首页 > 过刊浏览>2015年第39卷第10期 >1558-1568. DOI:10.11964/jfc.20150109690
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溶藻弧菌双组分调控系统KdpDE减毒活疫苗的构建及其免疫效果评价
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广东海洋大学 水产学院,广东海洋大学 水产学院,广东海洋大学 水产学院,广东海洋大学 水产学院,广东省水产经济动物病原生物学及流行病学重点实验室

基金项目:

广东省海洋经济创新发展区域示范专项(GD2012-B01-004);国家科技支撑计划(2012BAD17B02、2012BAD17B03);广东高校国际合作创新平台(2013gjhz0008)


Construction of an attenuated two-component regulatory system KdpDE strain of Vibrio alginolyticus and evaluation of its immunogenic and protective effects as a vaccine
Author:
  • ZHANG Yanfei

    ZHANG Yanfei

    Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China;Zhaoqing Aquatic Technologies Popularizing Center, Zhaoqing 526060, China
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  • PANG Huanying

    PANG Huanying

    Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China
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  • JIAN Jichang

    JIAN Jichang

    Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China
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  • LU Yishan

    LU Yishan

    Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China
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  • TANG Jufen

    TANG Jufen

    Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China
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  • WU Zaohe

    WU Zaohe

    Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Zhanjiang 524088, China;Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang 524088, China
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Affiliation:

Fisheries College,Guangdong Ocean University,Fisheries College,Guangdong Ocean University,Fisheries College,Guangdong Ocean University,Fisheries College,Guangdong Ocean University,Guangdong Provincial key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals

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    摘要:

    为了研究溶藻弧菌双组分调控系统KdpDE减毒活疫苗对鱼体的免疫保护作用,本研究采用Overlap PCR和同源重组技术,构建了kdpD/kdpE(kdpDE)基因无标记基因框内敲除突变株。对野生株和缺失突变株的生物学特性及致病性进行了比较研究,发现kdpDE的缺失对溶藻弧菌的生长速率和胞外蛋白酶活性的影响不明显,但是突变株的泳动能力、生物被膜形成能力较野生株下降。斑马鱼致病性实验结果显示,突变株毒力下降78.5倍,表明减毒株构建成功,可以作为减毒活疫苗的候选菌株。突变株在鱼体内存活能力实验结果显示,注射免疫7 d后,鱼体内未检测到该菌。以突变株ΔkdpDE为抗原,分别用注射和浸泡的方式免疫斑马鱼,免疫后第28天用溶藻弧菌和哈维氏弧菌攻毒,评估该疫苗候选株及不同免疫方式对斑马鱼的保护效果。实验结果表明,免疫后的斑马鱼能够抵抗溶藻弧菌的感染,其中注射免疫组的免疫保护率达83.3%,浸泡免疫组次之(66.7%);同时,该减毒活疫苗能刺激斑马鱼对哈维氏弧菌产生交叉保护效应,其中注射免疫组的免疫保护率为65.5%,浸泡免疫组为55.2%。以上结果表明,kdpDE基因敲除突变株可能是抵抗溶藻弧菌感染的有效候选减毒活疫苗。

    Abstract:

    To study the immune protection of fish vaccinated by the live attenuated Vibrio alginolyticus vaccine,we constructed an in-frame deletion mutant of kdpD/kdpE (kdpDE) genes using overlap PCR and homologous recombination technology,to investigate the effects of this mutant on the biological characteristics.We then compared the differences of the biological characteristics and virulence between the gene deletion mutant and wild strain.Furthermore,the immunoprotection of the vaccine was analyzed by challenge test.The mutant ΔkdpDE showed no differences of ECPase activity and growth from the wild-type strain.But the decreases in swarming ability and biofilm formation were found.Virulence of the mutant declined by 78.5 times in zebrafish model.No bacteria survived in zebrafish 7 days later after injection vaccination.Furthermore,the mutant was used as an antigen to immunize zebrafish.To estimate immunoprotection and immune routes of the live attenuated vaccine,the zebrafish were challenged by V.alginolyticus and V.harveyi at 28 days after immersed and injected vaccination.The relative percentage survival(RPS) value of injection group was 83.3%,and the RPS value of immersion group was 66.7%.The results showed that the ΔkdpDE of V.alginolyticus has a high immunogenicity.In addition,the vaccination led to cross-protection from V.harveyi and the RPS values of injection group and immersion group were 65.5% and 55.2% respectively.These results indicate that the kdpDE mutant is an effective vaccine candidate against V.alginolyticus infection.

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张燕飞,庞欢瑛,简纪常,鲁义善,汤菊芬,吴灶和.溶藻弧菌双组分调控系统KdpDE减毒活疫苗的构建及其免疫效果评价[J].水产学报,2015,39(10):1558~1568

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历史
  • 收稿日期:2015-01-26
  • 最后修改日期:2015-07-10
  • 录用日期:2015-10-28
  • 在线发布日期: 2015-10-28
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