鰤鱼诺卡氏菌疫苗对石斑鱼免疫效果研究

doi:10.13304/j.nykjdb.2022.0653

摘要/Abstract

摘要：

为探究鰤鱼诺卡氏菌灭活疫苗对石斑鱼的免疫效果，通过免疫基因表达量分析疫苗对石斑鱼的免疫保护。利用鰤鱼诺卡氏菌制备福尔马林灭活全菌疫苗，通过腹腔注射法对珍珠龙胆石斑鱼进行免疫，利用荧光定量法检测免疫后该鱼的肾脏、肝脏和脾3个器官中TLR2、MyD88、TNF-α、IL-1β免疫基因表达量的变化。结果显示，注射灭活疫苗后，TNF-α、IL-1β基因表达量在肾脏组织中较高，MyD88基因表达量在脾组织中较高，TLR2基因表达量在肝脏组织中较高；除脾组织中的TNF-α基因相对表达量出现下降趋势外，其余基因的表达量在所有器官中都呈现出上调的趋势。研究结果为探索珍珠龙胆石斑鱼与诺卡氏菌的相互作用提供理论基础，为渔业疫苗开发和应用奠定基础。

关键词: 鰤鱼诺卡氏菌, 石斑鱼, 灭活疫苗, TLR2, MyD88, TNF-α, IL-1β

Abstract:

In order to explore the immune effect of inactivated vaccine of Nocardia seriolae on grouper， the immune protection of the vaccine on grouper was analyzed by the expression of immune gene. In this study， formalin-inactivated vaccine was prepared by this bacteria， and the pearl gentian grouper was immunized by intraperitoneal injection. The expression changes of TLR2， MyD88， TNF-α and IL-1β immune gene in the kidney， liver and spleen of the fish were detected by fluorescence quantitative method. The results showed that by injecting inactivated vaccine， the expressions of TNF-α and IL-1β genes were higher in kidney tissues， MyD88 gene’s expression was higher in spleen tissues， and TLR2 gene’s expression was higher in liver tissues. Except for a trend of decreasing relative expression of TNF-α gene in the tissue spleen， and the other genes showed up-regulation in all organs compared with that in blank group. Above results provided a theoretical basis for exploring the interaction between pearl gentian grouper and Nocardia seriolae， and laid a foundation for the development and application of fishery vaccines.

Key words: Nocardia seriolae, grouper, lumbar injection, TLR2, MyD88, TNF-α, IL-1β

中图分类号:

S941.51+6

高浩峰, 邵蓬, 武尊, 吴雅婷, 汪笑宇. 鰤鱼诺卡氏菌疫苗对石斑鱼免疫效果研究[J]. 中国农业科技导报, 2024, 26(2): 145-152.

Haofeng GAO, Peng SHAO, Zun WU, Yating WU, Xiaoyu WANG. Immune Effect of Nocardia seriolae Caviar Vaccine on Grouper[J]. Journal of Agricultural Science and Technology, 2024, 26(2): 145-152.

图/表 7

表1 RT-qPCR所用引物

Table 1 Primer were used to RT-qPCR

基因 Gene	引物序列 Primer sequence（5’-3’）
TLR2	GAACCAAGCATCGTGCCAGG
TLR2	CAACGCTATCCGAGCCCCAAG
MyD88	CGGGACAGAGGCTGAGAG
MyD88	GCTGGAGCAGACGGAGTG
IL-1β	TCTGGGCATCAAGGGCACACA
IL-1β	CCATGTCGCTGTTCGGATCGA
TNF-α	GTGTCCTGCTGTTTGCTTGGTA
TNF-α	CAGTGTCCGACTTGATTAGTGCTT
β-actin	TGCTGTCCCTGTATGCCTCT
β-actin	CCTTGATGTCACGCACGAT

表2 福尔马林在各个时间的灭活情况

Table 2 Inactivation of formalin at various times

灭活时间 Inactivation time	福尔马林含量Formalin content/%
灭活时间 Inactivation time	0.1	0.2	0.3	0.4	0.5	0.0
30 min	+	+	+	+	+	+
1 h	+	+	+	+	-	+
2 h	+	+	+	+	-	+
4 h	+	+	+	-	-	+
8 h	+	+	+	-	-	+
12 h	+	+	+	-	-	+
24 h	+	+	-	-	-	+

图1 注射组血清抗体效价注：同一时间点不同小写字母表示处理之间差异在P<0.05水平显著。

Fig. 1 Knetics of antibody activities in injectionNote： Different lowercase letters in same time point indicate significant differences between treatments at P<0.05 level.

图2 鰤鱼诺卡氏菌灭活疫苗免疫珍珠龙胆石斑鱼脾、肾、肝TLR2基因的相对表达量注：*表示与对照组差异在P<0.05水平显著，虚线所示为对照组相对表达量。

Fig. 2 Expression of TLR2 in spleen，kidney and liver after immunization with formalin inactivated Nocardia seriolaeNote： * indicates significant differences with the control at P<0.05 level， and the dotted line shows the relative expression of control group.

图3 鰤鱼诺卡氏菌灭活疫苗免疫珍珠龙胆石斑鱼脾、肾、肝MyD88基因的相对表达量注：*表示与对照组差异在P<0.05水平显著，虚线所示为对照组相对表达量。

Fig. 3 Expression of MyD88 in spleen，kidney and liver post immunization with formalin inactivated Nocardia seriolaeNote： * indicates significant differences with the control at P<0.05 level，and the dotted line shows the relative expression of control group.

图4 鰤鱼诺卡氏菌灭活疫苗免疫珍珠龙胆石斑鱼脾、肾、肝IL-1β基因的相对表达量注：*表示与对照组差异在P<0.05水平显著，虚线所示为对照组相对表达量。

Fig. 4 Expression of IL-1β in spleen，kidney and liver post immunization with formalin inactivated Nocardia seriolaeNote： * indicates significant differences with the control at P<0.05 level，and the dotted line shows the relative expression of control group.

图 5 鰤鱼诺卡氏菌灭活疫苗免疫珍珠龙胆石斑鱼脾、肾、肝TNF-α 基因的相对表达量注：*表示与对照组差异在P<0.05水平显著。虚线所示为对照组相对表达量。

Fig. 5 Expression of TNF-α in spleen，kidney and liver post immunization with formalin inactivated Nocardia seriolaeNote：* indicates significant differences with the control at P<0.05 level，and the dotted line shows the relative expression of control group.

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