Expression of recombinant interleukin 10 in Pichia pastoris and identification of its bioactivity

Objective To obtain recombinant human interleukin 10(rhIL-10) secretorily expressed in Pichia pastoris with bioactivity for studying its biological function and animal test. Methods IL-10 gene was inserted into the sites between XhoⅠ and EcoRⅠ downstream of α factor in the recombinant plasmid α/pUC18. Then, the fusion gene αIL-10 was digested and inserted into the sites between BamHⅠ and EcoRⅠ of expression plasmid pPIC9K of P. pastoris. After the recombinant plasmid IL-10/pPIC9K was analyzed to be correct construct with digestion and sequencing, it was linearized with digestion of SalⅠ and transformed into P. pastoris strain GS115. Recombinant yeast colonies grown in auxotrophic medium MD were in vivo screened with G418 for multicopy expression cassettes. Yeast transformants were proved to have the desired gene IL-10 with PCR, and then induced for expression of recombinant human IL-10 with methanol. Recombinant human IL-10 was induced to secretorily express with methanol. The desired protein was identified with SDS-PAGE and Western blot. IL-10 was quantitatively assayed with ELISA and its biological activity was analyzed with stimulating proliferation of MC/9 cells. Results Recombinant expression plasmid IL-10/pPIC9K was successfully constructed. Four transformants were screened with G418 and PCR. Induced with methanol, the expression level of IL-10 was (0.627±0.09)mg/L at flask fermentation, and its specific activity was 1.5×10~5 U/mg. Conclusion Recombinant human IL-10 has good biological activity secretorily expressed in P. pastoris.