文章摘要
李 烜,梁文飚,孙倍成,吴 华.人Survivin蛋白的表达?纯化及初步应用[J].南京医科大学学报,2008,28(10):1267~
人Survivin蛋白的表达?纯化及初步应用
The expression,purification and preliminary use of human Survivin protein
投稿时间:2008-05-22  
DOI:10.7655
中文关键词: Survivin  基因克隆  蛋白纯化  HCC
英文关键词: Survivin  cloning  protein purification  HCC
基金项目:
作者单位
李 烜 南京中医药大学第一附属医院普外科,江苏 南京 210029 
梁文飚 江苏省血液中心,江苏 南京 210042 
孙倍成 南京医科大学第一附属医院普外科,江苏 南京 210029 
吴 华 南京中医药大学中医临床系,江苏 南京 210046 
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中文摘要:
      目的:克隆?表达肿瘤特异性凋亡抑制因子(Survivin),并对其在人肝癌诊断中的意义进行研究?方法:从人白血病细胞系HL60提取总RNA,用RT-PCR 方法扩增出Survivin基因片段?将Survivin基因片段插入载体pMD-18T中,测序正确后亚克隆到原核表达载体pGEX-2TK中?IPTG诱导重组质粒pGEX-2TK-Survivin在大肠杆菌BL21中表达N端融合GST的目的蛋白,采用GST蛋白纯化系统进行纯化,经SDS-PAGE分析后,将所得产物用Thrombin裂解后进行Western blot鉴定?将获得的目的蛋白包板,用ELISA法初步检测了肝癌患者血清中抗Survivin蛋白的表达?结果:克隆了人Survivin基因?表达出相对分子质量约为16.2 ku的蛋白并进行了肝癌患者血清的检测应用?结论:成功克隆?表达和纯化了Survivin基因和融合蛋白,为进一步探讨以Survivin为基础的肿瘤诊断和治疗奠定了实验基础?
英文摘要:
      Objective:To clone the cDNA of human Survivin,study its expression in E. Coli. and demonstrat its significance in the diagnosis of hepatoma. Methods:Using the isolated total RNA from human leukemic cell line HL60 as a template,the human Survivin encoding cDNA was amplified by reverse transcription-polymerase chain reaction(RT-PCR) methods. The PCR product was cloned into prokaryotic expression vector pMD-18T and sequenced. The expression of recombinant plasmid pGEX-2TK-Survivin was induced in E.coli BL21 by IPTG, the expressed product was purified by GST protein purification system,then was cut GST from the expressed product useing Thrombin. After the identification of the Survivin protein by SDS-PAGE and Western blot,they were coated in ELISA plate to detect the expression in serum of hepatoma patient. Results:Human Survivin,an anti-apoptosis gene,express Surviving protein with a relative molecular mass of 16.2 ku was cloned and used preliminary in hepatoma patients. Conclusion:Human Survivin gene was cloned successfully;Survivin protein was successfully expressed,purified and preliminary used. It may provide preliminary labratory evidence based on Survivin for the diagnosis and therapy of carcinoma.
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