| EGFP-Arg7融合蛋白表达及在HeLa细胞中的转导活性 |
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| 引用本文： | 肖伟,华迪,汪雅雯,单含文,吕稼锋,赵健.EGFP-Arg7融合蛋白表达及在HeLa细胞中的转导活性[J].食品与药品,2010,12(5):158-161. |
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| 作者姓名： | 肖伟 华迪 汪雅雯 单含文 吕稼锋 赵健 |
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| 作者单位： | 肖伟,华迪,汪雅雯,赵健(生物反应器工程国家重点实验室,华东理工大学,上海,200237);单含文,吕稼锋(浙江日升昌药业有限公司,浙江东阳,322100) |
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| 摘 要： | 目的 构建pET-28a-EGFP-Arg7重组子,观察表达的融合蛋EGFP-Arg7在细胞内的转导活性.方法 构建EGFP-Arg7(阴性对照为EGFP)序列,与pET-28a连接后,转化Ecoli BL21(DE3),IPTG诱导表达,并经Ni2+.NTA纯化.纯化产物作用于HeLa细胞后用荧光显微镜观察其转导活性.结果 重组pET-28a-EGFP-Arg7经酶切鉴定和序列分析,证明构建正确.转化E.coil BL21(DE3)后,重组蛋白获得可溶性表达.纯化后的融合蛋白纯度达90%以上.重组蛋白EGFP-Arg7作用于HeLa细胞后用荧光显微镜可观察到强绿色荧光.结论 Arg7具有很好的转导活性,能携带与其连接的蛋白质穿过HeLa细胞膜.
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| 关 键 词： | 多聚精氨酸 蛋白转导域 HeLa细胞 |
Expression of EGFP-Arg7 Fusion Protein and Its Transduction Activity into HeLa Cells |
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| Affiliation: | XIAO Wei1,HUA Di1,WANG Ya-wen1,SHAN Han-wen 2,Lü Jia-feng2,ZHAO Jian1(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 construct an expression vector of pET-28a-EGFP-Arg7 and to investigate its transduction activity into cells.Methods EGFP-Arg7 gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-EGFP-Arg7 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 transduction activity into the HeLa cells was detected under the fluorescence... |
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| Keywords: | polyarginine protein transduction domain(PTD) HeLa cell |
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