尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果
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中国水产科学研究院珠江水产研究所

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广东省自然科学基金(2016A030313146);现代农业产业技术体系专项(CARS-46);广东省海洋与渔业局科技项目(A201601B05);广东省鱼病防治专项资金(20170070000600403);广州市科技计划项目(201707010312)


Preparation of live recombinant Lactococcus lactis vaccine expressing Sip protein of Streptococcus agalactiae isolated from tilapia (Oreochromis niloticus) and immunogenicity analysis
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Pearl River Fisheries Research Institute,Chinese Academy of Fisheries Science

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    摘要:

    链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×1010 CFU/mL)和低浓度组(2.24×109 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。

    Abstract:

    Streptococcosis is a serious disease that threatens the development of Oreochromis niloticus industry in China. In order to make a vaccine of Streptococcus agalactiae with high immune efficiency and simple operation, we constructed a recombinant shuttle-plasmid pNZ8124-Sip which could express Sip protein of S. agalactiae. The recombinant plasmid was electro-transferred into Lactococcus lactis NZ9000 after being identified by enzyme digestion and sequencing analysis. The SDS-PAGE was used to obtain the optimumly induced concentration of nisin and induction time. The Sip protein was purified by Ni-chelating affinity chromatography and tested by Western blot. O. niloticus was vaccinated orally by gavage with different concentration of the recombinant L. lactis NZ9000-pNZ8124-Sip. The ELISA was used to test the change of serum antibody. The relative percent survival (RPS) was obtained by artificial abdominal injection with S. agalactiae.The SDS-PAGE showed that the molecular weight of expressed protein was 48 ku, which is equal to the expected protein size. The recombinant protein mainly existed as soluble protein and inclusion bodies. The concentration of purified protein could reach 7.65 mg/mL. The optimal condition was induction with 100 ng/mL nisin for 6 h. Western blot results showed that the recombinant Sip protein could be specifically combined with the mouse anti-His tag antibody. Oral immunization showed that the serum antibody and the resistance to S. agalactiae were significantly improved in the middle concentration group (2.24×1010 CFU/mL) and the low concentration group (2.24×109 CFU/mL). The RPS of the middle concentration was 41.0%, which was the highest. This study can lay a foundation for research on oral vaccine of O. niloticus against S. agalactiae and has a broad prospect of application.

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蔡玉臻,刘志刚,卢迈新,可小丽,高风英,曹建萌.尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果[J].水产学报,2019,43(3):661~670

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  • 收稿日期:2017-11-09
  • 最后修改日期:2018-01-12
  • 录用日期:2018-03-04
  • 在线发布日期: 2019-03-15
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