重组人白细胞介素10在毕赤酵母中的表达及生物学活性测定

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

目的在毕赤酵母中分泌表达具有生物学活性的重组人白细胞介素10(rhIL-10),用于IL-10的生理功能研究和动物试验。方法通过PCR扩增IL-10基因,插入到重组质粒α/pUC18的α因子信号序列的XhoⅠ和EcoRⅠ酶切位点处,再将融合基因αIL-10重组到表达质粒pPIC9K的BamHⅠ和EcoRⅠ之间,SalⅠ酶切线性化重组质粒IL-10/pPIC9K,电穿孔转化毕赤酵母,营养缺陷型培养基MD和G418筛选含高拷贝表达盒的酵母转化子,PCR鉴定是否含有目的基因IL-10,甲醇诱导rhIL-10的表达,SDS-PAGE和Westernblot鉴定目的蛋白,用ELISA定量测定及MC/9细胞分析IL-10生物学活性。结果成功构建了重组表达质粒IL-10/pPIC9K,获得了4个含IL-10基因的高拷贝毕赤酵母转化子,甲醇诱导后,在摇瓶水平的IL-10表达量为(0.627±0.09)mg/L,比活性为1.5×105U/mg。结论毕赤酵母分泌表达的IL-10具有良好的生物学活性。

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 Xho Ⅰ and EcoR Ⅰ downstream of α factor in the recombinant plasmid α/pUC18. Then, the fusion gene αIL-10 was digested and inserted into the sites between BamH Ⅰ and EcoR Ⅰ 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 Sal Ⅰ 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.