哈维氏弧菌铁超氧化物歧化酶基因表达及免疫作用研究
EXPRESSION AND IMMUNOPROTECTIVE ANALYSIS OF IRON-COFACTORED SUPEROXIDE DISMUTASE FROM VIBRIO HARVEYI
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摘要: 哈维氏弧菌(Vibrio harveyi)是鱼虾等海水动物的重要病原菌。超氧化物歧化酶(Superoxide dismutase)通过催化超氧阴离子自由基(O2-)形成O2 和H2O2, 以保持细胞自由基产生和清除之间的平衡, 在病原菌适应环境及细菌致病性方面发挥重要作用。用PCR 从哈维氏弧菌基因组扩增得到600 bp 的目的片段, 序列分析表明与弧菌Fe-SOD 的序列相似性为91%—99%。将目的基因片段克隆到原核表达载体进行表达。SDS-PAGE电泳分析显示纯化的蛋白为单一条带, 分子量为27 kD。具有典型的Fe-SOD 吸收光谱, 对氯仿-乙醇和H2O2敏感, 表明纯化的蛋白属于Fe-SOD。用邻苯三酚自氧化法测得酶的最适pH 7, 最适温度20℃。该酶在pH6—8 的范围内稳定, 当温度超过40℃时酶的活力迅速丧失。纯化的重组蛋白免疫大菱鲆, 4 周后用致病性哈维氏弧菌进行人工感染试验, 对大菱鲆的免疫保护率为80.00%。Western blot 可以检测到免疫大菱鲆的血清中的特异抗体。Abstract: Vibrio harveyi is one of the important bacterial pathogens of marine animals. Superoxide dismutases, which abate and clear the effect of O2- via catalyse dismutation of superoxide into hydrogen peroxide and oxygen, belong to the anti-oxidation defense system and play a central role in protection against oxidative stress. An iron-cofactored superoxide dismutase gene was cloned by PCR amplification from the chromosomal DNA of V. harveyi SF1. The ORF of the sod gene consists of 600 bp, Sequence analysis showed that homologies of the amino acid sequence with those of Fe-SOD ranged from 91% to 99%. The sod gene was subcloned into pET26b (+) for experssion. SDS-PAGE confirmed that the purified protein was 27 kD. The purified SOD belonged to Fe-SOD based on the analysis of sensitivity to hydrogen peroxid, chlorofrom-ethanol and ultraviolet-visible spectroscopy. Pyorgallol autoxidation method was used to determine SOD activity of the purified protein. The purified SOD had the maximal activity at pH 7 and was stable over a range of pH 6—8. The optimal temperature for enzyme activity was 20 . The protein ℃ n was stable under 40℃. Turbots (Scophthalmus maximus) were immunized with 50 ug of the purified SOD. The immunized fish were then challenged with 0.1 mL of V. harveyi (3.9×108 CFU/mL) four weeks later. The relative percentage survivals (RPS) of the immunized group were 80.00%. Specific antibody could be detected in the sera of the immunized fish with Western blot analysis.
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Keywords:
- Vibrio harveyi /
- Superoxide dismutase /
- Expression /
- Purification /
- Characterization /
- Immunogenicity
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