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目的 研究影响人α 突触核蛋白 (α synuclein)基因在原核细胞中表达的常见因素 ,以提高其在原核细胞中的表达量。 方法 将重组质粒ProEXNACP转化进入到DH5α 大肠杆菌体内 ,观察诱导剂IPTG浓度、诱导时间、诱导温度、诱导培养基、诱导初始菌浓度及诱导中pH值变化等因素对蛋白表达产量的影响 ,用SDS PAGE梯度胶电泳分析 ,考马斯亮蓝染色进行比较研究 ,同时检测pH值 ,观察其变化。 结果 诱导剂IPTG浓度对诱导蛋白产生的影响不大 ,诱导过程中pH值变化较小 ,而诱导时间、温度、初始菌浓度和培养基的种类对诱导产生α 突触核蛋白有较大影响。其中 ,以LB培养基 ,37℃ ,180r min诱导 1～ 2h产生α 突触核蛋白的量最大。 结论 诱导剂IPTG诱导ProEXNACP/DH5α大肠杆菌产生α 突触核蛋白的量与诱导时间、温度、初始菌浓度和培养基的种类有明显的关系。初始菌A590 =0 5～ 0 8,LB培养基 ,37℃ ,大通氧量 (180r min以上 ) ,1～ 2h诱导可作为大量提取、纯化α 突触核蛋白的首选条件。
Objective To study the common factors influencing the expression of human α-synuclein gene in prokaryotic cells and to increase the expression of α-synuclein in prokaryotic cells. Methods The recombinant plasmid ProEXNACP was transformed into E. coli DH5α to observe the effect of IPTG concentration, induction time, induction temperature, induction medium, initial concentration of induced bacteria and pH during induction on the protein expression and yield. PAGE gradient gel electrophoresis analysis, Coomassie brilliant blue staining comparative study, at the same time the pH value was measured to observe the changes. Results The concentration of IPTG inducing agent had little effect on the induced protein production, but the pH value had little change during the induction process. The induction time, temperature, initial bacterial concentration and the medium type had a great influence on the induction of α-synuclein. Among them, the amount of α-synuclein produced by LB medium at 37 ° C for 180r min for 1 ~ 2h was the highest. Conclusion The induction of α-synuclein by ProTNACP / DH5α Escherichia coli induced by IPTG inducing agent has obvious relationship with induction time, temperature, initial bacterial concentration and the type of medium. The initial conditions of A590 = 0 5 ~ 0 8, LB medium, 37 ℃, oxygen uptake (more than 180r min), 1 ~ 2h induction can be used as a large number of extraction and purification of α-synuclein preferred conditions.