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Issue:ISSN 1000-7083
          CN 51-1193/Q
Director:Sichuan Association for Science and Technology
Sponsored by:Sichuan Society of Zoologists; Chengdu Giant Panda Breeding Research Foundation; Sichuan Association of Wildlife Conservation; Sichuan University
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Your Position :Home->Past Journals Catalog->2019 Vol.38 No.4

Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Copper Zinc Superoxide Dismutase from Micordera punctipennis
Author of the article:XIKERANMU Zilajiguli, MA Ji, TUSONG Kuerban, LIU Xiaoning*
Author's Workplace:Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
Key Words:Micordera punctipennis; extracellular copper-zinc superoxide dismutase; prokaryotic expression; polyclonal antibody
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
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