Study On CDNA Cloning Of Human Interieukin 10(hIL-10) And Expressing In Prokaryotic And Eukaryotic Cells

Interleukin-10(IL-10) is an important immunoregulatory cytokine produced by many cell populations. Its main biological function seems to be the limitation and termination of inflammatory responses and the regulation of differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen- presenting cells, mast cells, and granulocytes. However, very recent data suggest IL-10 also mediates immunostimulatory properties that help to eliminate infectious and noninfectious particles with limited inflammation. Numerous investigations, including expression analyses in patients, in vitro and animal experiments suggest a major impact of IL-10 in inflammatory, malignant, and autoimmune diseases. IL-10 overexpression or relative deficiency has been observed and is regarded to be of pathophysiological relevance in certain disorders. Recombinant human IL-10 has been produced and is currently being tested in clinical trials at abroad. This includes rheumatoid arthritis, inflammatory bowel disease, psoriasis, organ trans- plantation, and chronic hepatitis C. The results are hetero- geneous. They give new insight into the immunobiology of IL-10 and suggest that the IL-10 may become a therapeutic agent.There is still no domestic rhIL-10 product. Studies on over-expression of rhIL-10 are still on primary stage. In our research, cDNA encoding mature rhIL-10 was successfully cloned and was expressed by E.coli and yeast expression system respectively in order to lay a foundation for further rhIL-10 production. Achievements of this study are showed as followed: Firstly, A pair of specific primer was designed according to the published human IL-10 sequence searched from GenBank. With the pair of specific primer, cDNA encoding mature rhIL-10 was successfully cloned from LPS-stimulated PBMC by RT-PCR. The cloned cDNA was cloned into pGEM-T. Then recombinant pGEM-T-hIL-10 was purified for sequence analysis. The result revealed that cloned sequence was 100% homology to published hIL-10 cDNA sequence.Secondly, The cDNA encoding mature rhIL-10 was subcloned into the prokaryotic expression vector pET-28a(+) and identified with restriction enzyme digestion and sequencing. The correct recombinant was transformed into E.coli strain BL21(DE3) and induced by IPTG, the lysates of the bacteria were separated on SDS-PAGE. Thin layer scan analysis of the protein bands indicated that the interest protein accounted for 28.7% of total proteins of E.coli. Western blotting analysis showed the rhIL-10 could react with hIL-10 monoclonal antibody. After being primarily purified and refolded, the expressed protein displayed hIL-10 like activity: rhIL-10 could inhibit monocytes stimulated by LPS from expressing MHC class Ⅱ molecule in vitro. Thirdly, to obtain productive rhIL-10 with higher activity for large scale production, eukaryotic expression system-yeast was chosen to express rhIL-10 in our research. Yeast secretion expression plasmid pPICZαA-hIL-10 containing hIL-10 genes were successfully constructed. Then the linearized recombinant plasmids were electrotransformed into the competent yeast strain X-33 of Pichia pastoris, the positive transformants were selected and identified by PCR, SDS-PAGE and Western blotting. 10 yeast recombinant strains of X-33/ pPICZαA-hIL-10 were obtained, after 96 hour cultivation with 0.5% methanol, most rhIL-10 was acquired by 80% saturation of (NH4)2SO4. Thin layer scan analysis of the protein bands indicated that the interest protein accounted for 10% of the precipitated proteins. The expressed products were also purified by means of the immobilized metal affinity chromatography method, and the purity of the interest protein amounted to 94%. The purified rhIL-10 expressed by yeast was more active than that expressed by E.coli.