小胸鳖甲胞外铜锌超氧化物歧化酶重组蛋白的原核表达及多克隆抗体制备

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的主要抗氧化酶家族。基于原核表达系统,成功表达了拟步甲科Tenebrionidae小胸鳖甲Micordera punctipennis胞外铜锌SOD的重组蛋白(本文定义为Trx-His-MpecCu/Zn-SOD)。经Ni 2+亲和层析法纯化重组蛋白后,研究了重组蛋白的部分酶学性质。通过足垫加皮下注射法3次免疫小鼠后,分别用ELISA和Western blot的方法检测抗体效价和抗体特异性。结果表明,重组蛋白主要以包涵体形式存在,纯化后的重组蛋白浓度为1.33 mg·mL^-1,酶活力为27.52 U·mg^-1。Trx-His-MpecCu/Zn-SOD在25~45℃具有比较稳定的酶活性,在35℃最高,同时表现出比较广泛的酸碱耐受性(pH3~12),最适pH为9.0,表明重组蛋白的酶活性相对比较稳定。蛋白免疫法制备的鼠抗MpecCu/Zn-SOD多克隆抗体滴度高于1∶819 200。Western blot结果显示,该抗体能免疫结合重组蛋白Trx-His-MpecCu/Zn-SOD和小胸鳖甲体内天然MpecCu/Zn-SOD,但不能与黄粉虫Tenebrio molitor的总蛋白结合,说明制备的抗体效价较高且特异性较好。本研究结果为小胸鳖甲ecCu/Zn-SOD功能的深入研究奠定了基础。

Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Copper Zinc Superoxide Dismutase from Micordera punctipennis

Superoxide dismutases(SODs)are one of the major families of antioxidant enzymes that scavenge superoxide anion free radicals in living organisms.The fusion protein of an extracellular copper-zinc superoxide dismutase from Micordera punctipennis(defined as Trx-His-MpecCu/Zn-SOD),an insect in Tenebrionidae,was successfully expressed in the prokaryotic expression system.After purification of the target fusion protein by Ni-NTA purification system,the enzymatic properties of Trx-His-MpecCu/Zn-SOD were examined.Antibody titer and antibody specificity were determined by ELISA and Western blot after immunizing mice for 3 times using footpad and subcutaneous injection.The results showed that the fusion protein mainly existed in form of inclusion bodies.The concentration of purified recombinant protein was 1.33 mg·mL-1 with an enzyme activity of 27.52 U·mg-1.Trx-His-MpecCu/Zn-SOD had relatively stable enzyme activity in the temperature range of 25-45℃,and the highest enzyme activity was at 35℃.At the same time,it exhibited a wide range of acid-base tolerance(pH3-12)and the optimum pH was 9.0.This result indicated that the enzyme activity of Trx-His-MpecCu/Zn-SOD was relatively stable.The titer of mouse anti-MpecCu/Zn-SOD polyclonal antibody prepared by protein immunoassay was higher than 1∶819 200.Western blot analysis showed that the antibody could immunologically bind to Trx-His-MpecCu/Zn-SOD and natural ecCu/Zn-SOD protein of M.punctipennis,but not the total protein of Tenebrio molitor.The results revealed that the antibody had higher antibody titer and better immune-specificity.This study laid foundation for the in-depth study of the function of MpecCu/Zn-SOD.