Expression And Biological Activity Of Recombinant Human IL-17 Protein

Since its conception two decades ago, the Th1-Th2 paradigm has provided aframework for understanding Y cell biology and the interplay of innate and adaptiveimmunity. Naive T cells differentiate into effector T cells with enhanced functionalpotential for orchestrating pathogen clearance largely under the guidance of cytokinesproduced by cells of the innate immune system that have been activated by recognition ofthose pathogens. This secondary, education of post-thymic T cells provides a mechanismfor appropriately matching adaptive immunity to frontline cues of the innate immunesystem. Owing in part to the rapid identification of novel cytokines of the IL-17 andIL-12 families using database searches, the factors that specify differentiation of a neweffector T cell lineage-Th17-have now been identified, providing a new arm of adaptiveimmunity and presenting a unifying model that can explain many heretofore confusingaspects of immune regulation, immune pathogenesis, and host defense.This kind of T cells now named Th17 cells. It specifically secreted IL-17 cytokineand participated in a variety of immune responses in vivo.The changing of Th17 cellsquantity and IL-17 expression level, the alteration of proportion between Th17 cells andTh1 cells in vivo can suggests the degree of disease severity. Because of in-depthresearch on IL-17, We gradually realized its importance. However, in our country,theresearch on IL-17 is not enough and that affects our understanding to the passway of Tcells'activation. To address these issues,we depended on our good condition,started fromthe expression of protein,then gradually researched their biological characteristics.Thoseefforts established the foundation for the further research of IL-17 monoclonal antibodyand the mechanism which Thl7 cells and IL-17 make the role in autoimmune diseases. 1. Cloning the cDNA ecoding human IL-17 full-length and non-signal peptidefragmentThe published cDNA sequence for human IL-17 was used for specific pimer design.full-length of human IL-17 gene and non-signal peptide fragment of human IL-17 genewas amplified through RT-PCR from PHA-activated human peripheral blood T cells.Thendigested PQE3.0 and PCEP4 vectors and aquired IL-17 genes by double restrictionenzyme digest method.Next work was using lagase to ligate these products afterrecovery. The derived plasmid was verified by DNA sequence analysis and the vectornamed PQE3.0/IL-17 and PCEP4/IL-17 were transformed into TOP10 strain ofEscherichia coli for proliferation.After certified by digest and PCR,the derivedrecombinant PQE3.0 plasmid was transformed into M15 strain of Escherichia coli forfurther expression of fusion protein.2.Expression, purification and characterization of PQE3.0/IL-17 fusion protein inE.coliM15 expression strain was propagated in lura broth. A series of methods (lowtemperature induction, shorten of induction time, etc) was tried, to induce solubleIL-17/His fusion protein but failed. Fusion protein was mainly expressed as inclusionbody. Dynamics study indicated that the most high-level protein expression was after 5hour IPTG induction at 1mmol/L. The cell slurry was disrupted by sonication, and thenfusion protein was purified through HiTrap affinity chromatography column afterdenaturation, refolding and dialysis. Purity of protein is above 90% with 2mg/mlconcentration. Western blotting showed a single band of molecular mass 15KDa.3. Expression of IL-17 in eukaryotic expression systemExtracted the plasmid of PCEPE/IL-17,then transfected them into CHO cells, usinghygromycin screening method observed the positive clone cells under microscope.trypsin-ized these cells by trypsin,then transferred positive clones from 6-well plate to another24-well plate by sharp picker, continued to use the drug screening method until acquiredthe stabilizing protein expression clone.pipeted the cell culture supernatant and detected the expression of IL-17 by ELISA method of qualitative and quantitative.Theexperimental results were obtained fusion protein and the expression level was high.4. preliminary study of recombinant human IL-17 protein in the biological functionUseing the cervical carcinoma cell line HeLa as response cells, added differentconcentrations of IL-17/His, fusion protein and IL-17/Fc fusion protein.After 48 hoursreaction, detected the GM-CSF and IL-6 levels in these supernatants by ELISA.Comparing to the control group, Observed whether the two kinds of proteins' action havedose-dependent relationship in a certain dose range and compared the activity level ofthese two kinds of proteins.Taken together, we successfully expressed IL-17 recombinant protein inprokaryotic and eukaryotic expression system and preliminarily studied the biologicalactivity.The proteins with certain biological activity have laid the foundation for the nextphase of work. The research about hIL-17 anchor protein and hIL-17 monoclonalantibody will be continued next step.