College of Life Sciences, Liaoning Normal University, Dalian, 116029, Liaoning, China; Southern Laboratory of Ocean Science and Engineering, Guangdong, Zhuhai, 519000, China; Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
College of Life Sciences, Liaoning Normal University, Dalian, 116029, Liaoning, China; Liaoning Key Laboratory of Marine Animal Immunology & Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
Dev Comp Immunol. 2024 Jan;150:105076. doi: 10.1016/j.dci.2023.105076. Epub 2023 Oct 4.
The DM9-containing proteins have been identified as pattern recognition receptors (PRRs) to recognize invading pathogens and subsequently mediate downstream signal pathways, playing essential roles in innate immune responses of molluscs. In the present study, a novel DM9-containing protein (named as CgDM9CP-7) was identified from Pacific oyster Crassostrea gigas, which contained two tandem DM9 repeats similar to the previously identified CgDM9CPs. The mRNA transcripts of CgDM9CP-7 were found to be constitutively expressed in all the tested tissues including haemolymph, gill, hepatopancreas, mantle, adductor muscle and labial palp. The expression level of CgDM9CP-7 mRNA in haemocytes significantly up-regulated at 3 and 6 h after Vibrio splendidus stimulation, which was 5.67-fold (p < 0.01) and 4.71-fold (p < 0.05) of that in the control group, respectively, and it also increased significantly at 6 h (3.08-fold, p < 0.01) post lipopolysaccharide (LPS) stimulation. The protein of CgDM9CP-7 was mainly detected in membrane and cytoplasm of oyster haemocytes after V. splendidus stimulation. The recombinant CgDM9CP-7 protein (rCgDM9CP-7) displayed binding activities to MAN, LPS, PGN, Poly (I:C) as well as gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and fungi (Pichia pastoris and Yarrowia lipolytica). rCgDM9CP-7 was able to agglutinate Bacillus subtilis, V. splendidus, E. coli and S. aureus, inhibit their growth, and bind the recombinant protein CgMyd88-2 (K = 5.98 × 10 M) and CgMyd88s (K = 8.5 × 10 M) in vitro as well. The transcripts of CgIL17-1 (0.45-fold of the control group, p < 0.01), CgIL17-2 (0.19-fold, p < 0.05), CgIL17-3 (0.54-fold, p < 0.05), CgIL17-5 (0.36-fold, p < 0.05) and CgIL17-6 (0.24-fold, p < 0.01) in CgDM9CP-7-siRNA oysters decreased significantly at 6 h after V. splendidus stimulation. These results collectively indicated that CgDM9CP-7 was involved in the regulation of CgMyD88 and CgIL-17 expression in the immune response of oyster.
从太平洋牡蛎（Crassostrea gigas）中鉴定出一种新型含有 DM9 的蛋白（命名为 CgDM9CP-7），其包含两个串联的 DM9 重复序列，类似于先前鉴定的 CgDM9CPs。在所有检测的组织中，包括血淋巴、鳃、肝胰腺、套膜、闭壳肌和唇瓣，均发现 CgDM9CP-7 的 mRNA 转录物呈组成型表达。在灿烂弧菌刺激后 3 和 6 小时，血细胞中 CgDM9CP-7 mRNA 的表达水平显著上调，分别为对照组的 5.67 倍（p < 0.01）和 4.71 倍（p < 0.05），在脂多糖（LPS）刺激后 6 小时也显著增加（3.08 倍，p < 0.01）。在灿烂弧菌刺激后，CgDM9CP-7 蛋白主要在牡蛎血细胞的膜和细胞质中检测到。重组 CgDM9CP-7 蛋白（rCgDM9CP-7）显示与甘露糖（MAN）、脂多糖（LPS）、肽聚糖（PGN）、多聚肌苷酸（Poly（I:C））以及革兰氏阴性菌（灿烂弧菌和大肠杆菌）、革兰氏阳性菌（金黄色葡萄球菌和微球菌）和真菌（毕赤酵母和产朊假丝酵母）结合。rCgDM9CP-7 能够凝集枯草芽孢杆菌、灿烂弧菌、大肠杆菌和金黄色葡萄球菌，抑制它们的生长，并在体外与重组蛋白 CgMyd88-2（K = 5.98×10 -5 M）和 CgMyd88s（K = 8.5×10 -5 M）结合。在灿烂弧菌刺激后 6 小时，CgDM9CP-7 沉默牡蛎中 CgIL17-1（对照组的 0.45 倍，p < 0.01）、CgIL17-2（0.19 倍，p < 0.05）、CgIL17-3（0.54 倍，p < 0.05）、CgIL17-5（0.36 倍，p < 0.05）和 CgIL17-6（0.24 倍，p < 0.01）的转录物水平显著降低。这些结果共同表明，CgDM9CP-7 参与了牡蛎免疫反应中 CgMyD88 和 CgIL-17 表达的调节。
