多纹钱蝶鱼感染进程中海豚链球菌毒力基因表达特征及亚单位疫苗开发OA
Expression Characteristics of Virulence Genes of Streptococcus iniae in Infecting Selenotoca multifasciata and Development of Subunit Vaccines
海豚链球菌(Streptococcus iniae)是鱼类重要的病原菌,为筛选适宜的抗原蛋白并评价其免疫原性,本研究首先采用RT-qPCR方法,检测海豚链球菌感染多纹钱蝶鱼(Selenotoca multifasciata)过程中的细菌数量及7种毒力相关基因的体内诱导表达,随后通过大肠杆菌原核系统重组表达eno基因编码的α-烯醇化酶(Recombinant alpha-enolase,rENO),最后比较了海豚链球菌灭活疫苗和rENO亚单位疫苗对多纹钱蝶鱼的免疫保护效果.研究显示,肝脏细菌载量呈先升高后降低趋势,攻毒后48 h细菌载量达峰值;攻毒后48～72 h鱼的死亡率最高,呈急性感染特征.基因表达检测表明,毒力基因eno、sag、pgmA的相对表达量在感染后72 h显著高于48 h,在感染鱼体中的表达显著高于体外培养,以上结果表明,eno、sag和pgmA基因在海豚链球菌感染多纹钱蝶鱼进程中被诱导表达,可能发挥了关键毒力作用,其中eno基因在各时间节点及条件下的表达量最高且稳定.免疫后攻毒结果显示,海豚链球菌灭活疫苗和rENO亚单位疫苗的相对免疫保护率分别为94.12%和70.59%,表明rENO具备开发为海豚链球菌亚单位疫苗的应用潜力.综上所述,本研究筛选到了海豚链球菌感染多纹钱蝶鱼后诱导的3个重要毒力基因,并成功实现了eno基因在大肠杆菌原核系统中的重组表达,评价了亚单位疫苗的免疫效果,研究结果将为海豚链球菌的防控和疫苗开发提供参考.
Streptococcus iniae is an important pathogenic bacterium of fish.To screen suitable antigenic proteins and evaluate its immunogenicity,the RT-qPCR method was used to detect the bacterial load and the in vivo induced expression of 7 virulence-related genes of S.iniae in infecting Selenotoca multifasciata.The α-enolase(rENO)encoded by the eno gene was subsequently recombinantly expressed using a prokaryotic system in E.coli,and the immunoprotective effects of an inactivated S.iniae vaccine and an rENO subunit vaccine were finally compared in S.multifasciata.The bacterial load in liver tissue showed a tendency of increasing and then decreasing.At 48 hours post-challenge,the bacterial load reached its peak.The highest mortality rate of fish occurs 48～72 hours post-challenge,exhibiting characteristics of acute infection.Gene expression detection indicated that the relative expression levels of the virulence genes eno,sag,and pgmA at 72 hours post-infection were significantly higher than those at 48 hours.Additionally,their expression levels under the condition of fish body infection were significantly higher than those under in vitro culture conditions.These results indicated that the eno,sagI and pgmA genes are induced to express during infection,and may play key virulence roles.Among them eno had the highest and stable expression at all time points and conditions.The challenge test results after immunization showed that the relative percentage survival of the inactivated S.iniae vaccine and the rENO subunit vaccine were 94.12% and 70.59%,respectively.This indicated that rENO has the potential for development as a subunit vaccine against S.iniae.In summary,we screened three important virulence genes induced by S.iniae after infection of S.multifasciata,and successfully carried out the recombinant expression of the eno gene in a prokaryotic system of E.coli,and evaluated the immune effect of subunit vaccine.The results will provide references for the prevention and control of S.iniae and the development of vaccine.
王连慧;李扬;唐磊;茅云翔;莫照兰;李杰
中国海洋大学三亚海洋研究院,海南 三亚 572024中国海洋大学三亚海洋研究院,海南 三亚 572024中国海洋大学三亚海洋研究院,海南 三亚 572024||中国海洋大学海洋生命学院,山东 青岛 266003中国海洋大学三亚海洋研究院,海南 三亚 572024||中国海洋大学海洋生命学院,山东 青岛 266003||海南热带海洋学院水产与生命学院,海南 三亚 572022中国海洋大学三亚海洋研究院,海南 三亚 572024||中国海洋大学海洋生命学院,山东 青岛 266003中国水产科学研究院黄海水产研究所,山东 青岛 266071
农业科技
海豚链球菌毒力基因多纹钱蝶鱼蛋白表达亚单位疫苗
Streptococcus iniaevirulence geneSelenotoca multifasciataprotein expressionsubunit vaccine
《中国海洋大学学报（自然科学版）》 2026 (7)
53-62,10
国家重点研究发展计划项目(2022YFD2401203,2022YFD2401204)海南热带海洋学院崖州湾创新研究院重大科技计划项目(2022CXYZD001)资助 Supported by the National Key Research and Development Program(2022YFD2401203,2022YFD2401204)the Major Science and Technology Program of Yazhou Bay Innovation Research Institute of Hainan Tropical Oceanography Institute(2022CXYZD001)
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