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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 express biologically active recombinant human interleukin-10 (rhIL-10) in Pichia pastoris for physiological function studies and animal experiments of IL-10. Methods The IL-10 gene was amplified by PCR and inserted into the XhoⅠ and EcoRⅠ restriction sites of the α-factor signal sequence of the recombinant plasmid α / pUC18. The fusion gene αIL-10 was recombined into BamHI and EcoRI of the expression plasmid pPIC9K , Sal Ⅰ enzyme-digested linearized recombinant plasmid IL-10 / pPIC9K, electroporated into Pichia pastoris, auxotrophy medium MD and G418 screening yeast transformants containing high copy expression cassette, PCR identified whether the target gene IL-10, Methanol induced the expression of rhIL-10, and identified the target protein by SDS-PAGE and Western blot. The biological activity of IL-10 was analyzed by ELISA and MC / 9 cells. RESULTS: Recombinant plasmid IL-10 / pPIC9K was successfully constructed and four high-copy Pichia pastoris transformants containing IL-10 gene were obtained. After methanol induction, the expression level of IL-10 in shake flask was (0.627 ± 0.09 ) mg / L, the specific activity of 1.5 × 105U / mg. Conclusion Pichia pastoris secreted IL-10 has good biological activity.