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文章摘要
引用本文:
鱼类神经坏死病毒衣壳蛋白MCP和鮰爱德华氏菌外膜蛋白ompN1融合基因的原核表达
Prokaryotic Expression of Fusion Gene of Fish Nervous Necrosis Virus Capsid Protein MCP with Edwardsiella Ictaluri Outer Membrane Porin Protein ompN1
投稿时间:2020-02-28  修订日期:2020-04-03
DOI:
中文关键词: 病毒性神经坏死病  鮰爱德华氏菌  融合蛋白  包涵体
英文关键词: viral nervous necrosis, Edwardsiella ictaluri, fusion protein, inclusion bodies.
基金项目:国家自然科学基金资助项目(31760246);国家自然科学基金资助项目(31860726)
作者单位E-mail
张转丹 海南大学生命科学与药学院 728971926@qq.com 
肖正泮 海南大学生命科学与药学院  
武新丽 海南大学生命科学与药学院  
孙艳 海南大学生命科学与药学院  
罗应 海南大学生命科学与药学院  
吴昊 海南大学生命科学与药学院  
韦双双 海南大学生命科学与药学院  
裴业春 海南大学生命科学与药学院  
王大勇 海南大学生命科学与药学院 wangdy@hainanu.edu.cn 
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中文摘要:
      目前在针对鱼类神经坏死病毒的疫苗研究中,主要是将神经坏死病毒某些蛋白作为抗原进行注射免疫,但是传统的注射免疫并不能有效地激发黏膜免疫。本实验将鱼类神经坏死病毒的衣壳蛋白(MCP)与鮰爱德华氏菌的跨粘膜蛋白ompN1融合表达,拟制备能够抵抗神经坏死病毒的粘膜疫苗。笔者利用从 NCBI GenBank 库里获得的鱼类神经坏死病毒的外壳蛋白MCP和鮰爱德华氏菌的外膜蛋白ompN1的基因序列,将两者进行了序列优化与全基因合成,分别构建原核表达载体:MCP-ompN1 pET28a、MCP pET28a、ompN1 pET28a,并在大肠杆菌内分别诱导表达融合蛋白MCP-ompN1、MCP、ompN1后,再利用包涵体纯化及透析复性获得MCP-ompN1、MCP、ompN1蛋白。SDS-PAGE结果显示原核表达纯化得到了较纯的MCP-ompN1 融合蛋白,进一步的Western Blotting结果表明了纯化得到的MCP-ompN1 融合蛋白不仅具有MCP抗原性,还具有ompN1抗原性。本实验通过原核表达纯化得到了鱼类神经坏死病毒衣壳蛋白MCP和鮰爱德华氏菌外膜蛋白ompN1的融合蛋白MCP-ompN1,为进一步验证融合蛋白MCP-ompN1能否作为抵抗神经坏死病毒的粘膜疫苗奠定了基础。
英文摘要:
      At present, certain proteins of the nervous necrosis virus are injected into fish as vaccines,but traditional injection immunization cannot effectively stimulate mucosal immunity.In this experiment, the capsid protein (MCP) of fish nervous necrosis virus was fused with the transmucosal protein ompN1 of Edwardsiella ictaluri, to prepare a mucosal vaccine resisting the nervous necrosis virus. The gene sequences of the coat protein MCP of fish neuronecrosis virus and the outer membrane protein ompN1 of Edwardsiella ictaluri were obtained from the NCBI GenBank, and then the sequences were optimized and synthetized.Prokaryotic expression vectors were constructed respectively: MCP-ompN1 pET28a, MCP pET28a, ompN1 pET28a. Then the fusion proteins MCP-ompN1, MCP and ompN1 were respectively induced and expressed in Escherichia coli, and the proteins were purified by inclusion body purification and dialysis renaturation.As a result,SDS-PAGE results showed that the purified MCP-ompN1 fusion protein was obtained by prokaryotic expression and purification. Further Western Blotting results showed that the purified MCP-ompN1 fusion protein had both MCP antigenicity and ompN1 antigenicity.It is indicated that the MCP-ompN1 protein fused with the fish nervous necrosis virus capsid protein MCP and Edwardsiella ictaluri outer membrane protein ompN1 was obtained by prokaryotic expression and purification. The results can provide a base for futher study whether the fusion protein MCP-ompN1 can be used as a mucosal vaccine against nervous necrosis virus.
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