Molecular Cloning,characterization And Effects Of Recombinant AK,ES-15,TpMy,STP-1,EF-1?,MTF-12 Proteins Of Haemonchus Contortus On Goat Pbmcs

1.Arginine kinase from Haemonchus contortus decreased the proliferation and increased the apoptosis of goat PBMCs in vitroArginine kinase(AK),an important member of phosphagen kinase family has been extensively studied in various vertebrates and invertebrates.Immunologically,AKs are important constituents of different body parts,involved in various biological and cellular functions,and considered as immune-modulator and effector for pro-inflammatory cytokines.However,immunoregulatory changes of host cells triggered by AK protein of Haemonchus contortus,a parasitic nematode of ruminants,are still unknown.The current study was focused on cloning and characterisation of Hc-AK,and its regulatory effects on cytokines level,cell migration,cell proliferation,nitric oxide production and apoptosis of goat peripheral blood mononuclear cells(PBMCs)were observed.The full-length sequence of the Hc-AK gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and sub-cloned into the prokaryotic expression vector pET-32a.The biochemical characteristics of recombinant protein Hc-AK,which was purified by affinity chromatography,were performed based on the enzymatic assay.Binding of rHc-AK with PBMCs was confirmed by immunofluorescence assay(IFA).Immunohistochemical analysis was used to detect localisation of Hc-AK within adult worms sections.The immunoregulatory effects of rHc-AK on cytokine secretions,cell proliferation,cell migration,nitric oxide production and apoptosis were determined by co-incubation of rHc-AK with goat PBMCs.The full-length ORF(1,080 bp)of the Hc-AK gene was successfully cloned,and His-tagged AK protein was expressed in the Escherichia coli strain BL21.The recombinant protein of Hc-AK(rHc-AK)was about 58.5 kDa together with the fused vector protein of 18 kDa.The biochemical assay showed that the protein encoded by the Hc-ak exhibited enzymatic activity.Western blot analysis confirmed that the rHc-AK was recognised by the sera from rat(rat-antiHc-AK).The IFA results showed that rHc-AK could bind on the surface of goat PBMCs.Immunohistochemically,Hc-AK was localised at the inner and outer membrane as well as in the gut region of adult worms.The binding of rHc-AK to host cells increased the levels of IL-4,IL-10,IL-17,IFN-y,nitric oxide(NO)production and cell apoptosis of goat PBMCs,whereas,TGF-?1 levels,cell proliferation and PBMCs migration were significantly decreased in a dose dependent manner.Our findings suggested that rHc-AK is an important excretory and secretory(ES)protein involved in host immune responses and exhibit distinct immunomodulatory properties during interaction with goat PBMCs.2.Characterization of the Haemonchus contortus excretory/secretory antigen rHcES-15 and its effects on goat PBMCs in vitroThe small size excretory/secretory antigens of Haemonchus contortus parasite have intense interest among researchers for understanding the molecular basis of helminths immune regulation in term of control strategies.In this study,H.contortus ES antigen 15 kDa(HcES-15)was successfully cloned in an expression vector system and expressed protein was characterized for its immunoregulatory roles on host PBMCs.The HcES-15 gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR)from adult H.contortus cDNA,cloned into expression plasmid pET32a(+)and expression of the recombinant protein(rHcES-15)was induced by IPTG.Binding activity of rHcES-15 to goat peripheral blood mononuclear cells(PBMCs)was confirmed by immunofluorescence assay(IFA)and immunohistochemical analysis showed that H.contortus 15 kDa protein in outer and inner structure of adult worm,clearly indicated as parasite's ES antigen.The immunoregulatory role on cytokine production,cell proliferation,cell migration,nitric oxide(NO)production,apoptosis and phagocytosis were observed by co-incubatlocalized ion of rHcES-15 with goat PBMCs.The results showed that cytokines IL-4,IL-10,IL-17,the production of nitric oxide(NO),PBMC apoptosis and monocytes phagocytosis were all elevated after cells incubated with rHcES-15 at differential protein concentrations.We also found that IFN-?,TGF-?1,cells proliferation and migration were significantly suppressed with the interaction of rHcES-15 protein.Our findings indicated that low molecular ES antigens of H.contortus possessed discrete immunoregulatory roles,which will help to understand the mechanisms involved in immune evasion by parasite during host parasite interactions.3.Recombinant tropomyosin from Haemonchus contortus mediated suppressive effects on goat PBMCs in vitroThe fundamental phase in the functional immune regulation by excretory/secretory proteins(ESPs)is binding to the host PBMCs,during host-parasite interactions.In this research,the gene encoding Haemonchus contortus tropomyosin(Hc-TpMy),was successfully cloned and expressed in an expression vector system,and the IPTG induced recombinant protein was successfully recognized by the sera of rat experimentally infected with rHc-TpMy.The recombinant protein after host cell surface attachment was evaluated for immune functional analysis with goat peripheral blood mononuclear cells(PBMCs)in vitro.The immunofluorescence assay detected attachment of rHc-TpMy on the surface of host PBMCs.The immunohistochemical method highlighted the presence of Hc-TpMy protein in entire musculature of adult worm sections.Furthermore,immunoregulatory role of rHc-TpMy on cytokines expression,PBMC proliferation,migration,nitric oxide(NO)production,apoptosis and monocytes phagocytosis were observed.The results showed that expression of IL-4 and IFN-y cytokines,cell proliferation,nitric oxide production(NO)and migration were significantly suppressed by co-incubation of protein with goat PBMCs.However,the productions of IL-10,IL-17 and TGF-?1 cytokines,PBMCs apoptosis and monocytes phagocytosis were elevated at dose dependent manner.Our findings indicated that rHc-TpMy being an important ES binding protein exhibit distinct immuno-suppressive role on goat PBMCs and could provide better understanding of the immune evasion mechanism to control haemonchosis in future.4.Characterization of serine/threonine-protein phosphatase 1 from Haemonchus contortus and its effects on functions of goat PBMCs in vitroSerine/threonine protein phosphatases,as integral constituents of parasitic excretory/secretory proteins(ESPs)are assumed to be released during the host-parasite interactions.However,knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host PBMCs is scant.In the present study,an open reading frame(ORF)of serine/threonine protein phosphatase from H.contortus designated as HcSTP-1 was amplified and cloned using reverse transcription-PCR method.The 951 bp nucleotides sequence was encoded to a protein of 316 amino acid residues,conserved in characteristics motifs GDXHG,GDYVDRG,GNHE,HGG,RG,and H.The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on SDS-PAGE.Immunohistochemically,HcSTP-1 was found to be localized in both male and female adult worm sections.Using immunofluorescence assay(IFA),the binding activity of rHcSTP-1 was confirmed on surface of goat peripheral blood mononuclear cells(PBMCs),which resulted in expression of multiple cytokines and various immunomodulatory activities in vitro.The real time PCR results showed that mRNA level of IL-2,TGF-?1,IFN-? and IL-17(with 10?g/ml),were up-regulated and IL-10 was decreased,however,IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein.Further functional analysis showed that migratory activity of cells,intracellular nitrite production(NO)and apoptotic efficiency of PBMCs were elevated at significant level,whereas the proliferation of goat PBMCs and monocytes-associated MHC-? and MHC-? expressions were decreased significantly at concentration dependent fashion.Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells,which might represent a potential molecular target for controlling H.contortus infection in future.5.Characterization of Elongation factor 1 alpha of Haemonchus contortus and its effects on goat PBMCs in vitroThe Haemonchus contortus excretory and secretory proteins(HcESPs)interact extensively with host PBMCs to depress or modulate the efficiency of host immune system.In this study,an immunologically important ES protein from H.contortus called Elongation factor 1 alpha(HcEF-1?)was cloned and characterized to evaluate its immune regulatory characteristics on goat peripheral blood mononuclear cells(PBMCs).The recombinant HcEF-1? expressed in a prokaryotic expression system was purified and incubated with goat PBMCs to evaluate its effects on cytokines secretion,cell proliferation,cell migration,nitric oxide production and apoptotic proficiency in vitro.Results of Western blot showed that the recombinant EF-1? could be recognized by serum from goat infected with H.contortus.The immunofluorescence method detected that rHcEF-la could bind on the surface of PBMCs.Moreover,EF-1? was localized exclusively in outer and inner membranous structure as well as epithelial lining in gut region of the adult H.contortus clearly indicates as ES protein secreted at L4 to adult stage of parasite.Our study showed that the interaction of rHcEF-1? could induce goat PBMC to express cytokines IL-4,IL-17,IFN-? and TGF-?1 which in turn increased the cell migration,proliferation and PBMC apoptosis.Moreover,rHcEF-1? incubated with goat PBMC,downregulated the interleukin IL-10 and nitric oxide production(NO).The MHC-? molecule expressions in cells incubated with rHcEF-1? was increased significantly whereas,MHC-? could not compared to the control groups(PBS control and pET32a)which indicated that,this protein may potentially mediate Thl immune stimulation during host parasite interactions.6.Identification of a novel protein Methyltransferase-Type 12 from Haemonchus contortus and its effects on goat PBMCs in vitroA ubiquitously expressed excretory and secretory protein identified at L4+L5 developmental stages of Haemonchus contortus implicated in wide range of immune functional regulation on host PBMCs.Herein,we cloned a novel gene(764 base-pairs)from H.contortus cDNA designated as HcMTF-12 by using reverse transcriptase-polymerase chain reaction(RT-PCR)followed by prokaryotic expression in Escherichia coli BL21(DE3 strain).The recombinant His-tagged protein comprised of 254 amino acid residues was subsequently resolved at 12%sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and successfully localized within adult worm sections by immunohistochemical analysis indicated as ES protein of H.contortus.Western-blot recognized both native(MTF-12)and rHcMT-12 protein with sera containing antibodies both from rat and goat respectively.Analysis was further validated by immunofluorescence assay that confirmed rHcMTF-12 as membrane bound protein on surface of goat PBMCs.The immune functionality of rHcMTF-12 on cytokines transcription showed augmentation of IL-2,IL-4,IL-17 and IFN-? transcripts at considerable level dose dependently(P<0.001).The secretions of IL-10 and TGF-?1 were significantly decreased in PBMCs incubated with rHcMTF-12(P<0.001),however,IL-6 was not significantly different(P<0.662)when compared to control groups.Moreover,recruitment of cells and nitric oxide production by PBMCs were induced considerably whereas,the proliferation of cells were affected when incubated with rHcMTF-12 protein.Together all these outcomes suggested that MTF-12 from H.contortus significantly mediate immune functional regulation of PBMC,which might be a potential candidate for therapeutic interventions against haemonchosis.