Cloning,Expression And Effects Of Five Recombinant Proteins Of Haemonchus Contortus On The Functions Of Goat PBMC In Vitro

Haemonchus contortus,namely barber pole worm,a gastrointestinal nematode feeding on blood,mainly parasites in the abomasum,but occasionally in small intestine.Infections are associated with anaemia,anorexia,disorderly fur,decreased milk yield and death of host,which resulted in vast economic losses to livestock production.Meanwhile,due to the unlimited use of anthelmintics,the development of drug resistence and residue make it to be challenged around the world.Research about the molecular immune and pathogenic mechanism of H.contortus has been received increased attention recently.In a previous proteomic study,five proteins of Haemonchus contortus excretory and secretory products?HcESPs?,identified as proteins binding to goat peripheral blood mononuclear cells?PBMC?in vivo,included Glutaredoxin domain containing protein?Grx-1?,Transcription factor E2F dimerisation partner?TDP?domain containing protein?DP-1?,Crotonase domain containing protein?Crt-1?,Aromatic amino acid beta-eliminating lyase threonine aldolase domain containing protein?TA-1?,and Miro domain containing protein?Miro-1?.However,little is known about the effects of these proteins of H.contortus on the goat PBMC.Hence,in the present study,the effects of these proteins on the goat PBMC were explored,respectively.1 The cloning and sequence analysis of gene Grx-1,DP-1,Crt-1,TA-1 and Miro-1The gene Grx-1,DP-1,Crt-1,TA-1 and Miro-1 was amplified by PCR using specific primers,respectively.Then PCR product was cloned into pMD19-T vector,and the recombinants were identified by enzyme digestion and sequencing.The sequences of these genes were compared with that of H.contortus available in GenBank.Results showed that:the obtained sequence of Grx-1 with the length of 672 bp was found to encode a protein of 223 amino acids with a molecular mass of 26.313 kDa and theoretical pI of 8.77.Blast results showed that the identity of the inserted fragment was 100%to the Grx-1 of H.contortus?HCOI₀1871900?.The obtained sequence of DP-1with the length of 1851 bp was found to encode a protein of 616 amino acids with a molecular mass of 68.288 kDa and theoretical pI of 9.28.Blast results showed that the identity of the inserted fragment was 98%to the DP-1 of H.contortus?HCOI₀1892600?.The obtained sequence of Crt-lwith the length of 777 bp was found to encode a protein of 258 amino acids with a molecular mass of 28.873 kDa and theoretical pI of 6.9.Blast results showed that the identity of the inserted fragment was 98%to the Crt-1 of H.contortus?HCOI₀1053700?.The obtained sequence of TA-1 with the length of 1194 bp was found to encode a protein of 397 amino acids with a molecular mass of 43.818 kDa and theoretical pI of 7.9 Blast results showed that the identity of the inserted fragment was 95%to the TA-1 of H.contortus?HCOI₀1467900?.The obtained sequence of Miro-1 with the length of 894 bp was found to encode a protein of 297 amino acids with a molecular mass of 33.689 kDa and theoretical pI of 6.68.Blast results showed that the identity of the inserted fragment was 97%to the Miro-1 of H.contortus?HCOI₀2135300?.The phylogenetic trees of amino acid sequences of these proteins was built by using MEGA4.0 and the result of clado-gram showed that the similarity of these protein sequences with Ancylostoma ceylanicum,Dictyocaulus viviparus and Necator americanus was high when compared with other analogous protein sequences in other parasites or species.2 Expression of rGrx-1,rDP-1,rCrt-1,rTA-1 and rMiro-1By sub-cloning the fragment of these genes into the vector pET-32a?+?,the prokaryotic expression plasmid pET-32a/Grx-1,pET-32a/DP-1,pET-32a/Crt-1,pET-32a/TA-1 and pET-32a/Miro-1 were constructed.The recombinants was transformed into E.coli BL21 and induced by IPTG.The expression of recombinant protein?rGrx-1,rDP-1,rCrt-1,rTA-1 and rMiro-1?was analyzed by SDS-PAGE.These recombinant proteins were purified with Ni2+-nitrilotriacetic acid?Ni-NTA?column and refolded by utilizing linear gradient decreased urea.Then purified recombinant proteins were administered to SD rats to obtain the anti-recombinant proteins sera.SDS-PAGE analysis showed that these genes were successfully expressed mainly in the inclusion body,as a fusion protein with a relative molecular mass of 46 kDa,88 kDa,49 kDa,64 kDa and 54 kDa.Western blot results showed that these recombinant proteins were successfully recognized by the sera of goats experimentally infected with H.contortus,indicated that these proteins had immunogenicity.3 Binding of rGrx-1,rDP-1,rCrt-1,rTA-1 and rMiro-1 to goat PBMCPBMC were separated and cultured in vitro with rGrx-1/rDP-1/rCrt-1/rTA-1/rMiro-1 at a final concentration of 10 ?g/mL.The binding of these recombinant proteins to PBMC was detected by immunofluorescence antibody technique?IFA?.Results of IFA showed that rGrx-1,rDP-1,rCrt-1,rTA-1 and rMiro-1 all could bind to goat PBMC.4 Detection of changes in cytokine secretion of goat PBMC after incubated with rGrx-1/rDP-1/rCrt-1/rTA-1/rMiro-1PBMC were incubated with a serial concentration of rGrx-1/rDP-1/rCrt-1/rTA-1/rMiro-1?0,10,20 and 40 p.g/mL?or protein of empty pET-32a,and incubated at 37? and 5%CO2.After 24 h,cells were harvested for total RNA extraction followed by RT-PCR.The detection of cytokine transcription levels of IL-2,IL-4,IL-17,IL-10,IFN-y and TGF-?mRNA was performed by real-time qPCR.Results showed that compared to the control,40?g/mL of rGrx-1 significantly increased the production of TGF-p and decreased the production of IL-4 and IFN-y.10 and 20 ?g/mL of rDP-1 both significantly increased the secretion of IL-4 and TGF-P.10 and 20 ?g/mL of rCrt-1 significantly increased the secretion of IL-4;20 ?g/mL of rCrt-1 significantly decreased the secretion of IL-10,20 and 40 ?g/mL obviously inhibited the production of IL-17,and 40 ?g/mL significantly decreased the secretion of IL-2.20 ?g/mL of rTA-1 significantly increased the secretion of IL-4,IL-2 and IFN-?,and the secretion of IL-17 was obviously boosted in any test group stimulated with rTA-1.The expression of IL-17 was obviously boosted in the treatment stimulated with rMiro-1 at 10 and 40 ?g/mL,IL-4 was obviously at 20 and 40 ?g/mL,and IL-2 was obviously at 40 ?g/mL.5 Effects of five recombinant proteins on the cell proliferation and migration of goat PBMCCell proliferation and migration were tested by CCK-8 method and Millcell???Hanging Cell Culture Inserts after goat PBMC were stimulated with rGrx-1/rDP-1/rCrt-1/rTA-1/rMiro-1 at a final concentration of 0,10,20 and 40 ?g/mL for 72 h and 24 h,respectively.Results showed that,compared with the control,40 ?g/mL of rGrx-1 could significantly inhibit the cell proliferation and migration;10 ?g/mL of rDP-1 and 20 ?g/mL of rTA-1 both significantly enhance the cell proliferation and migration;rMiro-1 could activate the cell proliferation and migration in a dose dependent manner;10 and 20 ?g/mL of rCrt-1 might elevate the cell migration percentage,but 40 ?g/mL of rCrt-1 reduce the percentage,and no significant difference on the cell proliferation was observed between treatment group and the control.6 Effects of five recombinant proteins on the NO release of goat PBMC and monocyte phagocytosisNO release of PBMC and monocyte phagocytosis was examined by nitrate reductase assay and flow cytometer after goat PBMC/monocyte were treated with rGrx-1/rDP-1/rCrt-1/rTA-1/rMiro-1 at the dose of 0,10,20 and 40 ?g.mL-1 for 24 h and 48 h,respectively.Results showed that,compared to the control,20 and 40 ?g/mL rGrx-1 could significantly depressed the NO release of goat PBMC and monocyte phagocytosis;No significant change in the NO release of goat PBMC and monocyte phagocytosis were observed in the test group of rDP-1 and rCrt-1;Any dose of rTA-1 could significantly boost the production of NO,notably at the 20 ?g/mL,while markedly enhance the monocyte phagocytosis only at 40 ?g/mL;The production of NO was significantly increased in the treatment of rMiro-1 at 20 and 40 ?g/mL,while monocyte phagocytosis was obviously strengthened by the 40 ?g/mL of rMiro-1.