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目的:真核表达重组猪单链IL-12(pscIL-12)并鉴定表达的pscIL-12的生物学活性。方法:采用梭华-SofastTM将pcDNA3.1(+)-pscIL-12转入CHO-K1细胞,ELISA法测定细胞培养上清中pscIL-12的表达水平;MTT法检测细胞培养上清对NK细胞杀伤作用的影响;ELISA法测定细胞培养上清刺激人PBMC分泌IFN-γ的水平;流式细胞术(FCM)检测细胞培养上清对人淋巴母细胞增殖水平的作用。结果:融合基因pscIL-12在CHO-K1细胞中表达产物pscIL-12融合蛋白含量较高;生物学活性鉴定表明:pscIL-12融合蛋白可增强NK细胞活性、提高IFN-γ的产生和促进人淋巴母细胞的增殖。结论:转染pcDNA3.1(+)-pscIL-12质粒的CHO-K1细胞成功表达了具有良好生物学活性的pscIL-12。
OBJECTIVE: To eukaryoticly express recombinant porcine single chain IL-12 (pscIL-12) and identify the biological activity of the expressed pscIL-12. Methods: pcDNA3.1 (+) - pscIL-12 was transfected into CHO-K1 cells by Soxhlet-SofastTM. The expression of pscIL-12 in the cell culture supernatant was determined by ELISA. The effect of cell culture supernatant on NK cells The effect of cell culture supernatant on the secretion of IFN-γ by human peripheral blood mononuclear cells was detected by ELISA. The effect of cell culture supernatant on the proliferation of human lymphoblastoid cells was detected by flow cytometry (FCM). Results: The expression level of pscIL-12 fusion protein in CHO-K1 cells was higher than that of pscIL-12 fusion protein. The biological activity of pscIL-12 fusion protein showed that pscIL-12 fusion protein could enhance the activity of NK cells and enhance the production of IFN- Lymphoblast proliferation. CONCLUSION: CHO-K1 cells transfected with pcDNA3.1 (+) - pscIL-12 plasmid successfully expressed pscIL-12 with good biological activity.