| 一种新型凋亡素融合蛋白的克隆表达及活性测定 |
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| 引用本文： | 赵健,肖伟,范立强,王富军,吕稼锋,袁勤生,刘建文.一种新型凋亡素融合蛋白的克隆表达及活性测定[J].食品与药品,2009(7). |
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| 作者姓名： | 赵健 肖伟 范立强 王富军 吕稼锋 袁勤生 刘建文 |
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| 作者单位： | 华东理工大学生物反应器工程国家重点实验室;浙江日升昌药业有限公司; |
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| 摘 要： | 目的克隆表达EC-SOD3-凋亡素融合蛋白,并检测其生物活性。方法PCR扩增出apoptin序列,与表达载体EC-SOD3-pET-28a连接后在大肠杆菌BL21(DE3)中经IPTG诱导,表达产物进行Ni2+-NTA纯化和MTT活性检测。结果表达载体pET-28a-EC-SOD3-apoptin经酶切鉴定和序列分析,证明质粒构建正确,转化E.coliBL21(DE3)后,重组蛋白获得表达,Ni2+-NTA纯化后的凋亡素融合蛋白纯度达到85%以上,经噻唑蓝(MTT)法测定具有活性。结论成功构建了表达载体pET-28a-EC-SOD3-apoptin,并在大肠杆菌中表达了EC-SOD3-凋亡素融合蛋白,纯化的蛋白质具有诱导HeLa细胞凋亡的能力。
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| 关 键 词： | EC-SOD3-凋亡蛋白 克隆表达 纯化 噻唑蓝法 |
Cloning and Expression of New Apoptin Fusion Protein and Its Bioactivity |
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| Affiliation: | 1.State Key Laboratory of Bioreactor Engineering;East China University of Science and Technology;Shanghai 200237;China;2.Zhejiang Reachall Pharmaceutical Co.;Ltd.;Zhejiang Dongyang 322100;China |
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| Abstract: | Objective To clone and express apoptin-EC-SOD3 fusion protein and determine its bioactivity in vitro.Methods Apoptin gene was amplified by PCR and cloned into prokaryotic expression vector EC-SOD3-pET-28a.The constructed recombinant plasmid pET-28a-EC-SOD3-apoptin was transformed to E.coli BL21(DE3) for the expression under the induction of IPTG.The expressed protein was purified by Ni2+-NTA affinity chromatography and its activity was identified by MTT.Results PCR analysis proved that the recombinant plasm... |
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| Keywords: | apoptin-EC-SOD3 fusion protein cloning and expression purification MTT |
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