Recombinant Expression And Function Analysis Of Four Proteins Of Haemonchus Contortus Binding To Goat Peripheral Blood Mononuclear Cells

Haemonchus contortus is a common gastrointestinal nematode of ruminant animals like cattle and goat.They are mainly parasitic in abomasum to feed on host blood and bring great harm to our contry animal husbandry.Escretory sectory antigen are metabolic products of parasite,their effect on host immune system is not clear..In our laboratory,146 proteins of H.contortus ESPs binding to goat peripheral blood mononuclear cells were deceted.The study of their function is helpful to explain the mechanism of the interaction between H.contortus and host.In this paper,Cyclization recombination enzyme-actin like-6 protein(CREA6),CS domain containing protein(CS),NADH:ubiquinone oxidoreductase domain containing protein(NDUDC)and Hepatocellular carcinoma-associated antigen59(HCA59)were selected from above 146 proteins.Their genes were cloned and expressed,and the effect of four recombinant proteins on the immune function of goat PBMCs was studied.It isfundamental for further understanding the regulation of ESPs of H.contortus to the host immune system.1 Cloning and expression of four genes of Haemonchus contortus and western blot analysis of four recombinant proteinsFour proteins CREA6?CS?NDUDC and HCA59 were selected from Haemonchus contortus excretory and secretory products.Their open reading frams were amplified by RT-PCR,the length of gene fragments were 1125 bp,609 bp,438 bp and 426 bp.Then the prokaryotic expression plasmid of pET-32a-CREA6,pET-32a-CS,pET-32a-NDUDC and pET-32a-HCA59 were constructed successfully.After induced expression,purification and renaturation,four recombinant proteins with molecular weight of 42 kD,23 kD,17 kD and 16 kD were obtained.The antigenicity of proteins was analyzed by Western blot with serum from goat infected with H.contortus as primary antibody.The results showed that four recombinant proteins could be recongnized by serum from infected goat,it indicated that rCREA6,rCS,rNDUDC and rHCA59 have strong antigenicity.2 Four recombinant proteins of Haemonchus contortus binding to goat PBMCs and effect of proliferation on the PBMCsPeripheral blood mononuclear cells(PBMCs)were separated from goat blood sample and incubated with four recombinant proteins at 37? for 1h in vitro to check the binding of the protein to PBMCs by indirect immunofluorescence assay.Different concentrations(0,10,20,40 ?g/mL)of the recombinant protein were used to stimulate PBMCs,and the effects of cell were tested by CCK-8 kit.The results showed that four recombinant proteins could combine with PBMCs in vitro,and have the function of enhancing cell proliferation.3 Effects of recombinant proteins of Haemonchus contortus on the cytokines secretion of goat PBMCsPeripheral blood mononuclear cells(PBMCs)were separated from goat blood sample and cultured with four recombinant proteins of different concentrations(10,20,40 ?g/mL)in vitro.Total RNA of goat PBMCs was extracted,which were stimulated with four recombinant proteins of different concentration.Then the level of mRNA transcription of IL-2?IL-4?IL-10?IL-17?IFN-? and TGF-P was detected by real-time fluorescent quantitative PCR.The results showed that the mRNA transcription levels of IL-4 and IFN-y in rNDUDC group were different from the control group apparently(P<0.01);the mRNA transcription levels of IL-10?IL-17 were relatively increased in rHCA59 of 40?g/mL(P<0.05)?The expression level of IL-4 was increased significantly after stimulation with rCREA of 20 and 40 ?g/mL.The changes of mRNA transcription levels in rCS group were not significant(P>0.05).4 Effects of four recombinant proteins of Haemonchus contortus on NO secretion and cell migration of goat PBMCsPeripheral blood mononuclear cells(PBMCs)were separated from goat blood sample and cultured with four recombinant proteins of different concentrations(10,20,40 ?g/mL)in vitro,the supernatant was collected by centrifugation to check NO secretion in cells by total NO assay kit.The effect of recombinant proteins with different concentrations on cell migration was detected by migration chamber.The result revealed that rCREA6,rCS and rNDUDC could promote the NO secretion and cell migration of PBMCs,and rNDUDC was significantly higher than the former.After rHCA59 stimulation,the level of NO secretion was significantly increased,but the migration ability was inhibited,and the higher the protein concentration,the stronger the inhibition.5 Effects of four recombinant proteins of Haemonchus contortus on phagocytosis of goat monocytesPeripheral blood mononuclear cells(PBMCs)were separated from goat blood sample,monocytes were isolated from PBMCs because of their adherent growth.Four Monocytes were cultred with four recombinant proteins of different concentrations(10,20,40 ?g/mL)in vitro,then the effect of proteins on monocyte pha-gocytosis of FITC-dextran by flow cytometry was tested.The result indicated that The phagocytosis of monocytes were significantly enhanced by rNDUDC stimulation with different concentrations.rCREA6 of 40 ?g/mL could enhance cell phagocytosis to some extent.The change of monocytes phagocytosis was not obvious after rCS stimulation.After stimulation of rHCA59,the phagocytic ability of the monocytes was decreased,but the change was not significant.