中文摘要：

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℃,1mmol/L的IPTG诱导4h后,获得分子量约为18kDa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93ml/L和78mg/L。Tat-aFGF14-154及其对照蛋白aFGF14-154对Balb/c3T3细胞有明显促细胞增殖的作用,最佳作用浓度分别为1280ng/ml和160ng/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1000ng/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%。

英文摘要：

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 1 280ng/ml and 160ng/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.