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首页> 中文期刊> 《中国水产科学》 >罗非鱼无乳链球菌C5a肽酶（ScpB）的原核表达及其免疫原性

罗非鱼无乳链球菌C5a肽酶（ScpB）的原核表达及其免疫原性

         
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摘要

Streptococcus agalactiae is a major bacterial pathogen that has caused severe economic losses in many spe-cies of freshwater, marine and estuarine fish worldwide. ScpB is a highly conserved surface protein among all group B streptococcus (GBS) strains and is an attractive surface-exposed antigen for inclusion among vaccine components against GBS. In this study, the scpB gene was amplified from the genomic DNA of an S. agalactiae strain isolated from diseased tilapia farmed in Guangdong province, China. The scpB gene contains a 2 799 bp open reading frame (ORF), encoding 932 amino acids. The molecular mass of the deduced protein was 103 kD. Blast analysis showed that it shares high similarity with scpB sequences of human GBSs registered in GenBank. Prokaryotic expression vector pET32a (+) was used to construct a recombinant expression vector pET32a (+)-scpB, which was transformed into Escherichia coli BL21 (DE3). Colonies containing the recombinant expression vector pET32a (+)-scpB were selected and then induced to express using 0.5 mmol/L IPTG for 8 h at 37℃. The expressed recombinant protein was purified by nickel chelate affinity chromatography and ultrafiltration tube enrichment. SDS-PAGE analysis and western blotting showed a spe-cific protein band of about 121 kD. The recombinant strain could produce large amounts of ScpB protein, mainly in the form of an inclusion body. To analyze the immunogenicity of the recombinant protein, the purified fusion protein and Freund’s adjuvant were mixed according to a certain proportions to produce vaccines. Three immune dosages, 1μg/g(F1), 3μg/g(F3) and 5μg/g(F5) were used. Four weeks after immunization, tilapia were challenged by artificial infection with the GBS ZP-N strain, which was previously isolated and confirmed to be a tilapia pathogen by our labo-ratory. The recorded relative percent survivals (RPS) of the vaccinated groups ranged from 69.99%to 89%, of which group F5 has the highest RPS. The lysozyme activities, superoxide dismutase (SOD) activities and antibody levels (OD450nm) were tested weekly until the end of the experiment. The results showed that after immunization, there were significantly higher lysozyme activities, superoxide dismutase (SOD) activities and antibody activities(OD450) in the vaccinated fish compared with the control group (P<0.01). The results of this study showed that recombinant protein ScpB has a strong immunogenicity and protective effect, laying the foundation for further study of polypeptide vaccines for S. agalactiae.%以尼罗罗非鱼(Oreochromis niloticus)为鱼体样本,克隆了罗非鱼源无乳链球菌(Streptococcus agalactiae) scpB基因,构建了原核表达质粒pET32a(+)-scpB,转入大肠杆菌BL21(DE3)后, IPTG诱导表达,表达的重组蛋白经镍离子金属螯合柱纯化及超滤管浓缩后,进行SDS-PAGE和Western blot分析鉴定。纯化的重组蛋白以不同剂量(1μg/g、3μg/g、5μg/g)免疫尼罗罗非鱼后,每周检测各实验组鱼体血清非特异性免疫指标[溶菌酶活性、超氧化物歧化酶(SOD)活力]及抗体水平变化情况,并于免疫4周后以4倍LD50的剂量对其进行人工攻毒。结果显示,该重组蛋白在大肠杆菌中以包涵体的形式存在；对尼罗罗非鱼的相对免疫保护率为69.66%~89.00%,其中5μg/g 组的相对保护率最高；受免鱼体血清中溶菌酶活性、SOD活力和抗体水平较对照组有极显著提高(P<0.01),结果表明,重组蛋白ScpB具有较强的免疫原性和保护作用。本研究旨在为GBS多肽疫苗的研制奠定基础。

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  • 来源
    《中国水产科学》 |2014年第1期|169-179|共11页
  • 作者

    李庆勇; 可小丽; 卢迈新; 朱华平; 高风英; 刘志刚;

  • 作者单位

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

    上海海洋大学 水产与生命学院;

    上海 201306;

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

    中国水产科学研究院 珠江水产研究所;

    农业部热带亚热带水产资源利用与养殖重点实验室;

    广东;

    广州 501380;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类 鱼病学;
  • 关键词

    罗非鱼; 无乳链球菌; C5a肽酶; 原核表达; 免疫原性;

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