摘要
目的构建IL-24基因的真核表达载体,在毕赤酵母GS115中高效表达,研究重组N-糖基化IL-24蛋白体外诱导肿瘤细胞凋亡的活性。方法借助过渡质粒α/pUC18,将IL-24基因插入到质粒pPIC9K的BamHⅠ和EcoRⅠ之间,构建重组质粒IL-24/pPIC9K,转化毕赤酵母GS115分泌表达,Tricine-SDS-PAGE和Western blot鉴定目的蛋白,ELISA检测蛋白表达量,糖苷酶PNGaseF分析IL-24糖基化形式和程度。MTT法和形态学分析重组IL-24诱导MCF-7乳腺癌细胞凋亡的活性。结果成功构建重组表达质粒IL-24/pPIC9K,IL-24在毕赤酵母最高表达量为(81.31±14.46)mg·L-1。约70%的IL-24发生了N-糖基化。重组IL-24诱导MCF-7乳腺癌细胞凋亡,对正常人肺成纤维细胞NHLF没有影响。N-糖基化IL-24对MCF-7抑制率约高于去糖基化IL-24。结论毕赤酵母分泌形式的表达和适度的糖基化修饰都有利于目的蛋白IL-24的生物学活性,为后续的研究提供基础。
Aim To obtain recombinant human IL-24 secretorily expressed in Pichia pastoris, and study the activity of inducing tumor cells apoptosis of this N-glycosylation protein. Methods By the recombinant plasmid α/pUC18, the confirmed IL-24 gene was inserted between the sites of BamH I and EcoR I of expression plasmid pPIC9K. The recombinant plasmid IL- 24/pPIC9K was transformed into P. pastoris strain GSll5. Yeast transformants were induced for expression of recombinant human IL-24 with methanol. The desired protein was identified with Tricine-SDS-PAGE and Western blot. Amount of IL-24 was assayed with ELISA and the glyeosylation was analyzed by PNGase F. The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro.Results Recombinant expression plasmid IL-24/ pPIC9K was successfully constructed. 5 transformants were screened with G418 and PCR. Induced with methanol, the expression level of IL-24 was （81.31 ±14. 46） mg·L^-1 at flask fermentation, and 70 % IL- 24 generated N-glycosylation. Recombinant IL-24 in- duced apoptosis in MCF-7 cells, but not in NHLF. Conclusion The seeretorily expression of the N-gly- cosylation IL-24 protein in P. pastoris and the study of inducing tumor cells apoptosis lay the foundation for the further study of molecular mechanism of IL-24 on anti-tumors and the potential application.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2009年第7期915-919,共5页
Chinese Pharmacological Bulletin
基金
国家高技术研究发展计划(863计划)重点资助课题(No2001AA21516)
