Expression, Purification And Adsorption Of Thl7 Cell-related

Autoimmune disease (AID) refers to the body’s immune system responsing to the own antigen, which leads to the tissue damage and even the disease. Because the pathogenesis of autoimmune disease is complex, it has not been studied clearly and there is still no effective treatment for clinical treatment. The past time people thought that autoimmune diseases are related to the two subpopulations of Thl and Th2 cells about the T-helper cells, and it will cause the disease if these two subpopulations interact with loss of balance, but with further research, people found that it can not explain the cause for some diseases, and corresponding A new cell subsets(Th17 cell subsets) were found and can explain many doubts better. breaking tradition concept, this new cell subsets were found having the closer relationship with autoimmune diseases in recent years. The main effect cytokines of Th17-related are IL-17, IL-6 and TNF-a. from the view of the clinical treatment about autoimmune diseases, the detection and removal of Thl7 cell-related factors has more practical significance. In this paper, it consists of the expression and purification of main effect cytokines of Thl7 cells, and the adsorption evaluation of typical sorbent. Specific contents include:(1) Expression and purification of TNF-a. Recombinant plasmid of pET23a-TNF-a was constructed and transferred into a host BL21 (DE3), making protein expression by IPTG induction. The optimal conditions for protein expression were confirmed by optimizing conditions:LB cell culture medium,37℃,175 rpm activating 12 h, transferring to a big culture by 1% inoculum for 3 h, then with 0.1 mM IPTG inducing 4 h, the expression result of the target protein is about 1.2 mg/mL,56% of the total protein. The cells were sonicated, following by 0.2% PEI (w/w) precipitation and 40% saturated ammonium sulfate precipitation, Ni-NTA affinity resin chromatography. Finally, the purity of sample is over 95%.(2) Expression of IL-6, IL-17A/17F. Recombinant plasmid of pPIC9K-IL-6, pPIC9K-IL-17A and pPIC9K-IL-17F were constructed and transferred into the host GS115. Then the His+Mut+type of recombinants were screening by RDB media, MM/MD media, PCR.The positive recombinants were cultured with YPD media basing on 30℃,225 rpm for about 14-16 h, transferring into BMGY culture by 1% inoculum basing on 30℃,225 rpm until growing to OD600 of 2-6, and then transferring to the medium of BMMY to make the OD600 of medium reach 1,following with 25 ℃,225 rpm culturing 96 h, methanol inducing every 24 h with amount of a final concentration of 1%. Finally, the IL-6 expresses successfully,but the expression of IL-17A and 17F has not been confirmed successfully.(3) Evaluation of polystyrene adsorbent for Inflammatory cytokines. Through isotherms and adsorption kinetics, adsorbents were confirmed that they have the fast adsorption rate for TNF-a, about 2 h reaching the maximum adsorption. The affinity constant between polystyrene adsorbent and TNF-a 57 mL/mg, ie 9.7×105 L/mol, equilibrium dissociation constant(Kd) is 0.018 mg/mL, the theoretical maximum binding capacity (Qm) is about 845.49 μg/g, Polystyrene adsorbent also has the adsorption of IL-6.Through this paper, obtain the expression and purification method for the Th17 cells mainly related factors and explore adsorption properties of Inflammatory cytokines with polystyrene adsorbent, and lay a good foundation for the research of autoimmune diseases.