[1]杨珺,张家敏,黄卫平,等.N糖基化IL24的表达及体外诱导肿瘤细胞凋亡的研究[J].中国药理学通报,2009,(07):0.
 YANG Jun,ZHANG Jia min,HUANG Wei ping,et al.Expression of recombinant Nglycosylation interleukin 24 and study of inducing tumor cells apoptosis in vitro[J].Chinese Pharmacological Bulletin,2009,(07):0.
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N糖基化IL24的表达及体外诱导肿瘤细胞凋亡的研究()
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《中国药理学通报》[ISSN:/CN:]

卷:
期数:
2009年07期
页码:
0
栏目:
论著
出版日期:
2009-07-25

文章信息/Info

Title:
Expression of recombinant Nglycosylation interleukin 24 and study of inducing tumor cells apoptosis in vitro
作者:
杨珺1张家敏1黄卫平1张卫军2刘开云2邹全明2
1.浙江医学高等专科学校,浙江 杭州310053;2.第三军医大学临床微生物教研室,重庆400038
Author(s):
YANG Jun1ZHANG Jiamin1 HUANG Weiping1 ZHANG Weijun2 LIU KaiYun2ZOU Quanming2
1.Zhejiang Medical College, Hangzhou310053,China;2.Dept of Clinical Microbiology,Third Military Medical University, Chongqing400038,China
关键词:
白细胞介素24N糖基化毕赤酵母分泌表达肿瘤细胞凋亡
Keywords:
interleukin 24NglycosylationPichia pastorissecretory expressiontumor cellsapoptosis
分类号:
R 32925;R 379;R 39212
文献标志码:
A
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PNGase F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。
Abstract:
AimTo 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 GS115. 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 glycosylation was analyzed by PNGase F.The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro.ResultsRecombinant 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 induced apoptosis in MCF7 cells, but not in NHLF.ConclusionThe secretorily expression of the Nglycosylation 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.

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备注/Memo

备注/Memo:
收稿日期:2009-03-09, 修回日期:2009-05-05基金项目:国家高技术研究发展计划(863计划)重点资助课题(No 2001AA21516)作者简介:杨珺(1972-),女,博士,副教授,研究方向:生物制药,Tel:057187692692,Email: jennyyangjun@sina.com;邹全明(1963-),男,博士,教授,博士生导师,研究方向:生物制药,通讯作者,Tel:02368752316,Email:w8301991@263.net
更新日期/Last Update: 2009-07-25