基因重组大肠杆菌表达血红素加氧酶-1及培养条件优化

Expression of heme oxygenase-1 in gene recombinant Escherichia coli and optimization of its cultivation

摘要:

【背景】血红素加氧酶-1 (HO-1)具有抗氧化应激、抗凋亡和抗纤维化等多种生理效应，有望成为一种新型药物应用于临床疾病的治疗。【目的】构建表达HO-1的基因重组大肠杆菌(Escherichia coli)，并优化其表达培养条件，实现HO-1高产率的表达。【方法】PCR法克隆集胞藻(Synechocystis sp.) PCC 6803的HO-1基因(ho1)，构建重组质粒pET-28a-ho1，转化大肠杆菌BL21(DE3)菌株，单因素实验优化表达培养基的种类、诱导剂添加时间、诱导培养时间、诱导剂浓度和诱导培养温度。【结果】构建了表达HO-1的基因重组大肠杆菌BL21(DE3)/pET-28a-ho1菌株，用甘油(GY)培养基培养至菌体浓度OD600约为0.8时，加入终浓度为0.1 mmol/L的IPTG诱导，30 °C诱导培养6 h，HO-1的表达量最高，Ni-NTA柱分离纯化得到的HO-1收率占细胞总蛋白的10.9%。【结论】获得了可溶性表达HO-1的基因重组大肠杆菌及其较佳的培养条件，为进一步研究集胞藻来源的HO-1的酶学性质和应用奠定了基础。

Abstract:

[Background] Heme oxygenase-1 (HO-1) has many physiological effects, such as anti-oxidative stress, anti-apoptosis and anti-fibrosis, and is expected to become a new drug for the treatment of clinical diseases. [Objective] A gene recombinant Escherichia coli was constructed for expressing HO-1, and its culture conditions were optimized to achieve the high yield of HO-1. [Methods] The gene of HO-1 (ho1) was cloned from the cell of Synechocystis sp. PCC 6803, and was recombined with the plasmid of pET-28a. The recombinant plasmid (pET-28a-ho1) was transformed into E. coli BL21(DE3). The single factor experiment was applied to optimize the type of medium, inducer adding time, cultivation time, inducer concentration and cultivation temperature for the expression of HO-1. [Results] A gene recombinant E. coli strain, BL21(DE3)/pET-28a-ho1, was successfully constructed for expressing HO-1. When the strain was cultured in glycerol medium (GY), and as the cell OD600 was about 0.8, IPTG with the final concentration of 0.1 mmol/L was added, the highest yield of HO-1 could be obtained after induction cultivation for 6 h at 30 °C. Separation of HO-1 by Ni-NTA column accounted for 10.9% yield of the total cell protein. [Conclusion] A gene recombinant E. coli strain, and its optimal cultivation conditions were achieved for the expression of soluble HO-1, which laid a foundation for further study on the enzymatic properties and application of HO-1 from Synechocystis sp.