实验海洋生物学重点实验室知识库

无脊椎动物缺乏适应性免疫，它识别“非己”主要通过模式识别受体 (PRRs) 识别入侵微生物的病原相关分子模式 (PAMPs) 以触发随后的先天免疫应答。本论文利用RACE、实时定量PCR、原核重组表达和RNA干扰等分子生物学技术，对三疣梭子蟹Portunus trituberculatus的C型凝集素 (PtCLec1和PtCLec2) 和纤维蛋白原相关蛋白 (PtFCN1、PtFREP1和PtFREP2) 等PRRs进行结构分析和免疫功能的研究。

C型凝集素 (C-type lectins) 是依赖Ca²⁺的糖识别蛋白的超家族，在先天免疫系统中起模式识别受体的作用。本研究克隆获得2条C型凝集素基因，分别命名为PtCLec1和PtCLec2。PtCLec1的cDNA全长为873 bp编码176个氨基酸，PtCLec2的cDNA全长为1175 bp编码267个氨基酸。PtCLec1和PtCLec2分别含有特异YPD基序和典型QPD基序的糖识别结构域 (CRD) 。PtCLec1主要在肝胰腺中高表达，PtCLec2主要在肠中高表达，且二者在溶藻弧菌、藤黄微球菌和毕赤酵母菌刺激后表达明显上调。重组蛋白rPtCLec1和rPtCLec2可以结合所有检测的PAMPs (脂多糖、肽聚糖和葡聚糖) 和微生物 (革兰氏阴性菌、革兰氏阳性菌和真菌)。在Ca²⁺存在下，rPtCLec1可以很强的凝集细菌和真菌且凝集活性受到D-半乳糖和脂多糖的抑制，rPtCLec2对细菌具有很强的凝集活性但对真菌无作用，且凝集活性受到D-半乳糖、D-甘露糖和脂多糖的抑制。同时，rPtCLec1和rPtCLec2对革兰氏阴性菌和革兰氏阳性菌具有明显的抑菌活性，能够在体外促进对溶藻弧菌的清除活性和在体内促进血细胞的吞噬作用。PtCLec1和PtCLec2基因敲降后均可显著抑制吞噬作用相关基因的表达。PtCLec1基因敲降后能够增强酚氧化酶原激活 (proPo) 系统相关基因、甘露糖结合凝集素 (PtMBL) 和抗菌肽 (AMPs)、以及Toll通路和IMD通路关键基因 (PtMyD88和PtRelish) 的表达。与之相反，PtCLec2基因敲降后proPo系统相关基因、补体系统相关基因 (PtTEP和Ptα2M1)、AMPs和IMD通路关键基因PtRelish表达显著下调。此外，PtCLec2基因敲降后JNK通路关键基因 (PtJNK) 和Toll通路关键基因 (PtTLR和PtPelle) 的表达明显上调。

纤维蛋白原相关蛋白 (Fibrinogen-related proteins，FREPs)，也被称为血纤蛋白原结构域免疫凝集素 (FBNs)，是一类普遍羧基端含纤维蛋白原类似结构域(fibdnogen-1ike FBG domain) 的蛋白。本研究克隆获得3条FREPs基因。PtFCN1的cDNA全长为2019 bp，其氨基酸序列含有一个FBG结构域。PtFREP1和PtFREP2的cDNA全长分别为2428 bp和1475 bp，其氨基酸序列均含有一个FBG结构域和卷曲螺旋结构域 (coiled-coil domain)。PtFCN1主要在胃中的表达量最高，PtFREP1主要在肠中高表达，而PtFREP2主要在肝胰腺和胃中高表达。经副溶血弧菌、金黄色葡萄球菌和白斑综合症病毒 (WSSV) 刺激后，血细胞中的FREPs被不同程度激活。利用RNA干扰技术敲降PtFCN1和PtFREP1基因后，血细胞中溶藻弧菌清除速率明显下降。这些结果表明，FREPs不仅能够作为免疫识别中的模式识别受体，而且能够作为调理素清除病原。

Due to the lack of adaptive immunity, invertebrates discriminate non-self mainly by pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) of invading microorganisms to trigger subsequent innate immune responses. In the present study, C-type lectins (PtCLec1 and PtCLec2) and Fibrinogen-related proteins (PtFCN1, PtFREP1 and PtFREP2) were identified from Portunus trituberculatus through RACE, quantitative real-time PCR, prokaryotic expression system and RNA interference techniques. We further investigated the structural characteristics and immune function of these molecules in crab immune system.

C-type lectins are a superfamily of Ca²⁺-dependent carbohydrate-recognition proteins that function as pattern recognition receptors (PRRs) in innate immune system. In this study, two novel C-type lectin were identified from the swimming crab P. trituberculatus (PtCLec1 and PtCLec2). The full-length cDNA of PtCLec1 was 873 bp encoding 176 amino acids. And the full-length cDNA sequence of PtCLec2 was 1175 bp encoding 267 amino acids. PtCLec1 and PtCLec2 contained a single carbohydrate-recognition domain with a special YPD motif and a typical QPD, respectively. The PtCLec1 and PtCLec2 transcripts were mainly expressed in hepatopancreas and intestine, respectively, and their relative expression levels were significantly up-regulated after the challenges of Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtCLec1 and PtCLec2 (rPtCLec1 and rPtCLec2) could bind all the tested pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and microorganisms, including V. alginolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, M. luteus and P. pastoris. The rPtCLec1 also exhibited strong activity to agglutinate bacteria and yeast in a Ca²⁺-dependent manner. The rPtCLec2 could agglutinate all the tested Gram-negative bacteria and Gram-positive bacteria in the presence of Ca²⁺, but exhibited no agglutination activity against fungi. The agglutinating activity of rPtCLec1 could be inhibited by D-galactose and LPS, and rPtCLec2 could also bind D-mannose. Moreover, rPtCLec1 and rPtCLec2 revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria, and promoted the clearance of V. alginolyticus in vivo and hemocyte phagocytosis in vitro. Knockdown of PtCLec1 and PtCLec2 could down-regulate the expression of phagocytosis-related genes, but the expression levels of prophenoloxidase (proPO) system-related genes, mannose-binding lectin (PtMBL), antimicrobial peptides (AMPs), Toll and IMD pathways key genes (PtMyD88 and PtRelish) were significantly enhanced in the PtCLec1-knockdown crabs. On the contrary, knockdown of PtCLec2 could significantly suppressed the expression of proPO system-related genes, complement-like genes (PtTEP and Ptα2M1) and AMP genes, as well as the key gene PtRelish involved in the IMD pathway. Otherwise, the expression of JNK and Toll signaling pathway key genes PtJNK, PtPelle and PtTLR were remarkably enhanced after knockdown of PtCLec2.

Fibrinogen-related proteins (FREPs), also known as fibrinogen-like domain immunolectins (FBNs), have a fibrinogen-related domain (FReD). In this study, we cloned three new FREPs from P. trituberculatus. The complete cDNA of PtFCN1 was 2019 bp encoding a FBG domain. The full-length cDNA of PtFREP1 and PtFREP2 were 2428 bp and 1475 bp, respectively, both encoding a FBG domain and a coiled-coil domain. PtFCN1 transcript was mainly detected in stomach. PtFREP1 was highest expressed in intestine, and PtFREP2 was mainly expressed in hepatopancreas and stomach. The temporal expression of FREPs in hemocytes showed different activation times after challenged with V. parahaemolyticus, S. aureus and WSSV. The clearance rate of V. alginolyticus in hemocytes was significantly decreased after PtFCN1-siRNA and PtFREP1-siRNA injection. All these results indicate that these FREPs might act as PRR in immune recognition and opsonin in pathogen elimination.