近江牡蛎C型凝集素CRD结构域PAMPs结合谱及抑菌活性

PAMPs Binding Spectrum and Antibacterial Activity of C-type Lectin CRD Domain from Crassostrea rivularis

[目的]揭示C型凝集素(CTL)在近江牡蛎固有免疫防御机制中的重要作用。[方法]采用基因克隆、蛋白表达等技术进行试验。[结果]克隆获得1种近江牡蛎CTL基因,其CDS区1350 bp,编码449个氨基酸,含1个CRD结构域,Ca^(2+)结合位点2关键氨基酸基序为QPS/WHD;CTL mRNA相对表达呈组织差异分布,肝胰腺、外套膜、血细胞表达量较高,其次为鳃、肌肉柱、卵巢,唇瓣表达量最少;CTL的CRD结构域体外重组蛋白具有LPS、PGN和glucan 3种PAMPs结合活性,能结合、抑制和杀死革兰氏阳性菌中的金黄色葡萄球菌、革兰氏阴性菌中的大肠杆菌和鳗弧菌,能结合、抑制但不能杀死毕赤酵母和耶罗维亚酵母。[结论]近江牡蛎CTL的CRD结构域具有较广泛的PAMPs结合谱和抑菌活性,在其固有免疫防御机制中可发挥识别、结合、抑制和杀死多种有害菌的重要作用。

[Objective]In order to reveal the important role of CTL in the innate immune defense mechanism of Crassostrea rivularis.[Meth-od]Gene cloning and protein expression else techniques were used in the experiment.[Result]The results showed that CTL was cloned by gene cloning.Its CDS region was 1350 bp,encodes 449 amino acids.CRD domain was found in CTL gene,with QPS/WHD key motif in Ca^(2+)bind-ing site 2.Quantitative real-time PCR was employed to analyze CTL mRNA level.Relative mRNA levels of CTL were the highest expression in hepatopancreas,mantle,haemocytes,followed by gill,muscle column,ovary.Relative mRNA levels of CTL was the lowest expression in lip flap.The CRD domain recombinant protein of CTL had the binding activity of LPS,PGN and glucan.The CRD domain recombinant protein of CTL could inhibit and kill Staphylococcus aureus which belong to gram positive bacteria,could inhibit Bacillus coli and Vibrio anguillarum which belong to gram negative bacteria,but could not inhibit and kill Pichia pastoris and Yarrowia lipolytica.[Conclusion]The CRD domain of CTL in Crassostrea rivularis has a wide range of PAMPs binding spectrum and antibacterial activity.It can play an important role in identif-ying,binding,inhibiting and killing a variety of harmful bacteria in its inherent immune defense mechanism.