生物技术通报 ›› 2016, Vol. 32 ›› Issue (8): 139-144.doi: 10.13560/j.cnki.biotech.bull.1985.2016.08.021

• 研究报告 • 上一篇    下一篇

大口黑鲈溃疡综合征病毒MCP基因的原核表达及重组蛋白的免疫效果初步分析

马冬梅1, 2, 邓国成1, 白俊杰1, 2, 江小燕1, 曹婷婷1, 蔡磊1   

  1. 1. 中国水产科学研究院珠江水产研究所 农业部热带亚热带鱼类选育与养殖重点开放实验室,广州 510380;
    2. 淡水水产健康养殖湖北省协同创新中心,武汉 430070
  • 修回日期:2015-11-04 出版日期:2016-08-25 发布日期:2016-08-25
  • 作者简介:马冬梅,女,博士,副研究员,研究方向:鱼类遗传育种;E-mail:madongmei2003@163.com
  • 基金资助:
    国家自然科学基金项目(31001107),“948”计划重点项目(2011-G12),国家科技支撑计划(2012BAD26B03),广东省省级科技计划项目(2015A020209035)

Prokaryotic Expression of MCP Gene from Largemouth Bass Ulcerative Syndrome Virus and the Immune Effect Analysis of Recombinant Protein

MA Dong-mei1, 2, DENG Guo-cheng1, BAI Jun-jie1, 2, JIANG Xiao-yan1, CAO Ting-ting1, CAI Lei1   

  1. 1. Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation(Ministry of Agriculture),Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380;
    2. Freshwater Aquaculture Collaborative Innovation Center of Hubei Province,Wuhan 430070
  • Revised:2015-11-04 Published:2016-08-25 Online:2016-08-25

摘要: 为了预防近年来流行于广东省养殖大口黑鲈(Micropterus salmoides)中的病毒性溃疡综合征,以大口黑鲈溃疡综合征病毒(Largemouth bass ulcerative syndrome virus,以下简称 LBUSV)的主要衣壳蛋白(MCP)作为目标抗原蛋白,将MCP基因开放阅读框插入到pBV220载体中,转化大肠杆菌DH5α,构建重组表达MCP蛋白的工程菌。重组菌经过42℃温度诱导和SDS-PAGE电泳,检测到在分子量51 kD处有一条特异表达蛋白带,重组蛋白约占重组菌体总蛋白的30%。重组MCP蛋白经洗涤、纯化、溶解、复性和透析后纯度达90%以上。将纯化后的重组蛋白与不完全弗氏佐剂混合、乳化后,免疫大口黑鲈,然后进行感染实验,感染后20 d疫苗保护率最高达67.7%。结果表明,该病毒MCP蛋白具有一定的免疫原性,可以作为制作重组疫苗的候选蛋白。

关键词: 大口黑鲈, 溃疡综合征病毒, 主要衣壳蛋白, 原核表达, 免疫效果

Abstract: For preventing the outbreak of viral ulcerative syndrome in largemouth bass(Micropterus salmoides)in Guangdong province,the ORF of major capsid protein(MCP)gene,as target antigen protein gene,from largemouth bass ulcerative syndrome virus(LBUSV)was inserted in vector pBV220,then transformed into Escherichia coli DH5α,and the recombinant engineering bacterium for expressing MCP was constructed. Following 42℃ temperature inducement and SDS-PAGE analysis,the recombinant bacterium was detected to produce a special expression protein with molecular weight about 51 kD,and the proportion of recombinant MCP protein was nearly 30% of total bacterial protein. After washed,purified,dissolved,renatured and dialyzed,the purity of the recombinant protein reached over 90%. The purified protein was mixed and emulsified with incomplete Freund's adjuvant,then was injected in largemouth bass as vaccine. The immunized fish were challenged with LBUSV,and the highest relative percentage of survival reached 67.7% after 20 days. The results indicate that the MCP of LBUSV has the immunogenicity and can be selected as candidate protein for recombinant vaccine.

Key words: largemouth bass(Micropterus salmoides), ulcerative syndrome virus, major capsid protein, prokaryotic expression, immune effect