TAT-aFGF融合蛋白在大肠杆菌中的表达及其生物活性测定

中国生物工程杂志

2010, Vol. 30

Issue (05): 11-17

研究报告

TAT-aFGF融合蛋白在大肠杆菌中的表达及其生物活性测定

林健聪¹,张敏静^1,3,苏志坚^1,2, 陈红霞¹,邱壮伟¹,娄国锋¹,项琪^1,2,3,黄亚东^1,2,3**

1.暨南大学生命科学技术学院医药生物技术研究开发中心广州 510632
2.广东省生物工程药物重点实验室广州 510632
3.基因工程药物国家工程研究中心广州 510632

Expression, Purification and Bioassay of Tat-aFGF Fusion Protein in Escherichia coli

LIN Jian-cong ¹,ZHANG Min-jing^1,3 ,SU Zhi-jian^1,2,CHEN Hong-xia¹,QIU Zhuang-wei¹,Lou Guo-feng¹, Xiang Qi^1,2,3,HUANG Yadong^1,2,3

1.Life Science and Techonology College of Jinan University，Biopharmaceutical Research & Development Center, Guangzhou 510632,China
2.Guang Dong Provincial Key Laboratory of Bioengineering Medicine，Guangzhou 510632，China
3.National Engineering Research Center of Genetic Medicine，Guangzhou 510632，China

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摘要：

构建表达Tat与人酸性成纤维细胞生长因子（acidic fibroblast growth factor, aFGF）融合蛋白的重组质粒pET3c–Tat-aFGF14-154和pET3c-Tat-aFGF27-154，并转化大肠杆菌BL21（DE3）中。37℃，l mmol/L的IPTG诱导4 hrs后，获得分子量约为18 kDa的融合蛋白，表达量分别占菌体总蛋白的42%和24%。利用阳离子交换（CM-Sepharose FF）、肝素亲和层析（Heparin-Sepharose CL-6B）以及分子筛（Sephadex G-25）联用的方法可获得纯度大于95%的目的蛋白，得率分别为93和78 mg/L。Tat-aFGF14-154及其对照蛋白aFGF14-154对Balb/c 3T3细胞有明显促细胞增殖的作用，最佳作用浓度分别为1280 ng/ml和160 ng/ml。而Tat-aFGF27-154 及其对照蛋白aFGF27-154则几乎没有促细胞增殖活性。免疫荧光分析结果表明，Tat-aFGF14-154及Tat-aFGF27-154均能穿过PC12、Balbc/3T3、HaCaT和海马神经元细胞的细胞膜，并主要定位于细胞浆中。此外，四种重组蛋白均能降低Aβ25-35对大鼠海马神经元细胞的毒性，在剂量为1000 ng/ml时，Tat-aFGF14-154、Tat-aFGF27-154、aFGF14-154及aFGF27-154细胞存活率分别为90.66%、81.87%、85.71%和78.02%，而Aβ25-35模型组的存活率仅为56.87%。

关键词： 穿膜肽; 人酸性成纤维细胞生长因子; 融合蛋白; 神经元

Abstract:

Acidic fibroblast growth factor (aFGF) is a potent neurotrophic factor. It can stimulate the reparation and regeneration of central and peripheral nerves after various injuries. Recently, an approach to deliver therapeutic peptides to the brain is the application of fusion proteins linked to so-called trans-activator transcription (TAT) protein, which can carry the therapeutic protein to permeate blood-brain barrier(BBB) and cell membranes. In this study, aFGF was linked to TAT protein by genetic engineering, and the soluble TAT-aFGF has been expressed successfully in E.Coli. DNA coded fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were constructed and cloned into vector pET-3c and the fusion proteins were expressed in E.coli BL21 (DE3). The fusion proteins Tat-aFGF14-154 and Tat-aFGF27-154 were puritified using the combination of CM-Sepharose FF, Heparin affinity chromatography and Sephadex G-25 and the purity were higher than 95%. The fusion proteins were confirmed as Tat-aFGF14-154 and Tat-aFGF27-154 by Western bolt and MALDI-TOF. The mitogenic activity assayed by MTT on Balb/c 3T3 cell showed that Tat-aFGF14-154 and aFGF14-154 had mitogenic activity to Balb/c 3T3 and the best concentration were 1280 ng/ml and 160 ng/ml,respectively.On the other hand, Tat-aFGF27-154 and aFGF27-154 had little mitogenic activity. Tat-aFGF14-154 and Tat-aFGF27-154 could transduce the membrane of PC12, hippocampal neurons, Balb/c 3T3 and HaCaT cells and were mainly located mainly in the cytoplasm detected by immunofluorescence. Four recombinant proteins could efficiently protect hippocampal neurons from toxicity induced by Aβ25-35.

Key words: Tat human acidic fibroblast growth factor fusion protein neurons

收稿日期: 2010-02-22 出版日期: 2010-05-25

基金资助:

“重大新药创制”国家科技重大专项(2009ZX09103749)、科技部中小型创新基金(2008A10905004)、国家自然科学基金(30670541)、广东省教育部产学研结合项目（2009B090300285）、广东省博士启动项目（9451093201003248）资助项目

通讯作者: 黄亚东 E-mail: tydhuang@jnu.edu.cn

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引用本文:

林健聪张敏静苏志坚陈红霞邱壮伟娄国锋项琪黄亚东. TAT-aFGF融合蛋白在大肠杆菌中的表达及其生物活性测定[J]. 中国生物工程杂志, 2010, 30(05): 11-17.

LIN Jian-Cong, ZHANG Min-Jing, SU Zhi-Jian, CHEN Gong-Xia, QIU Zhuang-Wei, LOU Guo-Feng, XIANG Qi, HUANG E-Dong. Expression, Purification and Bioassay of Tat-aFGF Fusion Protein in Escherichia coli. China Biotechnology, 2010, 30(05): 11-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I05/11

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