三角帆蚌HcCnAα基因克隆及维氏气单胞菌感染后的表达

The HcCnAα gene cloning in Hyriopsis cumingii and expression after infection with Aeromonas veronii

摘要:

钙调神经磷酸酶 (CN)属苏/丝氨酸蛋白磷酸酶，在软体动物的抗菌应答、免疫调节中发挥重要作用。本研究利用cDNA末端快速扩增 （RACE）技术克隆获得三角帆蚌CN基因α型催化亚基 （HcCnAα）的cDNA序列，并进行了生物信息学分析。构建pET30a-HcCnAα原核表达载体，将诱导表达、纯化、复性后蛋白免疫小鼠制备多克隆抗体。用蛋白质印迹法 （Western blot，WB）和荧光定量PCR （qPCR）技术分析健康状态和感染维氏气单胞菌GL2后HcCnAα在三角帆蚌不同组织中的表达情况。结果显示，HcCnAα cDNA全长2 737 bp，其中3′ UTR长131 bp，5′ UTR长1 154 bp，CDS区为1 452 bp，编码483个氨基酸，序列包含蛋白磷酸酶保守的PP2Ac结构域。WB和qPCR结果显示，HcCnAα在三角帆蚌各组织中广泛存在，并在闭壳肌、斧足、性腺、鳃中的表达量较高，在血液中的表达量最低。维氏气单胞菌GL2感染三角帆蚌后3~24 h，鳃中HcCnAα的表达量显著升高，6 h时闭壳肌中表达量也显著升高，感染后24 h，肝胰腺、斧足、血液、外套膜中的表达量显著升高。而感染后48 h，闭壳肌、鳃、外套膜、肝胰腺中的表达量显著降低。研究表明，三角帆蚌HcCnAα基因与其他物种CnAα基因高度相似，具有保守的PP2Ac结构域，在各组织中普遍表达，并参与了三角帆蚌在细菌感染后的免疫反应。本研究首次克隆了三角帆蚌HcCnAα基因并制备了多克隆抗体，揭示了HcCnAα基因参与三角帆蚌的抗感染免疫应答，为深入研究三角帆蚌的免疫调控机制和疾病防控策略奠定了基础。

关键词:三角帆蚌;维氏气单胞菌;钙调神经磷酸酶;多克隆抗体;细菌感染

Abstract:

Calcineurin (CN) is a threonine/serine protein phosphatase that plays an important role in the antimicrobial response and immune response in molluscs. In order to investigate the role of CN in the antimicrobial immune response of Hyriopsis cumingii, the α-type catalytic subunit of H. cumingii CN gene (HcCnAα) was cloned by rapid-amplification of cDNA ends (RACE) and its characteristics were analyzed by bioinformatics methods. With the pET30a-HcCnAα expression vector successfully constructed and the mice immunized after protein purification and ultrafiltration, polyclonal antibody with good specificity was obtained. The relative expression of HcCnAα in different tissues were analyzed by Western blot (WB) and quantitative real-time PCR (qPCR) in a fit state or infected by Aeromonas veronii GL2. The results showed that the full length of HcCnAα cDNA was 2 737 bp, including 131 bp of 3′ UTR, 1 154 bp of 5′ UTR, and it encoded a total of 483 amino acids. The sequence contained PP2Ac domains, which are conserved domains of protein phosphatase. WB and qPCR results showed HcCnAα was expressed in all the tissues of H. cumingii. The expression of HcCnAα was highest in adductor, higher in foot, gill, intestines, kidney, and gonad, and the lowest in blood. After infection with A. veronii GL2, the expression of HcCnAα was significantly up-regulated in gills at 3 to 24 h, and also in adductor at 6 h, in hepatopancreas, foot, blood, and mantle at 24 h, but the expression significantly decreased in adductor, gill, mantle, and hepatopancreas at 48 h. This study suggested that the HcCnAα gene of H. cumingii was highly similar to the CnAα of other species with conserved PP2Ac domains; it was expressed in all tissues, and played a role in immune responses after the bacterial infection. This study successfully accomplished the cloning of HcCnAα genes for the first time and the polyclonal antibody was obtained, supporting the involvement of HcCnAα in the immune response after the bacterial infection in H. cumingii, and thus established a solid groundwork for future investigations in immune regulation, disease prevention and control strategies for H. cumingii.

Key words:Hyriopsis cumingii;Aeromonas veronii;calcineurin;polyclonal antibody;bacterial infection