文章摘要
程文芳,郝 波,张国新,施瑞华.幽门螺杆菌Tipα重组蛋白的表达?纯化及鉴定[J].南京医科大学学报,2011,(2):199~202
幽门螺杆菌Tipα重组蛋白的表达?纯化及鉴定
Construction,expression and purification of recombinant H.polyri Tip-α protein
投稿时间:2010-08-24  
DOI:10.7655
中文关键词: Tipα蛋白  基因表达  蛋白纯化
英文关键词: Tip-α protein  gene expression  purification
基金项目:江苏省高校自然科学研究项目(08KJ3320003)
作者单位
程文芳 南京医科大学第一附属医院消化科, 江苏 南京 210029 
郝 波 南京医科大学第一附属医院消化科, 江苏 南京 210029 
张国新 南京医科大学第一附属医院消化科, 江苏 南京 210029 
施瑞华 南京医科大学第一附属医院消化科, 江苏 南京 210029 
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中文摘要:
      目的: 克隆幽门螺杆菌(H.polyri,HP)的Tipα基因,构建含有Tipα的重组载体,在大肠杆菌表达并纯化以获得Tipα蛋白?方法:从HP的26695菌株扩增位于HP0596的Tipα基因,将Tipα基因与pET28a载体(His标签)同时经BamHI和XhoI酶切?纯化及连接后,构建含有Tipα的重组载体pET28a-Tipα,重组载体转化至大肠杆菌并表达,表达产物以Ni-NTA 亲和层析纯化,Western blot进一步鉴定?结果:克隆Tipα基因,经测序证实与Genbank对比,二者同源性为100%?大肠杆菌表达产物,纯化后SDS-PAGE显示相对分子量为23 ku?Western blot证实纯化产物与抗Tipα抗体特异性结合?结论:成功克隆并表达出HP的Tipα蛋白,为进一步研究Tipα的功能奠定基础?
英文摘要:
      Objective:To construct a recombinant vector containing Tip-α of H.polyri, and transfect it in E.coli to purify recombinant protein. Methods:By PCR technique, Tip-α cDNA was amplified, and then cloned into the pET28a vector. The recombinant plasmid was transformed into E.coli. The correct clone was identified by sequence analysis. The expression product was purified by nick-nitrilotriacetic acid(Ni-NTA) metal-affinity chromatography and analyzed by SDS-PAGE and Western blot methods. Results: Enzyme digestion analysis and sequencing assay showed that Tip-α gene had been successfully inserted into the vector. SDS-PAGE showed a 23 ku protein identified by Western blot analysis. Conclusion:A recombinant plasmid containing Tip-α has been constructed and expressed successfully.
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