Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Copper Zinc Superoxide Dismutase from Micordera punctipennis
Abstract：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.
2019,38(4): 387-393 收稿日期：2019-01-09
分类号：Q78
基金项目：国家自然科学基金项目（31360527）
作者简介：孜拉吉古丽·西克然木(1987-),博士研究生,研究方向:昆虫低温分子生物学,E-mail:864971439@qq.com
*通信作者:刘小宁,E-mail:liuxn0103@sina.com
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