【摘要】 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。更多还原

【Abstract】 Objective: To establish the yeast expression system for oncostatin M (OSM). Methods:The cDNA encoding human OSM was inserted into pPIC9K vector and transformed into Pichia pastoris. The correct phenotype of recombinants was screened through MD and MM auxotrophic culture medium. The OSM mRNA transcription in the P.pastoris and molecular weight were identified with Northern blot and Western blot. MTT method was used to test the activity of OSM in inhibiting the growth of human melanoma cells. Results: Established P.pastoris system could accomplish human OSM secreting expression. The recombinant OSM with a 30 000 molecular weight amounted to 70% of the total protein in medium supernatant. The OSM showed an inhibitory effect on the growth of A375 cells in vitro. Conclusions:The P.pastoris system of OSM was proved effective with a high expressing level. The secreted recombinant OSM present in supernatant of culture medium would be easy to be purified. It may lay the foundation for the large scale preparation of OSM in future. [更多还原