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海豚链球菌simApgmA真核表达质粒对尼罗罗非鱼免疫保护的研究

马丽 吴金英 高凇泽 孙彩云 李文笙

马丽, 吴金英, 高凇泽, 孙彩云, 李文笙. 海豚链球菌simA和pgmA真核表达质粒对尼罗罗非鱼免疫保护的研究[J]. 南方水产科学, 2020, 16(3): 38-46. doi: 10.12131/20190163
引用本文: 马丽, 吴金英, 高凇泽, 孙彩云, 李文笙. 海豚链球菌simApgmA真核表达质粒对尼罗罗非鱼免疫保护的研究[J]. 南方水产科学, 2020, 16(3): 38-46. doi: 10.12131/20190163
Li MA, Jinying WU, Songze GAO, Caiyun SUN, Wensheng LI. Study on immunoprotection of eukaryotic expression plasmids of Streptococcus iniae simA and pgmA in Oreochromis niloticus[J]. South China Fisheries Science, 2020, 16(3): 38-46. doi: 10.12131/20190163
Citation: Li MA, Jinying WU, Songze GAO, Caiyun SUN, Wensheng LI. Study on immunoprotection of eukaryotic expression plasmids of Streptococcus iniae simA and pgmA in Oreochromis niloticus[J]. South China Fisheries Science, 2020, 16(3): 38-46. doi: 10.12131/20190163

海豚链球菌simApgmA真核表达质粒对尼罗罗非鱼免疫保护的研究

doi: 10.12131/20190163
基金项目: 现代农业产业技术体系专项资金“国家特色鱼淡水鱼产业技术体系”(CARS-46);广州市科技计划重点项目 (201904020043);现代农业人才支撑计划项目 (2016—2020)
详细信息
    作者简介:

    马丽:马 丽(1994—),女,硕士研究生,研究方向为鱼类疫苗。E-mail: mali48@mail2.sysu.edu.cn

    通讯作者:

    吴金英(1963—),女,博士,副教授,从事鱼类生理学和免疫学研究。E-mail: lsswjy@mail.sysu.edu.cn

  • 中图分类号: Q 819

Study on immunoprotection of eukaryotic expression plasmids of Streptococcus iniae simA and pgmA in Oreochromis niloticus

  • 摘要:

    尼罗罗非鱼 (Oreochromis niloticus) 是世界水产养殖业中的重要经济鱼类,但在养殖生产中易受海豚链球菌 (Streptococcus iniae) 感染而致病致死,使用疫苗是一种相对理想的防感染措施。该研究采用海豚链球菌simApgmA基因构建的真核表达载体作为DNA疫苗,肌肉注射罗非鱼评估疫苗保护效果。免疫后在DNA和RNA水平上,在注射鱼体内检测到2个目的基因。首次免疫后第7至第28天,鳃、肝、肾脏、头肾中疫苗组的白介素1 (Interleukin,IL-1β) 与肿瘤坏死因子 (Tumor Necrosis Factor,TNF-α) 表达量高于PBS对照组;疫苗组的抗体滴度、血清抗菌活性显著 (P<0.05) 高于PBS对照组。攻毒后,注射pcDNA3.1-pgmA、pcDNA3.1-simA、pcDNA3.1-pgmA与pcDNA3.1-simA等比例混合疫苗的相对保护率 (Relative percent survival,RPS) 分别为60.7%、49.9%和75.0%。结果表明所制备的疫苗具有免疫保护效果,可作为候选疫苗。

  • 图  1  海豚链球菌sn03株SiM蛋白进化树

    Figure  1.  Phylogenetic tree of SiM protein in S. iniae sn03

    图  2  海豚链球菌sn03株PGM蛋白进化树

    Figure  2.  Phylogenetic tree of PGM protein in S. iniae sn03

    图  3  首次免疫后28 d目的基因在DNA水平上的组织分布

    a-1、a-2、a-3分别为肝、脾、头肾simA分布;M. Marker 3;1. pcDNA3.1-simA组;2. 混合疫苗组;3、4分别为PBS组、pcDNA3.1 (+)组;5. 阳性对照;6. 阴性对照b-1、b-2、b-3分别为肝、脾、头肾pgmA分布;M. DS2000;1. 阳性对照;2. 阴性对照;3、4分别为pcDNA3.1−pgmA组及混合疫苗组;5. PBS组;6. pcDNA3.1 (+)组

    Figure  3.  Tissue distribution of target gene at DNA level at 28 d after first immunization

    a-1, a-2, a-3. Expression maps of liver, spleen and head kidney, respectively; M. Marker3; 1. pcDNA3.1-simA group; 2 Mixture vaccine group; 3–4. PBS group and pcDNA3.1(+) group, respectively; 5. Positive control; 6. Negative control b-1, b-2, b-3. Expression maps of liver, spleen and head kidney, respectively; M. DS2000; 1. Positive control; 2. Negative control; 3–4. pcDNA3.1-pgmA and mixture vaccine group respectively; 5. PBS group; 6. pcDNA3.1 (+) group

    图  4  首次免疫后28 d目的基因在RNA水平上的组织分布

    a-1、a-2、a-3分别为肝、脾、头肾simA分布;M. Marker 3;1. 阴性对照;2. 阳性对照;3. PBS组;4. pcDNA3.1 (+)组;5、6分别为混合疫苗组、pcDNA3.1 (+)-simA组 b-1、b-2、b-3分别为肝、脾、头肾pgmA分布;M. Marker 3;1. pcDNA3.1-pgmA组;2. 混合疫苗组;3. PBS组;4. pcDNA3.1 (+)组;5、6分别为阳性对照、阴性对照组

    Figure  4.  Tissue distribution of target gene at RNA level at 28 d after first immunization

    a-1, a-2, a-3. Expression maps of liver, spleen and head kidney, respectively; M. Marker3; 1. Positive control; 2. Negative control; 3. Mixture vaccine group; 4. pcDNA3.1-simA group; 5–6. PBS group and pcDNA3.1 (+) group, respectively;b-1, b-2, b-3. Expression maps of liver, spleen and head kidney, respectively; M. Marker 3; 1. pcDNA3.1-pgmA group; 2. Mixture vaccine group; 3. PBS group; 4. pcDNA3.1 (+) group; 5–6. Positive control and negative control, respectively

    图  5  血清抗体滴度和抗菌活性

    n=3; *. 差异显著 (P<0.05,独立样本t检验);后图同此

    Figure  5.  Antibody titer and antibacterial activity in serum

    n=3; *. Significant difference (P<0.05, Inndependent t-test); the same case in the following figures

    图  6  首次免疫28 d后鳃 (a)、肝 (b)、肾脏 (c) 和头肾 (d) 中免疫相关基因相对表达水平

    **. 差异极显著 (P<0.01)

    Figure  6.  Relative expression of immune genes in parotid (a), liver (b), kidney (c) and head kidney (d) at 28 d after first immunization

    **. Very significant difference (P<0.01)

    图  7  罗非鱼免疫攻毒后存活状况 (n=30)

    Figure  7.  Survival status of tilapia after injection with S. iniae

    表  1  免疫相关基因定量引物

    Table  1.   Immune genes' primers for real-time PCR

    名称
    Name
    序列
    Sequence
    IL-1β-F CAGGTGGTGGAAGTGTGTCA
    IL-1β-R GGTCAAACTGGAGCGCAAAC
    TNF-α-F CCTGCTGTTTGCCTGGTACT
    TNF-α-R GCTTTGCTGCTGATCCGTTT
    β-actin-F TCAGCAAGCAGGAGTACGATG
    β-actin-R AGCTGAAGTTGTTGGGCGTT
    下载: 导出CSV

    表  2  simApgmA基因生物信息学分析

    Table  2.   Bioinformatics analysis of simA and pgmA genes

    分析项目
    Analysis item
    simA基因
    simA gene
    pgmA基因
    pgmA gene
    ORF区长度
    Length of ORF
    1 566个 碱基 1 716个 碱基
    GC碱基含量
    GC base content
    36.59% 38.69%
    编码氨基酸的数目
    Number of coding amino acids
    521 571
    蛋白分子量预测
    Molecular weight of protein
    57.5 kD 62.4 kD
    蛋白分子式预测
    Protein molecular formula
    C2481H4159N705O827S12 C2804H4352N714O876S10
    等电点
    Isoelectric point
    5.78 4.68
    保守性
    Conservatism
    与QMA0248株相似度为99% 与DGX07株相似度为100%;与QMA0248株相似度为99%
    下载: 导出CSV

    表  3  DNA疫苗对罗非鱼的相对保护率

    Table  3.   Relative protection rate of DNA vaccine against tilapia

    组别
    Group
    前处理
    Pre-processing
    总尾数
    Total
    死亡尾数
    Death number
    死亡率
    Mortality/%
    相对保护率
    RPS/%
    1 PBS 30 28 93.3
    2 pcDNA3.1-simA 30 14 46.7 49.9
    3 pcDNA3.1-pgmA 30 11 36.7 60.7
    4 混合疫苗 30 7 23.3 75.0
    5 pcDNA3.1(+) 30 25 83.3 10.7
    下载: 导出CSV
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  • 收稿日期:  2019-08-20
  • 修回日期:  2020-01-06
  • 录用日期:  2020-02-13
  • 网络出版日期:  2020-09-28
  • 刊出日期:  2020-06-05

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