Eukaryotic Expression Of Human Interleukin-35and Study Of Biological Function

In2007, IL-35(Interleukin-35) was found by Liew’ and Vignali’group, It is a heterodimer consisting of Epstein-Barr virus-induced gene3(EBB) and the p35subunit of IL-12.IL-35is a member of IL-12family, and its structure is homology with other IL-12family members. IL-35signals through a heterodimeric receptor composed of a specific component, IL12-Rβ2and the common chain, gp130. IL-35signals through a unique heterodimer of receptor chains IL-12Rβ2and gp130or homodimers of each chain. IL-35is a suppressive cytokine secreted by the regulatory T cells, it can also convert naive T cells into IL-35-producing induced regulatory T cells (iTr35cells).We obtained a high purity rhIL-35[rhIL-35-IgG1(Fc)] by genetic engineering, and protein purification techniques, and studied its biological activity. This thesis includes three parts:Firstly, the construction and expression of rhIL-35. The recombined plasmid of IL-35, pSTEP2-IL35-LFc, was constructed and transfected into HEK293T cells. Then rhIL-35-IgG1(Fc)was expressed and purified by affinity chromatography, and examined by SDS-PAGE and Western blot. Secondly, the biological activity of rhIL-35was investigated. The study found IL-35-Fc was capable of binding gp130of the receptor chain, and its neutralization activity on gp130was investigated on M1mouse myeloid leukemia cells. In addition, the neutralization activity of IL-35to LIF on M1mouse myeloid leukemia cells has also been investigated. Thirdly, the biological effect of rhIL-35on T cells was explored. The study explored the rhIL-35induction of mouse splenocytes and thymocytes after co-stimulation of anti-CD3and anti-CD28, and PBMC after the stimulation of anti-CD3. The results showed that there was no significant effect.In conclusion, high purity and active rhIL-35-Fc protein was produced. The study also demonstrated the binding function and neutralization activity of IL-35to gpl30and LIF on M1mouse myeloid leukemia cells. Recombinant human IL-35did not affect T cell proliferation after co-stimulation of anti-CD3and anti-CD28. Neither an increase nor a decrease in comparison to the untreated T cells was observed. Even high doses of recombinant human IL-35did not reveal differences in T cell proliferation. Recombinant human IL-35did not induce or inhibit T cell differentiation to CD4+T cell and CD4+CD25+T cell.