| TAT细胞穿透肽融合小鼠SurvivinT34A重组蛋白的原核表达纯化及其细胞转导效能研究 |
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| 引用本文： | 于海涛,陈科达,倪崖,阎辉.TAT细胞穿透肽融合小鼠SurvivinT34A重组蛋白的原核表达纯化及其细胞转导效能研究[J].细胞生物学杂志,2011(7). |
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| 作者姓名： | 于海涛 陈科达 倪崖 阎辉 |
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| 作者单位： | 温州医学院检验医学院;浙江省医学科学院; |
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| 基金项目： | 国家自然科学基金(No.30873024); 浙江省科技厅重大科技专项(No.2008C13031-1)资助项目~~ |
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| 摘 要： | 为研究细胞穿透肽TAT融合小鼠存活素T34A(survivinT34A)重组蛋白(TAT-msvT34A)转导细胞的效能,该实验将表达TAT-msvT34A的原核表达载体pTAT-msvT34A转化大肠杆菌表达株E.coli BL21(DE3),异丙基硫代半乳糖苷(IPTG)诱导,表达的融合蛋白主要以包涵体形式存在。通过亲和层析、离子交换柱层析、分子筛等步骤纯化,得到TAT-msvT34A融合蛋白的纯度可达98%。纯化的融合蛋白用异硫氰酸荧光素(FITC)标记(FITC-TAT-msvT34A)后,分别转导HepG2、TC-1、B16及HEK293细胞株,流式细胞仪检测显示,较低浓度的重组蛋白即对HepG2、TC-1、B16及HEK293等细胞株具有较高的转导效能,在100 nmol/L时的转导效率均可达到60%以上,作为对照,FITC标记的牛血清白蛋白(FITC-BSA)在100 nmol/L时对上述细胞株的转导效率极低,结果具有显著性差异(P<0.01)。荧光显微镜观察可见,50 nmol/L和100 nmol/L的TAT-msvT34A融合蛋白对HepG2细胞的转导效率均可达50%,而对照FITC-BS...
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| 关 键 词： | 抑存活素 TAT细胞穿透肽 原核表达 凋亡 肿瘤 |
Prokaryotic Expression and Purification of the Recombination Protein Containing Cell-penetrating TAT Peptide and Mouse SurvivinT34A Mutant:Investigation of Its Transduction Efficiency in Cells |
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| Yu Haitao,Chen Keda,Ni Ya.Prokaryotic Expression and Purification of the Recombination Protein Containing Cell-penetrating TAT Peptide and Mouse SurvivinT34A Mutant:Investigation of Its Transduction Efficiency in Cells[J].Chinese Journal of Cell Biology,2011(7). |
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| Authors: | Yu Haitao Chen Keda Ni Ya |
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| Institution: | Yu Haitao~(1,2),Chen Keda~2,Ni Ya~(1,Yan Hui~(2*) (1 School of Medical Lab Science,Wenzhou Medical College,Wenzhou 325035,China,2 Zhejiang Academy of Medical Sciences,Hangzhou 310013,China) |
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| Abstract: | The expression and purification of the recombination protein TAT-msvT34A consisting of mice survivinT34A mutant fused with cell-penetrating TAT peptide and its transduction efficiency in several tumor and non-tumor cell lines were investigated.In this study,the prokaryotic expression vector pTAT-msvT34A was transformed into expression strain of E.coli BL21-DE3 and subsequently the transformed bacteria were induced by IPTG for expression of the recombinant protein.It was shown that the recombinant fusion pro... |
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| Keywords: | survivin TAT cell-penetrating peptide prokaryotic expression apoptosis tumor |
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