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一种新型凋亡素融合蛋白的克隆表达及活性测定 被引量：3

Cloning and Expression of New Apoptin Fusion Protein and Its Bioactivity
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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。 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 Ni^2＋ -NTA affinity chromatography and its activity was identified by MTT. Results PCR analysis proved that the recombinant plasmid pET-28a-EC- SOD3-apoptin was correctly constructed. The expressed soluble fusion protein was consistent with that expected. Its purity was over 85 % after being purified with Ni^2＋ -NTA agarose and the purified protein showed bioactivity by MTF assay. Conclusion The recombinant plasmid for prokaryotic expression of pET-28a-EC-SOD3-apoptin can be constructed and the apoptin-EC-SOD3 fusion protein can be expressed successfully in E. coli BL21 （ DE3 ）. MTT result indicates that HeLa cells are highly sensitive to the purified apoptin-EC-SOD3.
作者 赵健 肖伟 范立强 王富军 吕稼锋 袁勤生 刘建文
机构地区 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
出处 《食品与药品》 CAS 2009年第4期7-11,共5页 Food and Drug
关键词 EC—SOD3-凋亡蛋白 克隆表达 纯化 噻唑蓝法 apoptin-EC-SOD3 fusion protein cloning and expression purification MTT
分类号 Q591.2 [生物学—生物化学]
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参考文献12

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同被引文献29

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食品与药品
2009年 第4期

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