| 溶藻弧菌HY9901血红素结合蛋白HutB的原核表达及纯化 |
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| 引用本文： | 薛丽丽,;庞欢瑛,;黄郁葱,;吴灶和,;简纪常,;周泽军.溶藻弧菌HY9901血红素结合蛋白HutB的原核表达及纯化[J].广东海洋大学学报,2014(3):47-51. |
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| 作者姓名： | 薛丽丽 ;庞欢瑛 ;黄郁葱 ;吴灶和 ;简纪常 ;周泽军 |
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| 作者单位： | [1]广东海洋大学水产学院,广东湛江524088; [2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088; [3]广东省水产经济动物病害控制重点实验室,广东湛江524088; [4]仲恺农业工程学院,广东广州510225 |
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| 基金项目： | 国家科技支撑计划（2012BAD17802、2012BAD17803）;广东省自然科学基金（S2013040014562） |
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| 摘 要： | 为探究溶藻弧菌(Vibrio alginolyticus)HY9901血红素结合蛋白HutB作为疫苗候选抗原的可能性,采用PCR方法扩增V.alginolyticus HY9901 hutB基因全长序列,克隆到pMD18-T载体,经双酶切、连接后,定向插入到pET-28a(+)中,构建原核表达载体pET-HutB,转入大肠杆菌BL21(DE3)中进行IPTG诱导表达。SDS-PAGE验证后,优化诱导表达条件和纯化条件,并进行Western-blot分析。结果表明,HutB蛋白在E.coli中诱导表达的最优条件:0.4 mmol/L IPTG,37℃诱导4h,表达的蛋白分子量与预期大小相符,主要以包涵体的形式存在,300 mmol/L咪唑洗脱时效果最佳,能与鼠抗His-tag单抗特异性结合。
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| 关 键 词： | 溶藻弧 hutB基因 原核表达 优化 纯化 |
Prokaryotic Expression and Purification of the Recombinant Hemin-Binding Protein（HutB） of Vibrio alginolyticus Strain HY9901 |
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| Institution: | XUE Li-li , PANG Huan-ying , HUANG Yu-cong , WU Zao-he, JIAN Ji-chang , ZHOU Ze-jun (1. Fisheries College of Guangdong Ocean University,Zhanjiang 524088, China; 2.Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemilogy for Aquatic Economic Animals, Zhanjiang 524088, China; 3.Key Laboratory of Disease Controlling for Aquatic Economic Animals of Guangdong Higher Education Institutions, Zhanjiang 524088, China; 4.Zhongkai University of Agriculture and Engineering , Guangzhou 510225, China) |
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| Abstract: | To investigate the prokaryotic expression and explore the possibilities as a vaccine candidate antigens of HutB from V.alginolyticus HY9901, the hutB gene was cloned by PCR, and then followed by digestion, connection, the prokaryotic expression vector pET-HutB was constructed through directional inserting into pET-28a（＋）, then transformed into Escherichia coli BL21（DE3） with IPTG induction. After verification of SDS-PAGE the inducted expression and purification conditions was optimized further. The results showed that： The optimization condition of inducible expression for HutB protein in E.coli was optimazed at 37℃ for 3 hours, with 0.4mmol/L IPTG. The best elution concentration of imidazole was 300mmol/L, and the expressed protein existed in the form of inclusion body. The recombinant HutB fusion protein could be combined with mouse anti-His-Tag antibody. |
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| Keywords: | Vibrio alginolyticus hutB gene prokaryotic expression optimization purification |
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