| 刘曦,张帅,刘志纯,等.α-突触核蛋白的异源表达及其与Pectinolide G的相互作用研究[J].中国海洋药物,2020,39(1):31-37. |
| α-突触核蛋白的异源表达及其与Pectinolide G的相互作用研究 |
| Heterologous expression of α-synuclein and its interaction with Pectinolide G |
| 投稿时间：2019-03-19 修订日期：2019-04-15 |
| DOI： |
| 中文关键词: α-synuclein，异源表达，帕金森疾病，表面等离子共振，分子模拟 |
| English Keywords:α-synuclein protein heterologous expression Parkinson’s disease SPR molecular simulation |
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| 中文摘要: |
| 目的 体外获得人源α-突触核蛋白并研究其与海洋小分子Pectinolide G的实际结合情况 方法 采用基因重组的方法构建α-突触核蛋白的原核重组表达载体并在BL21(DE3)中诱导蛋白表达,从诱导温度、时间和诱导剂浓度三方面对蛋白表达进行优化,并确立硫酸铵纯化目的蛋白的具体条件,通过表面等离子共振技术和分子对接技术,探究人源α-synuclein与Pectinolide G的结合情况。结果 通过基因重组将α-synuclein目的基因与PET30a原核表达载体构建成融合载体,确立该蛋白的最佳诱导表达条件为：0.5mM IPTG,37 ℃, 5 h,最佳纯化条件为: 95℃加热破碎后菌液,50%饱和硫酸铵溶液沉淀目的蛋白,质谱法确认其分子量为14.6KD,符合人源α-synuclein理论分子量。最后,利用纯化得到的α-synuclein通过表面等离子共振(SPR)和计算机分子模拟技术,确认了其可与Pectinolide G较好的结合。结论 通过原核重组表达获到人源α-synuclein,并确认了其与海洋小分子Pectinolide G的结合力和结合模式 |
| English Summary: |
| Objective Obtain human α-synuclein in vitro and study its actual binding to marine small molecule Pectinolide G. Method The prokaryotic recombinant expression vector of α-synuclein was constructed by genetic recombination and transformed into BL21(DE3). Protein expression was optimized from three aspects of induction temperature, induction time and inducer concentration. The specific conditions for purifying the target protein by ammonium sulfate was established. To explore the combination of human α-synuclein and Pectinolide G, surface plasmon resonance technology and molecular docking technology were used. Results The recombinant plasmid of α-synuclein and PET30a was constructed into a fusion vector by gene recombination. The optimal expression conditions of the protein were 0.5 mM IPTG, 37 °C, 5 h. The optimal purification conditions were: after 95 °C heating the crushed bacterial solution, the target protein was precipitated by 50% saturated ammonium sulfate solution. Besides, the molecular weight was confirmed to be 14.6 KD by mass spectrometry, which was in accordance with the theoretical molecular weight of human α-synuclein. Finally, the purified α-synuclein was confirmed to have better binding to Pectinolide G by surface plasmon resonance (SPR) and computer molecular simulation techniques. Conclusion Human α-synuclein was obtained , and its binding ability and binding mode to Pectinolide G were confirmed. |
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