Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

IF 3.1 4区化学 Q2 CHEMISTRY, MULTIDISCIPLINARY Chemical Research in Chinese Universities Pub Date : 2008-11-01 DOI:10.1016/S1005-9040(09)60024-6

Hai-hong ZHANG, Xi-zhen ZHANG, Dong-hai ZHAO, He-liang SHI, Yong-hui YU, Yong-ge WU, Xiang-hui YU, Wei KONG

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引用次数: 2

Abstract

Survivin, a novel member of inhibitor of apoptosis(IAP) protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass(M_r) of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni²⁺ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.

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人Survivin的构建、表达及其多克隆抗体的制备

Survivin是凋亡抑制剂(IAP)蛋白家族的新成员，在癌症中异常表达，但在正常分化的成人组织中检测不到。survivin的癌症特异性表达，加上其在抑制细胞死亡和调节细胞分裂中的重要性，使其成为癌症的有用诊断标志物和癌症治疗的潜在靶点。RT-PCR从293细胞总RNA中扩增Survivin cDNA，将其克隆到原核表达载体pRSET-B中。重组质粒pRSET-B-Surv在大肠杆菌BL21中表达，表达的融合蛋白相对分子质量(Mr)约为21000。重组蛋白经Ni2+亲和层析柱纯化，SDS-PAGE和Western blot鉴定。将纯化后的重组蛋白注射家兔，获得高效价的抗survivin多克隆抗体。

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来源期刊

Chemical Research in Chinese Universities 化学-化学综合

CiteScore

5.30

自引率

6.50%

发文量

152

审稿时长

3.0 months

期刊介绍： The journal publishes research articles, letters/communications and reviews written by faculty members, researchers and postgraduates in universities, colleges and research institutes all over China and overseas. It reports the latest and most creative results of important fundamental research in all aspects of chemistry and of developments with significant consequences across subdisciplines. Main research areas include (but are not limited to): Organic chemistry (synthesis, characterization, and application); Inorganic chemistry (bio-inorganic chemistry, inorganic material chemistry); Analytical chemistry (especially chemometrics and the application of instrumental analysis and spectroscopy); Physical chemistry (mechanisms, catalysis, thermodynamics and dynamics); Polymer chemistry and polymer physics (mechanisms, material, catalysis, thermodynamics and dynamics); Quantum chemistry (quantum mechanical theory, quantum partition function, quantum statistical mechanics); Biochemistry; Biochemical engineering; Medicinal chemistry; Nanoscience (nanochemistry, nanomaterials).