创伤弧菌微球口服疫苗的制备及其免疫效果研究

doi:10.19640/j.cnki.jtau.2023.06.008

天津农学院学报 ›› 2023, Vol. 30 ›› Issue (6): 41-50.doi: 10.19640/j.cnki.jtau.2023.06.008

创伤弧菌微球口服疫苗的制备及其免疫效果研究

何学欣, 武尊, 吴雅婷, 高浩峰, 周文礼, 邵蓬^通信作者

天津农学院水产学院,天津 300392

收稿日期:2022-11-16 出版日期:2023-12-31 发布日期:2024-02-05
通讯作者: 邵蓬（1982—）,男,副教授,博士,主要从事渔业资源与环境、水生动物病害防控等方面的研究。E-mail：shaopeng@tjau.edu.cn。
作者简介:何学欣（1984—）,女,硕士在读,主要从事水生动物疾病防控方面的研究。E-mail：287029732@qq.com。

Investigation on the immune effect of Vibrio vulnificus inactivated vaccine in fish

He Xuexin, Wu Zun, Wu Yating, Gao Haofeng, Zhou Wenli, Shao Peng^{Corresponding Author}

College of Fisheries, Tianjin Agricultural University, Tianjin 300392, China

Received:2022-11-16 Online:2023-12-31 Published:2024-02-05

摘要/Abstract

摘要： 为了研究创伤弧菌灭活疫苗的免疫效果,本研究将实验室保存的创伤弧菌菌株EPL0201经0.3%甲醛灭活,采用海藻酸钠包裹灭活菌液制备了创伤弧菌微球疫苗,通过口服途径免疫健康的珍珠龙胆石斑鱼（Pearl gentian grouper）,在免疫后第28天用1×10⁷ cfu/mL创伤弧菌腹腔注射珍珠龙胆石斑鱼,检测了免疫后珍珠龙胆石斑鱼部分免疫相关基因的表达趋势和攻毒后的免疫保护率。创伤弧菌微球口服疫苗的粒径为（34.55±5.33）μm,包覆率为90.5%。测其安全性良好。实时定量PCR（quantitative real-time PCR,qRT-PCR）结果显示,与对照组相比,各免疫基因先后出现不同的表达变化。TLR信号通路上的基因TLR1、TLR2、TLR3、TLR5和M?y?D88及炎性细胞因子IL-1β、IL-12p40和TNF-α在肾脏、脾脏、肝脏和肠组织中都参与免疫应答。TGF-β只在脾脏中有表达。免疫基因在各个组织中的表达变化不同,其中,表达量最高的是IL-1β在脾脏中,最高值为对照组的72.37倍。同一种免疫基因在鱼体不同器官和组织中的峰值表达量时间不同,同一组织中不同免疫基因的峰值表达量也不相同,说明各组织中的基因表达变化有差异性。攻毒试验结果显示,免疫保护率为61.3%。本研究结果显示创伤弧菌微球口服疫苗能够引起免疫基因参与免疫应答反应,对珍珠龙胆石斑鱼具有免疫保护作用,也为微球口服疫苗防治创伤弧菌引起的疾病提供试验数据支持。

关键词: 创伤弧菌, 微球口服疫苗, 珍珠龙胆石斑鱼, 免疫效果

Abstract: In order to study the immune effect of inactivated Vibrio vulnificus vaccine, this study prepared Vibrio vulnificus microsphere vaccine by inactivating the Vibrio vulnificus strain EPL0201 stored in the laboratory with 0.3% formaldehyde and wrapping the inactivated bacteria solution with sodium alginate. Healthy pearl gentian grouper was immunized by oral route, and was injected intraperitoneally with 1×10⁷ cfu/mL Vibrio vulnificus to challenge the pearl gentian grouper. The expression trend of some immune related genes of the pearl gentian grouper after immunization and the immune protection rate after challenge were detected. The results showed that the particle size of Vibrio vulnificus microsphere oral vaccine was（34.55±5.33）μm. The coverage rate was 90.5%. Its safety was good. Quantitative real-time PCR（QRT-PCR）results showed that, compared with the control group, each immune gene had different expression changes successively. They participated in immune response in kidney, spleen, liver and intestine which involved the genes TLR1, TLR2, TLR3, TLR5, and Myd88 in the TLR signaling pathway and inflammatory cytokines IL-1β, IL-12P40 and TNF-α. The expression of immune genes in different tissues was different, and the highest expression level of IL-1β was in spleen, and the highest value was 72.37 times that of the control group. The peak of peak expression of the same immune gene in different organs and tissues of fish was different, and the peak expression of different immune genes in the same tissue was also different, which indicated that the gene expression changes in different tissues were different. The results of challenge experiment showed that the immune protection rate was 61.3%. The results of this study showed that Vibrio vulnificus oral vaccine can induce immune genes to participate in the immune response, and has immune protection effect on pearl Gentian grouper. It also provides experimental data support for the prevention and treatment of diseases caused by Vibrio vulnificus with microsphere oral vaccine.

Key words: Vibrio vulnificus, oral microsphere vaccine, pearl gentian grouper, immune effect

中图分类号:

S942.5

何学欣, 武尊, 吴雅婷, 高浩峰, 周文礼, 邵蓬. 创伤弧菌微球口服疫苗的制备及其免疫效果研究[J]. 天津农学院学报, 2023, 30(6): 41-50.

He Xuexin, Wu Zun, Wu Yating, Gao Haofeng, Zhou Wenli, Shao Peng. Investigation on the immune effect of Vibrio vulnificus inactivated vaccine in fish[J]. Journal of Tianjin Agricultural University, 2023, 30(6): 41-50.

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: http://xuebao.tjau.edu.cn/CN/10.19640/j.cnki.jtau.2023.06.008

http://xuebao.tjau.edu.cn/CN/Y2023/V30/I6/41

参考文献

[1] BAKER-AUSTIN C,OLIVER J D.Vibrio vulnificus:new insights into a deadly opportunistic pathogen[J]. Environmental Microbiology,2018,20(2):423-430.
[2] JONES M K,OLIVER J D.Vibrio vulnificus:disease and pathogenesis[J]. Infection and Immunity,2009,77(5):1723-1733.
[3] 王青柏,李良秋,徐鹏,等. 创伤弧菌生物学特性和检测技术研究进展[J]. 安徽农业科学,2019,47(5):33-36.
[4] 刘秀珍,邹晓理,莫小燕,等. 海水网箱养殖石斑鱼病原菌研究[J]. 热带海洋,1994,13(1):81-86.
[5] 王际英,张德瑞,马晶晶,等. 珍珠龙胆石斑鱼肌肉营养成分分析与品质评价[J]. 海洋湖沼通报,2015(4):61-69.
[6] 陈信忠. 石斑鱼病毒性神经坏死病研究[D]. 厦门:厦门大学,2006.
[7] 王玉堂. 疫苗及其在水生动物疾病预防中的应用 (三)[J]. 中国水产,2018(9):88-90.
[8] 薛淑霞,魏俊利,王雪惠,等. 一种迟缓爱德华氏菌微球口服疫苗及制备方法:CN109395070A[P].2019-03-01.
[9] 曹兵兵,高昉,姜勇,等. 创伤弧菌致病机制研究进展[J]. 温州医科大学学报,2018,48(2):145-149.
[10] KIM Y R,LEE S E,KOOK H,et al.Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells[J]. Cellular Microbiology,2008,10(4):848-862.
[11] 胡秀彩,王英杰,吕爱军,等. 半滑舌鳎创伤弧菌分离鉴定及其荧光定量PCR方法的建立[J]. 水产学报,2019,43(5):1359-1368.
[12] CHEN C Y,CHAO C B,BOWSER P R.Infection of Tilapia Oreochromis sp. by Vibrio vulnificus in freshwater and low-salinity environments[J]. Journal of the World Aquaculture Society,2006,37(1):82-88.
[13] 许悦,王印庚,周永灿,等. 海南地区养殖石斑鱼皮肤溃疡病病原的分离、鉴定、药敏分析及病理学研究[J]. 海南大学学报(自然科学版),2018,36(2):153-163.
[14] 张小江,任燕,常藕琴,等. 斜带石斑鱼口服PELA-OmpK微球疫苗的示踪及免疫效果[J]. 中国水产科学,2008(5):837-844.
[15] 袁高亮,朱雷,张杰,等. 渔用口服疫苗在水产养殖上的应用[J]. 河南水产,2019(2):27-29,33.
[16] 任燕,张小江,常藕琴,等. PELA-OmpK微球疫苗的部分特征及其对鲫鱼的口服免疫效果[J]. 中国生物制品学杂志,2011,24(11):1306-1309.
[17] 王兴丽,汪开毓,王二龙,等. 斑点叉尾鮰源海豚链球菌Srr蛋白海藻酸钠-壳聚糖口服疫苗的制备及其免疫效果研究[J]. 水生生物学报,2018,42(1):39-46.
[18] 张运铎. 海藻酸钠功能特性应用研究进展[J]. 河南科技,2011(17):56-57.
[19] RAUTA P R,NAYAK B,DAS S.Immune system and immune responses in fish and their role in comparative immunity study:A model for higher organisms[J]. Immunology Letters,2012,148(1):23-33.
[20] WERTS C,TAPPING R I,MATHISON J C,et al.Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism[J]. Nature Immunology,2001,2(4):346-352.
[21] 朱兴华. 短蛸Toll样受体信号通路相关基因表达研究[D]. 上海:上海海洋大学,2021.
[22] 李青梅. 多个免疫相关基因在草鱼中肾的表达[D]. 杨凌:西北农林科技大学,2013.
[23] 林克冰,葛辉,林琪,等. 斜带石斑鱼TLR5S基因结构及功能分析[J]. 厦门大学学报(自然科学版),2013,52(1):109-115.
[24] 祝璟琳,邹芝英,李大宇,等. 异源无乳链球菌胞外产物灭活疫苗对罗非鱼的免疫效果[J]. 中国水产科学,2022,29(2):325-340.
[25] COOPER A M,KHADER S A.IL-12p40: an inherently agonistic cytokine[J]. Trends in Immunology,2007,28(1):33-38.
[26] ZWC A,LLKA B,FEI Z A,et al.Two types of TNF-α and their receptors in snakehead(Channa argus):Functions in antibacterial innate immunity[J]. Fish & Shellfish Immunology,2020,104:470-477.
[27] 胡元红. 黄颡鱼TNF-α、TNFR1和TNFR2基因的克隆、表达及其功能研究[D]. 武汉:华中农业大学,2021.
[28] 王尚念. 草鱼IRAK-4介导IL-1β细胞信号转导研究[D]. 成都:电子科技大学,2015.
[29] PANDIT N P,SHEN Y B,XU X Y,et al.Differential expression of interleukin-12 p35 and p40 subunits in response to Aeromonas hydrophila and Aquareovirus infection in grass carp,Ctenopharyngodon idella[J]. Genetics and Molecular Research,2015,14(1):1169-1183.
[30] REYES-CERPA S,MAISEY K, REYES-LPEZ F,et al.Fish cytokines and immune response[M]//TURKER H. New Advances and Contributions to Fish Biology. Santa Clara:InTech,2012:22.
[31] 杨潇. 草鱼白细胞介素1β诱导表达的负调控机制及其在炎症反应中的意义[D]. 成都:电子科技大学,2014.

创伤弧菌微球口服疫苗的制备及其免疫效果研究

Investigation on the immune effect of Vibrio vulnificus inactivated vaccine in fish

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 1

编辑推荐

Metrics

本文评价