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目的:构建用于表达具有Tat序列的新型神经营养因子MANF(mesencephalic astrocytederived 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TatMANF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。
OBJECTIVE: To construct a recombinant plasmid for expressing Tat-MANF (Tat-MANF) fusion protein with Tat sequence. The recombinant protein was expressed by prokaryotic expression system and its biological activity was tested. Methods: The TAT-MANF fusion gene was constructed by using MANF cDNA as template and TAT sequence and His-tag and appropriate restriction enzyme sites. After inserted into the expression vector Pet22b +, it was transformed into E. coli BL21 for expression and purification, and the expressed recombinant protein was identified by SDS-PAGE and Western blotting. To verify the biological activity of Tat-MANF, neurogenic glioblastoma cells (SH-SY5Y) were induced to toxicity with 30 μmol / L 6-hydroxydopamine (6-OHDA) while adding 2 μg / ml of TAT-MANF And control MANF protein, 24h after flow cytometry cell apoptosis rate. Brain blood vessel barrier was simulated with B-endo3 in vitro and co-incubated with FITC-labeled TatMANF for 4h, and observed under a fluorescence microscope. Results: The TAT-MANF fusion gene was successfully constructed. The expression product was consistent with the size of the target protein and could bind with MANF antibody. Recombinant protein could reduce the apoptosis of SH-SY5Y cells induced by 6-OHDA. After incubated with Tat-MANF-FITC and B-end3 cells for 4h, the cells showed obvious fluorescence. Conclusion: The obtained recombinant protein Tat-MANF has neuronal protection and transmembrane function, which lays a material foundation for the further study of Parkinson's disease in vivo.