亚洲玉米螟酚氧化酶原的原核表达和性质分析

Prokaryotic expression and characterization of prophenoloxidase from the Asian corn borer,Ostrinia furnacalis( Lepidoptera: Pyralidae)

【目的】昆虫体内酚氧化酶原(PPO)是一种重要的天然免疫蛋白,参与昆虫的体液免疫和细胞免疫过程。本研究采用原核表达体系,大量表达可溶且具有活性的重组PPO蛋白,可用于各种酚氧化酶(PO)抑制剂的筛选,从而为创制抑制昆虫免疫系统的新型杀虫剂提供条件。【方法】利用从亚洲玉米螟Ostrinia furnacalis 5龄幼虫体内克隆获得PPO基因,构建了pET-28b-PPO原核表达载体,在大肠杆菌Escherichia coli中重组表达了亚洲玉米螟PPO蛋白;采用Ni-NAT亲和层析柱快速纯化目的蛋白,进行了Western杂交鉴定;测定分析了重组PPO蛋白激活为PO后的酶学性质以及不同金属离子(Mg2+,Cu2+和Fe2+)对PPO二级结构的影响。【结果】融合蛋白PPO得到了表达和纯化。重组PPO蛋白激活为PO后最适反应温度为30℃,最适pH为7.2,以L-DOPA为底物时PO催化反应的Vmax为140.8 U/mg·min,Km为2.96 mmol/L。Fe2+存在的情况下重组PPO蛋白中β-折叠结构成分显著增加至53.7%±4.6%,α-螺旋结构成分则显著下降至2.6%±1.2%(P<0.05);有Mg2+存在的情况下,重组PPO蛋白中β-折叠结构成分显著下降,α-螺旋结构成分稍有上升。有Cu2+存在的情况下,重组PPO蛋白中β-折叠结构成分显著下降为10.0%±1.6%,而α-螺旋结构成分则上升至35.3%±6.9%。【结论】结果说明不同金属离子对重组PPO蛋白的二级结构有显著影响。

【Aim】Prophenoloxidase（ PPO） is an important immune protein in insects,which is involved in insect humoral immunity and cellular immunity. In our study,the prokaryotic expression system was applied to express soluble recombinant PPO with activity on a large scale. The recombinant PPO was used to screen various phenoloxidase（ PO） inhibitors so as to create novel insecticides targeted on the insect immune system. 【Methods】Using the PPO gene cloned from the 5th instar larvae of the Asian corn borer,Ostrinia furnacalis（ Guenée）,the prokaryotic expression vector,pET-28b-PPO,was constructed,and the recombinant PPO protein was expressed in Escherichia coli. The fusion protein was purified from the supernatant of the lysis of E. coli cells with Ni-NAT affinity chromatography column,and identified with Western blotting. Enzymatic characteristics of phenoloxidase（ PO） activated from the recombinant PPO with cetylpyridinium chloride（ CPC） and the effects of metal ions,such as Mg2 ＋,Cu2 ＋and Fe2 ＋on the secondary structure of the recombinant PPO were also assayed and analyzed. 【Results】The recombinant PPO protein was expressed and purified. The suitable temperature for PO obtained throughthe activation of the recombinant PPO was 30℃,and the suitable pH was 7. 2. The kinetic parameters calculated for substrate oxidation were Vmax= 140. 8 U /mg·min and Km = 0. 92 mmol /L for L-DOPA.The content of β-sheet in the recombinant PPO increased dramatically to 53. 7% ± 4. 6% when Fe2 ＋ions existed. However,the content of α-helix decreased significantly to 2. 6% ± 1. 2%（ P 〈 0. 05）. The content of β-sheet in the recombinant PPO reduced clearly and the content of α-helix increased slowly when Mg2 ＋ions existed. The content of β-sheet in the recombinant PPO declined remarkably to 10. 0% ±1. 6% when Cu2 ＋ions were present,but the content of α-helix increased significantly to 35. 3% ±6. 9%. 【Conclusion】The results suggest that the secondary structure of the recombinant PPO is affected notably by different metal ions.