[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.
点击复制

人survivin的克隆、在大肠杆菌中的表达及活性鉴定()
分享到:

《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:
survivincloninggene expressionpurification
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:

[1] Li F, Ambrosini, Chu EY. Control of apoptosis and mitotic spindle checkpoint by survivin[J]. Nature, 1998, 396: 580-4.
[2] Adida C, Crotty PL, Mcgrath J, et al. Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse proliferation[J]. Am J Pathol, 1998, 152(1): 43.
[3] Carter BZ, Milella M, Altieri DC, et al. Cytokine-regulated expression of survivin in myeloid leukemia[J]. Blood, 2001, 97: 2784-90.
[4] Suzuki A, Ito T, Kawano H, et al. Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death[J]. Oncogene, 2000, 19: 1346-53.
[5] Shin S, Sung BJ, Cho YS, et al. Anti-apoptosis protein human survivin is a direct inhibitor or Caspase -3 and -7[J]. Biochemistry,2001, 40:1117-23.
[6] Olie RA, Simoes AP, Baumann B, et al. A novel antisense oligonucleotide tatgeting survivin expression induces apoptosis and sensitizes lung cancer cell to chemotherapy[J]. Cancer Res, 2000,60 (11): 2805-9.
[7] Grazia, Ambrosini, Colette A, et al. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting[J]. J Biol Chem, 1998, 273: 11177-82.
[8] 卢圣栋.现代分子生物学实验技术[M].北京:中国协和医科大学出版社,1990.400-3.
[9] Lu CD, Altieri DC, Tanigana N. Nobuniko Tanigana. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas[J]. Cancer Res,1998, 58(9): 1808-12.
[10] 张智清,姚立红,侯云德.含pRpL启动子的原核高效表达载体的组建及其应用[J].病毒学报[Vins Acta(China)],1990,6:111-5.
[11] 熊绍虎,余磊,谭海燕,等.重组人骨形态发生蛋白-2在大肠杆菌中的表达及纯化[J].第一军医大学学报,2002,22(5):413-6.Xiong SH, Yu L, Tan HY, et al. Expression and purification of recombinent bone morphogenetic protein-2 in E.coli[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22(5): 413-6.
[12] 周最民,郭亚兵,王战会,等.人微球蛋白基因在大肠杆菌中的稳定、高效表达[J].第一军医大学学报,2002,22(11):986-91.Zhou ZM, Guo YB, Wang ZH, et al. Efficient and constant expression of cloned human β2-microglobulin gene in E.coli[J]. J First Mil Med Univ/Di Yi Jun Yi Da Xue Xue Bao, 2002, 22 (11):986-91.

相似文献/References:

[1]刘宇虎,钟东,柳娟,等.干扰素诱导的跨膜蛋白-1基因的扩增、克隆及蛋白表达[J].南方医科大学学报,2005,(10):1221.
 LIU Yu-hu,ZHONG Dong,LIU Juan,et al.PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 gene[J].Journal of Southern Medical University,2005,(08):1221.
[2]李荣,郑航,罗荣城.HER2基因的克隆及其在MCF-7细胞中的表达[J].南方医科大学学报,2005,(10):1264.
 LI Rong,ZHENG Hang,LUO Rong-cheng.Cloning and expression of the HER2 gene in MCF-7 cells[J].Journal of Southern Medical University,2005,(08):1264.
[3]张革,王烈峰,汪华侨,等.人Aβ42重组抗原的融合表达及Aβ抗体的检测[J].南方医科大学学报,2005,(02):160.
 ZHANG Ge,WANG Lie-feng,WANG Hua-qiao,et al.Fusion expression of human Aβ42 recombinant protein and detection of Aβ antibody[J].Journal of Southern Medical University,2005,(08):160.
[4]宋远斌,何思杰,余楠,等.柯萨奇病毒A16型VP1~VP4基因克隆及其表达产物的抗原相关性分析[J].南方医科大学学报,2012,(12):1713.
[5]洪权,吴镝,陈香美,等.人尿酸转运蛋白基因的克隆与序列分析[J].南方医科大学学报,2005,(06):623.
 HONG Quan,WU Di,CHEN Xiang-mei,et al.Cloning and sequence analysis of human uric acid transporter gene[J].Journal of Southern Medical University,2005,(08):623.

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