were separated and cultured in vitro with a final concentration 5μg/ mL of the recombinant enzymes. The binding of the recombinant enzymes to PBMCs were observed by immunofluorescence antibody technique.The result showed that recombinantenzymes HC29 and GAPDH could bind to PBMCs from goats in vitro.2 Effects on cytokine expression in goat peripheral blood lymphocyte induced by six recombinant enzymesIn order to study the effects of recombinant enzymes on the cytokines(IL-2,IL4,IL-10,IL-17,IFN-γ,TGF-β) of goat peripheral blood mononuclear cells. In this study, we use the recombinant enzymes HC29, GAPDH, ENO, TPI, ALD and GDHto incubate with goat PBMCs for 24 hours. Then mRNAs were extracted and quantitied by Real Time PCR to test the expression of cytokines after the incubation. The results showed that the mRNA transcription levels of IL-2, IL-4, IL-10, IL-17 and IFN-y were different from control group apparently in GAPDH group (P<0.01); the mRNA transcription levels of IL-10 and IFN-y were different from control group apparently in HC29 group (P<0.01); the mRNA transcription levels remained steady in other groups (P>0.05)3Effects of six recombinant enzymes of Haemonchus contortus on the proliferationof goat PBMCsRecombinant enzymes HC29, GAPDH, ENO, TPI, ALD and GDH were incubated with goat PBMCs for 24 hours. Then use Cell Counting Kit-8 to detect the changes of cells number. Results showed that all of the recombinant enzymes had the effect on PBMCs. Especially, in the group of HC29 (10μg/mL,20μg/mL,40μg/mL), cell proliferation were observed significantly obvious compared to the control group (P< 0.01); In the GAPDH group (10μg/mL,20μg/mL,40μg/mL), the results were obvious compared to the control group; the other recombinant enzymes were not obvious compared to the control group (P> 0.05).4Effect of six recombinant enzymes from Haemonchus contortus on phagocytosis and NO secretion from goats macrophageIn order to study the effects of recombinant enzymes on the macrophage from goats.The phagocytosis and NO secretion of macrophage were tested after incubation with the recombinantenzymes.The results showed recombinant enzymes HC29 and GAPDH strengthened macrophage phagocytosis and boosted NO production, especially HC29. Whereas the other enzymes didn’t show the obvious function.