[1]别俊,孙茂盛,孙强明,等.人survivin的克隆、在大肠杆菌中的表达及活性鉴定[J].南方医科大学学报,2004,(08):913-916.
　BIE Jun,SUN Mao-sheng,SUN Qiang-ming,et al.Cloning, expression and identification of human survivin in E.coli[J].Journal of Southern Medical University,2004,(08):913-916.
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人survivin的克隆、在大肠杆菌中的表达及活性鉴定()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2004年08期
页码:
913-916
栏目:
出版日期:
2004-08-01

文章信息/Info

Title:
Cloning, expression and identification of human survivin in E.coli
作者:
别俊1;  孙茂盛2;  孙强明2;  郑建平3;  孙雯佳2
1. 昆明医学院研究生部, 云南, 昆明, 650031;
2. 中国医学科学院中国协和医科大学医学生物学研究所分子生物室, 云南, 昆明, 650118;
3. 昆明市延安医院, 云南, 昆明, 650023
Author(s):
BIE Jun1;  SUN Mao-sheng2;  SUN Qiang-ming2;  ZHENG Jian-ping3;  SUN Wen-jia2
1. Graduate School of Kunming Medical College, Kunming 650031, China;
2. Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences & Union Medical University, Kunming 650118, China;
3. Kunmimg Yan’an Hospital
关键词:
surivivin; 克隆; 基因表达; 纯化
Keywords:
survivin; cloning; gene expression; purification
分类号:
Q78
摘要:
目的 在原核中高效表达及活性鉴定重组人survivin蛋白分子。方法 应用RT-PCR的方法得到survivin的cDNA,并构建成pBV220-survivin原核表达质粒,将其导入E.coliBl21(Gold)中,诱导表达survivin蛋白。通过DEAESepharoseFastFlow离子交换柱和SephacrylS-200分子筛二步柱纯化以获取目的蛋白。用Westernblotting检测表达产物。结果 PT-PCR产物经测序分析与预期结果完全一致,表达质粒在E.coli Bl21(Gold)中得到高效表达,表达的survivin蛋白占总蛋白含量的30%以上,表达产物以包涵体的形式存在。通过离子交换柱和分子筛二步柱纯化及复性后获得的目的蛋白,其纯度达到95%以上。Westernblotting证明表达产物具有特异性。结论 获得了较高纯度的survivin蛋白,为进一步深入研究该蛋白的细胞凋亡调控功能提供了工具。
Abstract:
Objective To efficiently express and identify recombinant human survivin in E.coli. Methods Survivin cDNA was amplified by reverse transcriptional (RT)-PCR and cloned into the prokaryotic expression vector pBV220, followed by expression of the recombinant plasmid in E.coli strain BL21 (Gold). To obtain survivin protein, DEAE-Sepharose Fast-Flow ion exchange chromatography and Sephacryl S-200 gel filtration were performed. Western blot analysis was used for detecting the expressed product. Results Survivin protein was expressed in E.coli in the form of inclusion body at the expression level over 30% of the total cell protein. After ion exchange chromatography and gel filtration, the recombinant protein reached a purity over 95% and exhibited specific reaction with mouse anti-human antibody. Conclusion Survivin protein with high purity can be obtained by the method described above to facilitate further study of the anti-apoptosis function of survivin.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2003-8-26。
作者简介:别俊(1976-),男,昆明医学院在读硕士研究生,电话:0871-8334401,E-mail:bj520307@sina.com
通讯作者:孙茂盛,中国医学科学院医学生物研究所副所长,电话:0871-8334326,E-mail:maoshs@public.km.yn.cn
更新日期/Last Update: 1900-01-01