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目的 :构建抑瘤素M (oncostatinM ,OSM)的酵母表达体系。方法 :将人OSMcDNA连入pPIC9K酵母表达载体 ,转染毕赤酵母 (Pichiapastoris) ,用MD和MM营养缺陷型培养基筛选具有正确表型的重组转化子 ,用RNA印迹法和蛋白质印迹法鉴定酵母转化菌株中OSMmRNA转录和OSM相对分子质量。MTT法鉴定OSM蛋白对人黑素瘤细胞生长的抑制活性。结果 :构建的毕赤酵母体系可实现人OSM分泌表达 ,表达量占外分泌蛋白的 70 %以上。表达蛋白相对分子质量约 30 0 0 0 ,对黑素瘤细胞生长可产生明显抑制作用。结论 :OSM的酵母分泌表达体系表达量可观 ,表达蛋白集中于培养液上清 ,便于纯化 ,是日后进行OSM大规模制备的可行途径。
Objective: To construct yeast expression system of oncostatin M (OSM). METHODS: Human OSM cDNA was ligated into pPIC9K yeast expression vector and transfected into Pichia pastoris. The recombinant transformants with the correct phenotype were screened with MD and MM auxotrophic medium, and the yeast was identified by Northern blotting and Western blotting OSM mRNA transcription and OSM relative molecular mass in transformed strains. The inhibitory activity of OSM protein on human melanoma cell growth was identified by MTT assay. Results: The constructed Pichia pastoris system can express human OSM in more than 70% of the exocrine protein. The relative molecular mass of the expressed protein is about 30 000, which can significantly inhibit the growth of melanoma cells. CONCLUSION: The expression level of OSM in yeast secretion system is considerable, and the expression of protein concentrated in the culture supernatant for purification is a feasible way for the large-scale preparation of OSM in the future.