免疫球蛋白是鱼类适应性免疫中主要的效应因子之一, 研究鱼类免疫球蛋白对鱼类疾病的免疫防控尤为重要。本研究利用 PCR 与 RACE 技术克隆获得青鲫(Carassius auratus indigentiaus)分泌型免疫球蛋白 Z (secretory immunoglobulin Z, sIgZ)重链基因 cDNA 全长序列 (基因序列登录号: MZ274344.1), 青鲫 sIgZ 重链基因 cDNA 序列全长 2083 bp, 其开放阅读框长 1668 bp, 编码一条由 556 个氨基酸组成的肽链, 相对分子质量为 61.59 kD, 理论等电点为 7.03。青鲫 sIgZ 重链基因结构由 1 个可变区(VH), 4 个恒定区(CH1-CH4)以及 1 个分泌型的尾部(secretory tail, Sec tail)构成。在 GenBank 中进行同源序列检索, 青鲫 sIgZ 氨基酸序列与其他鱼类 IgZ 的相似性依次为团头鲂 (Megalobrama amblycephala) (63.17%)、草鱼(Ctenopharyngodon idella) (60.82%)、鲤(Cyprinus carpio carpio) (52.98%)、虹鳟(Oncorhynchus mykiss)(37.06%)和金头鲷(Sparus aurata)(35.01%)。利用 MEGA 5.1 软件进行多重序列比对分析, 采用邻接法构建遗传系统进化树, 发现青鲫 sIgZ 基因与同属鲤科(Cyprinidae)鱼类的 IgZ 聚为一支, 青鲫 sIgZ 与团头鲂的 sIgZ 亲源关系最近。通过 qPCR 检测青鲫不同组织中 sIgZ 基因的表达, 发现青鲫头肾中 sIgZ 基因的表达量最高, 中肾、脾和肝脏次之。利用嗜水气单胞菌(Aeromonas hydrophila)对青鲫进行浸泡感染, sIgZ 在头肾、脾脏、中肾、肝、肠、鳃和皮肤中的表达水平在感染的 28 d 内先升高后降低, sIgZ 的相对表达量在黏膜免疫组织(肠和皮肤)中达到峰值的时间要早于系统免疫组织(头肾和脾脏), 肠和皮肤中 sIgZ 在峰值时的相对表达量显著高于头肾中 sIgZ 在峰值时的相对表达量, 实验结果显示, 青鲫 sIgZ 在肠和皮肤黏膜免疫组织中可能发挥重要的免疫功能。

Immunoglobulin is one of the major effector factors in the adaptive immunity of fish. Thus, the study of immunoglobulin is crucial for the prevention and control of disease in fish. In this study, the complete cDNA sequence of secretory immunoglobulin Z heavy chain gene (GenBank accession no. MZ274344.1) of Carassius auratus indigentiaus was cloned by PCR and RACE. The complete cDNA sequence of C. auratus indigentiaus sIgZ heavy chain gene was 2083 bp, and its open reading frame (ORF) was 1668 bp, which encoded a peptide chain composed of 556 amino acids, with a relative molecular weight of 61.59 kD and theoretical isoelectric point of 7.03. The sIgZ heavy chain gene structure of C. auratus indigentiaus is composed of one variable region (V), four constant regions (CH1-CH4), and one secretory tail (Sec tail). According to the homologous sequence retrieval in GenBank, the similarity of sIgZ amino acid sequence between C. auratus indigentiaus and other fish was as follows: Megalobrama amblycephala (63.17%), Ctenopharyngodon idellus (60.82%), Cyprinus carpio (52.98%), Oncorhynchus mykiss (37.06%), and Sparus aurata (60.82%). Constructing a phylogenetic tree using the neighbor-joining method in MEGA 5.1, sIgZ of C. auratus indigentiaus was found to cluster into one branch with the IgZ of Cypriniformes, wherein the genetic evolution relationship between C. auratus indigentiaus and M. amblycephala was the closest. qPCR analysis revealed that C. auratus indigentiaus sIgZ gene expression was highest in the head kidney, followed by mesonephron, intestine, spleen, and gonad. After artificial infection with Aeromonas hydrophila, the C. auratus indigentiaus sIgZ mRNA levels first increased and then decreased in different tissues within 28 d. The relative expression levels of sIgZ in mucosal immune tissues (intestine and skin) peaked earlier than that in systemic immune tissue (head kidney and spleen), wherein the relative expression of sIgZ in the intestine and skin at peak was significantly higher than that in the head kidney. These results showed that sIgZ of C. auratus indigentiaus may play an important immune function in mucosal immune tissue (e.g., intestine and skin).

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湖南省科技厅自然科学基金项目(2022JJ30033)； 国家自然科学基金项目(32172965； 31972835)