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| 牙鲆(Paralichthys olivaceus)β诺达病毒衣壳蛋白重组表达及复性条件优化
组表达及复性条件优化 |
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蒋嫣冉,张亦陈,刘逸尘,耿绪云,孙金生
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天津市动植物抗性重点实验室/天津师范大学生命科学学院,天津市动植物抗性重点实验室/天津师范大学生命科学学院,天津市动植物抗性重点实验室/天津师范大学生命科学学院,天津市水生动物疫病预防控制中心,天津市动植物抗性重点实验室/天津师范大学生命科学学院；
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| 摘要: |
| β诺达病毒利用其缺乏校正功能的聚合酶进行基因组复制, 易导致突变, 因而具有广泛的宿主感染性, 而且可引发致死率极高的病毒性神经坏死症, 需要有针对性地研究检测及防御方法。本研究以感染牙鲆(Paralichthys olivaceus)的β诺达病毒为对象, 利用引物设计, 将His标签编码序列连接到完整病毒衣壳蛋白C端, 构建原核表达载体; 采用SDS-PAGE及质谱对表达产物进行分离和鉴定; 重组衣壳蛋白经镍离子亲和柱纯化后进行复性条件的正交优化。结果表明4个肽段经质谱鉴
定与预期一致; 纯化产物产量可达36mg/L; 4°C条件下, 复性缓冲液中尿素和PBS的最适浓度为0.8mol/L和0.05mol/L。本研究建立的制备方案可为研制相关疫苗以及开发针对该病毒的快速检测产品等提供有价值的参考。 |
| 关键词: 牙鲆 β诺达病毒 重组表达 复性优化 |
| DOI：10.11693/hyhz20140200045 |
| 分类号:P735 |
| 基金项目:国家科技支撑计划项目, 2011BAD13B04号, 2011BAD13B07 号。国家863项目, 2012AA092205号, 2012AA10A401号; 国家973 项目, 2012CB114405号 |
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| EXPRESSION AND REFOLDING OPTIMIZATION OF RECOMBINANT CAPSID PROTEIN OF BETANODAVIRUSISOLATED FROM PARALICHTHYS OLIVACEUS |
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JIANG Yan-Ran,ZHANG Yi-Chen,LIU Yi-Chen,GEN Xu-Yun and SUN Jin-Sheng
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Tianjin Key Laboratory of Animal and Plant Resistance/College of Life Science, Tianjin Normal University,Tianjin Key Laboratory of Animal and Plant Resistance/College of Life Science, Tianjin Normal University,Tianjin Key Laboratory of Animal and Plant Resistance/College of Life Science, Tianjin Normal University,Tianjin Center for Control and Prevention of Aquatic Animal Infectious Disease,Tianjin Key Laboratory of Animal and Plant Resistance/College of Life Science, Tianjin Normal University; Tianjin Center for Control and Prevention of Aquatic Animal Infectious Disease
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| Abstract: |
| Betanodavirusreplicated their genome with RNA dependent RNA polymerase (RdRp). However, the proofreading deficiency of RdRp may lead to mutation that could infect many economic fish species and cause viral nervous necrosis. In this study, an expression vector was constructed for the capsid protein (CP) of a betanodavirus infecting Paralichthys olivaceus; a His Tag for purification of recombined CP was introduced to the C termini by primer designing; product was separated and verified by SDS-PAGE and mass spectrum analysis, and the refolding conditions were optimized in orthogonal experiments. Results show that the 4 peptide segments of recombined CP reoccurred in mass spectrum. The production of purified product per liter could reach 36mg, the optimum concentrations of urea and PBS (phosphate buffer solution) in refolding buffer for recombined CP were 0.8mol/L and 0.05mol/L, respectively. The outcome of this study would help develop effective products for the detection and defense against betanodavirus. |
| Key words: Paralichthys olivaceus Betanodavirus recombinant expression optimization of refolding |