首页 >  广东海洋大学学报 >  凡纳滨对虾含酪氨酸酶结构域血蓝蛋白的免疫功能

2022, 42(5): 1-8. doi: 10.3969/j.issn.1673-9159.2022.05.001

凡纳滨对虾含酪氨酸酶结构域血蓝蛋白的免疫功能

1. 广东海洋大学食品科技学院/现代生化中心, 广东 湛江 524088;

2. 中山大学生命科学学院有害生物控制与资源利用国家重点实验室, 广东 广州 510275;

3. 南方海洋科学与工程广东省实验室(珠海), 广东 珠海 519000;

4. 广东海洋大学深圳研究院, 广东 深圳 518108

收稿日期:2022-06-08
修回日期:2022-06-08

基金项目:   广东省基础与应用基础研究基金项目(2021A1515110900);中山大学中央高校基本科研业务费专项资金(22qntd2615) 

关键词: 凡纳滨对虾 , 含酪氨酸酶结构域血蓝蛋白 , 免疫刺激 , 副溶血弧菌 , 免疫应答

Immune Function of Tyrosinase-Domain Containing Hemocyanin(TDCH)from Litopenaeus vannamei

1. College of Food Science and Technology/Modern Biochemistry Experimental Center, Guangdong Ocean University, Zhanjiang 524088, China;

2. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China;

3. Southern Marine Science and Engineering Guangdong Laboratory(Zhuhai), Zhuhai 519000, China;

4. Shenzhen Institute of Guangdong Ocean University, Shenzhen 518108, China

Received Date:2022-06-08
Accepted Date:2022-06-08

Keywords: Litopenaeus vannamei , tyrosinase-domain containing hemocyanin , immune stimulation , Vibrio parahaemolyticus , immune response

【目的】克隆凡纳滨对虾(Litopenaeus vannamei)一种新的含酪氨酸酶结构域血蓝蛋白(Tyrosinase-domain containing hemocyanin,TDCH),研究其免疫功能。【方法】根据NCBI上公布的tdch基因序列设计克隆引物,通过RT-PCR方法获得tdch的编码区;采用生物信息学方法分析tdch的结构特征;使用荧光定量PCR技术分析tdch在凡纳滨对虾12 个组织中的表达,以及在副溶血弧菌(Vibrio parahaemolyticus)、金黄色葡萄球菌(Staphhylococcu saureus)、黑曲霉菌(Aspergillus niger)、白斑综合征病毒、脂多糖及聚肌苷酸-聚胞苷酸[Poly(I:C)]刺激后在鳃和血细胞中的表达变化;利用RNAi 技术敲降tdch 在对虾中的表达,研究其在对虾抗副溶血弧菌免疫中的作用。【结果与结论】克隆获得tdch 的编码区全长,编码551 氨基酸的蛋白,理论分子质量为63.37 ku,等电点为7.74;tdch除含有血蓝蛋白结构域以外,还含有酪氨酸酶Tyrosinase 结构域;tdch 在对虾肌肉、肝胰腺、鳃、胃、表皮、触角、心脏、肠、幽门盲囊、眼柄和神经索中均有表达,眼柄中表达最高,在幽门盲囊中表达最低。Poly(I:C)刺激后,tdch在血细胞中的表达显著升高,在鳃组织中表达变化不明显;其余5 种免疫原刺激后,tdch 在鳃和血细胞中的表达均显著升高,沉默tdch 的表达可导致副溶血弧菌感染对虾存活率显著下降,表明tdch 参与了抗副溶血弧菌免疫应答。

【Objective】To clone a novel tyrosinase-domain containing hemocyanin (TDCH) from Litopenaeus vannamei and study its immune function.【Method】The specific primers were designed based on the tdch gene sequence retrieved from the NCBI Genbank database, and the coding region of tdch was amplified by RT-PCR; The structural characteristics of the TDCH protein were analyzed by bioinformatics; The tdch expression in 12 tissues of L.vannamei and its changes in gills and hemocytes from shrimp challenged by Vibrio parahaemolyticus, Staphylococcus aureus, Aspergillus niger, white spot syndrome virus (WSSV), lipopolysaccharide (LPS) and virus mimics Poly (I:C) were analyzed by real-time quantitative PCR; The RNAi strategy was used to knock down the expression of tdch in L.vannamei to study its effect on immunity against V.parahaemolyticus.【Result and Conclusion】The full length of the coding region of tdch was obtained, which encoded a 551-amino acid protein with a molecular weight of 63.37 ku and a theoretical isoelectric point of 7.74.In addition to hemocyanin domains, the TDCH protein also contained a tyrosinase domain.The expression of tdch was the highest in the eyestalk of L.vannamei and the lowest in the pyloric caecum.After stimulation with Poly (I:C), the expression of tdch in hemocytes increased significantly, but there was no significant change in gills, while the expression of tdch in gills and hemocytes increased significantly after stimulation with other five immunogens.Silencing of tdch could significantly reduce the survival rate of Vibrio parahaemolyticus-infected L.vannamei, indicating that tdch was involved in the immune response against V.parahaemolyticus infection.

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凡纳滨对虾含酪氨酸酶结构域血蓝蛋白的免疫功能