文章摘要
唐鹿群,张 蕾,郭人花,刘 丰,苏 川,束永前.人端粒酶逆转录酶与热休克蛋白70融合表达载体的构建及原核表达[J].南京医科大学学报,2007,(8):825~828
人端粒酶逆转录酶与热休克蛋白70融合表达载体的构建及原核表达
Construction and identification of recombinant vector encoding hTERT and HSP70 genes
投稿时间:2006-12-09  
DOI:10.7655
中文关键词: 热休克蛋白70  人端粒酶逆转录酶  融合基因  原核表达
英文关键词: heat shock protein 70  human telomerase reverse transcriptase  fusion gene  prokaryotic expression
基金项目:
作者单位
唐鹿群 南京医科大学第一附属医院肿瘤科,江苏 南京 210029 
张 蕾 南京医科大学江苏省现代病原生物学重点实验室,江苏 南京 210029 
郭人花 南京医科大学第一附属医院肿瘤科,江苏 南京 210029 
刘 丰 南京医科大学江苏省现代病原生物学重点实验室,江苏 南京 210029 
苏 川 南京医科大学江苏省现代病原生物学重点实验室,江苏 南京 210029 
束永前 南京医科大学第一附属医院肿瘤科,江苏 南京 210029 
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中文摘要:
      目的:构建热休克蛋白70(HSP70)与人端粒酶逆转录酶融合表达载体,在大肠杆菌进行表达?方法:利用PCR方法扩增hTERT基因片段(532aa~637aa),双酶切后插入原核表达载体pET32a的NcoⅠ和EcoRⅠ位点之间,形成pET32a-hTERT质粒?通过PCR方法扩增人HSP70全长cDNA,双酶切后插入pET32a-hTERT的EcoRⅠ和HindⅢ位点之间,构建hTERT与HSP70的N端融合基因原核表达质粒pET32a-hTERT-HSP70?含有该质粒的大肠杆菌BL21经异丙醛-B-D-硫代半乳糖苷(IPTG)诱导表达?结果:成功的扩增了HSP70基因与hTERT基因片段;测序结果表明成功的构建了pET32a-hTERT-HSP70原核表达载体?含有该质粒的大肠杆菌BL21经诱导后表达约110 kD的蛋白?结论:HSP70与人端粒酶逆转录酶融合表达质粒构建成功,并成功获得了融合蛋白hTERT-HSP70?
英文摘要:
       Objective:To construct the fusion gene of human hTERT and human heat shock protein 70(HSP70), and expressed it in E.coli. Methods:The hTERT and HSP70 genes were amplified by PCR. The hTERT gene was digested by NcoⅠand EcoRⅠ enzyme and inserted into the plasmid pET32a to construct pET32a-hTERT plasmid. Then the HSP70 gene was cloned into the expression vector pET32a-hTERT between EcoRⅠ and HindⅢ site to construct the prokaryotic expression plasmid pET32a-hTERT-HSP70. The E.coli BL21 contained the plasmid was induced by IPTG. Results:The HSP70 gene and hTERT gene segments were amplified from their templates; the DNA sequencing result showed that the prokaryotic fusion expression plasmid pET32a-hTERT-HSP70 was successfully constructed. The E.coli BL21 contained the plasmid could express a Mr 110 kD protein after induced by IPTG. Conclusion:The successful expression of fusion protein of human hTERT and heat shock protein 70 enables the further research of the HSP70 protein as a adjuvant-free carrier.
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