| Objective:To search the best condition of expression and purification of recombinant human interleukin-31 and to use the protein induce pruritus of mice.To evaluate the dependablity between recombinant human interleukin-31 and pruritus and how to the protein cause pruritus.Methods:(1) Culturing engineering bacteria E. coli BL21(DE3),SDS-PAGE assay expressd amount about different induced concentration of revulsivum,temperature and time.(2) E.coli cells were broken by ultrasonic,the inclusion bodies were washed and solvated in urea,further purified by sephadex G-75 and Ni-NTA agarose F.F.,SDS-PAGE assay purify effect.Using Western blot and amino acid sequence appraise the protein.(3) To dialyse and concentrate the protein after purification,and determine its concentration through use Bradford Protein Assay Kit.(4) Injecting the rhIL-31 protein to BALB/C mice by different pathways and dosages;observing the behavior and skin of the mice,and then collecting peripheral blood for observing the white blood cells,skin for stain the skin histological sections by HE,and serum for determine the levels of IL-6,IL-8,MIP-3βand L-selectin.Results: (1) After SDS-PAGE assay,the most expression of pET-32a/rhIL-31 in E.coli BL21(DE3) was that induced by 1.0 mmol/L IPTG,at 37℃and after 5 hours.(2) Purity of rhIL-31 in soluble inclusion bodies purified by Ni-NTA agarose F.F was better than sephadex G-75.(3) Western blot and amino acid sequence assay shows that we obtained human interleukin 31 protein.(4) The renaturation rhIL-31 can induce pruritus in BALB/C mice in a dose-dependent manner.Analysis of mice peripheral blood shows that the property of white blood cells rise to some degree in tested groups.(5) Comparison of the tested and controlled groups separately shows that the levels of IL-6, IL-8,MIP-3βand L-selectin in the serum of the tested groups was significantly higher than the controlled ones.Conclusion:(1) the most expression of pET-32a/rhIL-31 in E.coli BL21(DE3) was induced by 1.0 mmol/L IPTG,at 37℃after 5 hours.(2) Purity of rhIL-31 in soluble inclusion bodies were purified by Ni-NTA agarose F.F was better than sephadex G-75.(3) The renatured rhIL-31 can induce pruritus.The amount of white blood cells in tested groups was significantly higher and the levels of IL-6,IL-8, MIP-3βand L-selectin in the serum of the tested groups was higher than the controlled ones which suggest that IL-31 mainly induces pruritus by induces several cytokine and chemokine. |
