| 溶藻弧菌HY9901转运蛋白TolB的原核表达及条件优化和纯化 |
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| 引用本文： | 周泽军,庞欢瑛,丁燏,简纪常,吴灶和.溶藻弧菌HY9901转运蛋白TolB的原核表达及条件优化和纯化[J].中国农学通报,2013,29(11):55-59. |
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| 作者姓名： | 周泽军 庞欢瑛 丁燏 简纪常 吴灶和 |
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| 作者单位： | 1. 广东海洋大学水产学院,广东湛江524088;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;广东省教育厅水产经济动物病害控制重点实验室,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;广东省教育厅水产经济动物病害控制重点实验室,广东湛江524088;仲恺农业工程学院,广州510225 |
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| 基金项目： | 国家重点基础研究发展计划"海水养殖鱼类病原弧菌分子致病机制的研究"，国家自然科学基金项目"溶藻弧菌毒力蛋白的鉴定及功能研究"，广东省教育厅育苗基金"溶藻弧菌Ⅲ型分泌系统关键蛋白Ysc0的功能研究" |
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| 摘 要： | 为研究溶藻弧菌(Vibrio alginolyticus) HY9901转运蛋白TolB作为疫苗候选抗原的可能性，根据已发表的溶藻弧菌HY9901转运蛋白TolB序列(JQ846501)，设计1对带酶切位点的引物，PCR扩增tolB基因，然后经酶切、连接等步骤，构建tolB基因的原核表达载体pET-TolB，并对其IPTG浓度、诱导时间、温度、洗脱浓度进行优化，以期获得较大量的目的蛋白。结果表明：IPTG诱导后经SDS-PAGE与Western-blot分析，成功表达了溶藻弧菌HY9901株TolB蛋白，分子量与预期相符，且表达的蛋白以包涵体的形式存在；TolB蛋白在大肠杆菌中诱导表达的优化条件为：0.2 mmol/L IPTG，37℃诱导4 h；最佳咪唑洗脱浓度为400 mmol/L。这些结果为进一步研究目的蛋白的免疫原性和疫苗制备奠定基础。
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| 关 键 词： | 花色苷 花色苷 |
| 收稿时间： | 2012/10/7 0:00:00 |
| 修稿时间： | 2012/10/22 0:00:00 |
Purification and Optimization of Prokaryotic Expression of Translocation Protein TolB Gene from Vibrio alginolyticus Strain HY9901 |
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| Zhou Zejun
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Pang Huanying
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Ding Yu
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Jian Jichang
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Wu Zaohe.Purification and Optimization of Prokaryotic Expression of Translocation Protein TolB Gene from Vibrio alginolyticus Strain HY9901[J].Chinese Agricultural Science Bulletin,2013,29(11):55-59. |
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| Authors: | Zhou Zejun Pang Huanying Ding Yu Jian Jichang Wu Zaohe |
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| Abstract: | To investigate the possibility of TolB from Vibrio alginolyticus HY9901 as a candidate antigen for vaccine production, the tolB gene was amplified by using a pair of primer according to the published tolB gene sequence (JQ846501), and then followed by digestion, connection; the prokaryotic expression vector pET-TolB of tolB gene was constructed. Then the IPTG concentration, induction time, temperature, elution concentration of the pET-TolB was optimized to obtain a relatively large amount of the target protein. The result showed that, the TolB protein was successfully expressed after being inducted with IPTG and SDS-PAGE, Western-blot analysis. The molecular weight of TolB protein was in line with expectation, and the expressed protein existed in the form of inclusion body. The optimization condition of inducible expression for TolB protein in Escherichia coli was optimized at 37℃ for 4 hours, with 0.2 mmol/L IPTG. The best elution concentration of imidazole was 400 mmol/L. These results laid a foundation for further studies on the protein immunogenecity and vaccine preparation. |
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| Keywords: | purification |
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