Tat-MANF融合蛋白的制备及生物活性研究

目的：构建用于表达具有Tat序列的新型神经营养因子MANF（mesencephalic astrocyte-derived neurotrophic factor）融合蛋白（Tat-MANF）的重组质粒；利用原核细胞表达系统表达该重组蛋白，并检测其生物学活性。方法：以MANF cDNA为模板，利用PCR技术在上下游分别添加TAT序列和His标签及合适的限制性酶切位点，构建TAT-MANF融合基因。插入表达载体Pet22b⁺后，转化大肠杆菌BL21进行表达和纯化，用SDS-PAGE及Western印迹鉴定表达的重组蛋白。为了验证Tat-MANF的生物学活性，用30μmol/L浓度的6-羟多巴胺（6-OHDA）对神经母胶质瘤细胞（SH-SY5Y）进行毒性诱导，同时加入2μg/ml的TAT-MANF及对照MANF蛋白，24h后用流式细胞仪检测细胞的凋亡率。用脑微血管内皮细胞（B-endo3）体外模拟血脑屏障，与FITC标记的Tat-MANF共孵育4h，荧光显微镜下观察。结果：成功构建TAT-MANF融合基因，表达产物与目的蛋白大小相符，能与MANF抗体发生结合反应。重组蛋白可减少由6-OHDA导致的SH-SY5Y细胞凋亡，Tat-MANF-FITC与B-end3细胞共孵育4h后，可见细胞内明显荧光。结论：获得的重组蛋白Tat-MANF具有神经细胞保护作用及跨膜功能，为进一步开展帕金森症的体内治疗研究奠定了物质基础。

Expression of Tat-MANF Fusion Protein and Its Bioactivity Analysis

Objective: To express Tat-MANF fusion protein and evaluate its biological activities. Methods: Tat-MANF fusion gene is constructed by PCR method to add the TAT sequence in the N-terminus and a His tag in the C-terminus of the MANF cDNA respectively. The Tat-MANF cDNA is inserted into the pET22b⁺ expression plasmid and prokaryotic expression system (BL21) is used to express the fusion protein. After purification, Tat-MANF protein is detected by SDS-PAGE and Western blot analyses. In order to evaluate biological activities of Tat-MANF, 6-Hydroxydopamine (6-OHDA) was used to induce apoptosis in human dopaminergic neuroblastoma cells. Tat-MANF is added to the cell-culture medium and the neuroprotection effect is detected by flow cytometry after 24h. B-endo3 cells were used as an in vitro blood-brain barrier model, in which B-endo3 cells are incubated with the FITC-labeled Tat-MANF and examined under microscopy after 4 hours. Results: Tat-MANF-His recombinant protein is successfully expressed in prokaryotic expression system. Western blot revealed that Tat-MANF-His recombinant protein can be detected by both anti-MANF and anti-His antibodies. Flow cytometry demonstrates that Tat-MANF significantly inhibit 6-OHDA-induced neuronal cell apoptosis similar to MANF. After 4 hours co-incubation with Tat-MANF-FITC, intracellular fluorescent signal is observed in the B-endo3 cells, indicating that Tat-MANF is able to cross cell membrane of brain vascular endothelial cells, a key component of BBB(blood-brain barrier). Conclusion: The recombinant Tat-MANF fusion protein is neuroprotective and may be able to cross blood-brain barrier, which may provide a novel therapy to Parkinson's Disease.