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 and Edwardsiella Ictaluri Outer Membrane Porin Protein OmpN1

作　　者：张转丹 肖正泮 武新丽 孙艳 罗应 吴昊[1] 韦双双[1] 裴业春[1] 王大勇[1] ZHANG Zhuandan;XIAO Zhengpan;WU Xinli;SUN Yan;LUO Ying;WU Hao;WEI Shuangshuang;PEI Yechun;WANG Dayong(School of Life and Pharmaceutical Sciences,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命科学与药学院,海口570228

出　　处：《热带生物学报》2020年第2期145-155,共11页Journal of Tropical Biology

基　　金：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)。

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。笔者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;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,将两者进行序列优化与全基因合成,分别构建原核表达载体:MCP-ompN1 pET28a和MCP pET28a和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MCPompN1,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,Western Blotting结果表明,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。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 the fish nervous necrosis virus was fused with the transmucosal protein ompN1 of Edwardsiella ictaluri to prepare a mucosal vaccine against the nervous necrosis virus.The gene sequences of the coat protein MCP of the fish nervous necrosis virus and the outer membrane protein ompN1 of E.ictaluri were obtained from the NCBI GenBank,and then optimized and synthesized,and the synthesized genes were used to construct prokaryotic expression vectors respectively:MCP-ompN1 pET28a,MCP pET28a,ompN1 pET28a.The fusion proteins MCP-ompN1,MCP and ompN1 were induced and expressed in Escherichia coli,respectively,and they were purified through inclusion body purification and refolded through dialysis.SDSPAGE results showed that the purified MCP-ompN1 fusion protein was obtained by prokaryotic expression and purification.Further Western blotting showed that the purified MCP-ompN1 fusion protein had both MCP antigenicity and ompN1 antigenicity.It is indicated that the MCP-ompN1 protein into which the fish nervous necrosis virus capsid protein MCP was fused with E.ictaluri outer membrane protein ompN1 was obtained by prokaryotic expression and purification.Further study is needed to observe whether the fusion protein MCPompN1 can be used as a mucosal vaccine against nervous necrosis virus.

关 键 词：病毒性神经坏死病 鮰爱德华氏菌 融合蛋白 包涵体 

分 类 号：Q786[生物学—分子生物学] Q943