实验海洋生物学重点实验室知识库

目前有关蟹类免疫防御的研究大多数都集中在成体，胚胎如何保护自己免受病原体攻击这一发育免疫学中的关键问题却鲜有报道。本研究报道了我国重要的水产养殖蟹类三疣梭子蟹（Portunus trituberculatus）早期胚胎的免疫功能并探讨其免疫机制。实验表明，三疣梭子蟹受精卵蛋白提取液对革兰氏阴性菌溶藻弧菌、革兰氏阳性菌藤黄微球菌和金黄色葡萄球菌、真菌毕赤酵母菌的生长过程具有明显的抑制作用，而对革兰氏阴性菌铜绿假单胞菌和大肠杆菌抑制效果不明显或无抑制效果。三疣梭子蟹受精卵蛋白提取液对溶藻弧菌、大肠杆菌以及毕赤酵母菌具有明显的凝集活性和损伤作用，而对铜绿假单胞菌、藤黄微球菌和金黄色葡萄球菌无凝集和损伤作用。酶活实验表明，溶菌酶和Cu/Zn-SOD的活力随胚胎发育时期呈现逐渐上升的趋势。ELISA实验结果表明，补体系统的关键分子C3和C4存在于受精卵中，与以上酶活实验结果不同，C3和C4随胚胎发育时期呈现先下降后上升趋势且受精卵和卵裂期含量明显高于囊胚期，推测其可能是重要的母源免疫分子。补体凝集素途径的激活需要Ca²⁺参与，金属离子螯合实验显示，在受精卵蛋白提取液中添加Ca²⁺螯合剂后，其抑菌活性明显降低，而补充足量的Ca²⁺后，其抑菌活性在一定程度上得到了恢复，相反抑制替代途径再补充其所需的Mg²⁺不能使抑菌活性得到恢复。以上研究结果表明，三疣梭子蟹的受精卵蛋白提取液具有抑菌、凝菌和菌损伤活性，溶菌酶、Cu/Zn-SOD和补体凝集素途径在早期胚胎的免疫保护中发挥重要作用。

补体凝集素途径关键分子甘露糖结合凝集素（MBL）和纤维胶凝蛋白（ficolin）作为关键的模式识别受体（PRR）在先天免疫反应中起重要作用。本研究从三疣梭子蟹中克隆出了一种新的甘露糖结合凝集素（命名为PtMBL）和新的纤维胶凝蛋白（命名为Ptficolin）。PtMBL基因的cDNA全长为1208bp，开放阅读框（ORF）长度为732bp，编码含有244个氨基酸的蛋白质。经过多序列比对显示PtMBL与其他物种中的MBL具有较低的氨基酸相似性，但它含有具有QPD基序的保守碳水化合物识别结构域（CRD）。PtMBL主要在眼柄和鳃中检测到且经溶藻弧菌，藤黄微球菌和毕赤酵母菌刺激后，血细胞中PtMBL被不同程度的激活。重组PtMBL蛋白显示出针对革兰氏阴性菌和革兰氏阳性菌的抑菌活性并且可以结合并凝集细菌和真菌（具有Ca²⁺依赖性）。此外，D-半乳糖和D-甘露糖都可以抑制凝集活性，这表明PtMBL具有更广泛的病原体相关分子模式（PAMPs）识别谱。Ptficolin基因的cDNA全长为2183bp，开放阅读框（ORF）长度为963bp，编码含有321个氨基酸的蛋白质。经过多序列比对显示Ptficolin与其他无脊椎动物中的ficolin具有较低的氨基酸相似性，但与其他已经鉴定出的无脊椎动物的ficolin具有保守的信号肽和FBG结构域。Ptficolin主要在肝胰腺、眼柄和胸神经节中检测到且经溶藻弧菌，藤黄微球菌和毕赤酵母菌刺激后，血细胞中Ptficolin可产生快速响应。这些结果共同表明，PtMBL和Ptficolin不仅可以作为免疫识别中的模式识别受体，而且可以作为蟹类的先天免疫反应中潜在的抗菌蛋白发挥作用。

At present, most research focuses on defenses of adult crabs. However, few studies were conducted to elucidate how the developing embryos protect themselves from pathogen attacks before the immune system is fully mature. This study reported the immune function of the early embryos of Portunus trituberculatus, an important aquaculture crab in China, and explored its immune mechanism. The results showed that the extracts of fertilized egg from P. trituberculatus had significant inhibitory effects on the growth of Gram-negative bacteria Vibrio alginolyticus, Gram-positive bacteria Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, but not on Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. The protein extracts from fertilized eggs of P. trituberculatus had obvious agglutinating and damaging effect on V. alginolyticus, E. coli and P. pastoris, but had no agglutinating and damaging effects on P. aeruginosa, M. luteus and S. aureus. The activity of lysozyme and Cu/Zn-SOD increased with the development of embryo. ELISA results showed that the C3 and C4, key molecules of complement system, existed in fertilized eggs. Different from the above enzymatic activity experiments, C3 and C4 decreased first and then increased with the development of embryos, and the content of fertilized eggs and cleavage stage was significantly higher than that of blastocyst stage. It is speculated that C3 and C4 may be important maternal immune molecules. The activation of complement agglutinin pathway requires the participation of Ca²⁺. Metal ion chelating experiments show that the antimicrobial activity of protein extracts of crab decreased significantly after adding Ca²⁺ chelating agent, while the antimicrobial activity of protein extracts was restored after supplementing sufficient Ca²⁺. On the contrary, the antimicrobial activity of crab fertilized oocyte protein extracts could not be restored by supplementing Mg²⁺ by alternative inhibition pathway. The results showed that the protein extracts of P. trituberculatus had inhibiting microbial growth, agglutination and bacterial damage activities. Lysozyme, Cu/Zn-SOD and complement lectin pathways played an important role in the immune protection of early embryos.

The key molecules of the complement lectin pathway, mannose-binding lectin (MBL) and ficolin, are key model recognition receptors (PRRs) that play an important role in the innate immune response. In this study, a new mannose-binding lectin (designated as PtMBL) and a new ficolin (designated as Ptficolin) were cloned from P. trituberculatus. The complete cDNA of PtMBL gene was 1,208 bp in length with an open reading frame (ORF) of 732 bp that encoded 244 amino acid proteins. PtMBL shared lower amino acid similarity with other MBLs, yet it contained the conserved carbohydrate-recognition domain (CRD) domain with QPD motif and was clearly member of the collectin family. PtMBL transcripts were mainly detected in eyestalk and gills. The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria. It could also bind and agglutinate (Ca²⁺-dependent) both bacteria and yeast. Furthermore, the agglutinating activity could be inhibited by both d-galactose and d-mannose, suggesting the broader pathogen-associated molecular patterns (PAMPs) recognition spectrum of PtMBL. The complete cDNA of Ptficolin gene was 2,183 bp in length with an open reading frame (ORF) of 963 bp that encoded 321 amino acid proteins. Ptficolin shared lower amino acid similarity with other invertebrates ficolins, yet it contained the conserved FBG domain. Ptficolin transcripts were mainly detected in hepatopancreas, eyestalk and thoracic ganglia. The temporal expression of Ptficolin in hemocytes showed rapid activation after challenged with V. alginolyticus, M. luteus and P. pastoris. These results together indicate that PtMBL and Ptficolin could serve as not only a PRR in immune recognition but also a potential antbacterial protein in the innate immune response of crab.