【作者】 张峘；

【导师】 宋林生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 补体系统作为固有免疫的组成部分和免疫效应系统,在无脊椎动物防御过程中发挥了重要作用。但目前对无脊椎动物补体样系统的研究刚刚起步,对软体动物补体样成分的研究几近空白。本研究采用RACE、RT-PCR和基因原核重组表达等分子生物学技术开展了对栉孔扇贝(Chlamys farreri)含硫酯蛋白(thioester-containing protein, CfTEP)、含C1q结构域蛋白(C1 q-domain-containing protein, CfC1qDC)、C型凝集素(C-type lectin, Cflec-3, Cflec-4和Cflec-5)和海湾扇贝(Argopecten irradians) C型凝集素(AiCTL-6)、纤维蛋白原相关蛋白(fibrinogen-related protein, AiFREP)等基因的克隆、表达和功能研究。CfTEP、CfC1qDC、Cflec-3、Cflec-4、Cflec-5、AiCTL-6和AiFREP的cDNA全长分别为4616、777、2256、2080、1412、1063和990 bp。它们的开放阅读框编码的蛋白均含有一段信号肽,并分别含有α2-巨球蛋白结构域、C1q结构域、三个或四个或一个C型凝集素结构域和纤维蛋白原样结构域。在健康扇贝中,CfTEP和Cflec-4仅在健康扇贝的肝胰腺和性腺中表达,而其它基因在各个检测组织中均有表达。鳗弧菌刺激可显著诱导这些基因的表达。CfTEP基因组DNA通过转录后选择性剪接可形成七种不同的转录本,它们在雌雄扇贝性腺中对不同细菌的刺激呈现出不同的表达模式。原核重组的CfC1qDC蛋白具有较强的脂多糖结合活性；重组的Cflec-3蛋白对施氏假单胞菌具有钙离子依赖的、甘露糖结合型的凝集作用；重组的Cflec-5蛋白对毕赤酵母具有钙离子非依赖的、甘露糖结合型的凝集作用；重组的AiCTL-6蛋白对大肠杆菌、藤黄微球菌和金黄色葡萄球菌具有钙离子依赖的凝集作用；重组的AiFREP蛋白对人、鸡血红细胞以及鳗弧菌、大肠杆菌和藤黄微球菌具有钙离子依赖的凝集活性,并且其凝血活性可被含乙酰基的糖类所抑制。本研究结果表明,CfTEP、CfC1qDC、Cflec-3、Cflec-4、Cflec-5、AiCTL-6和AiFREP作为扇贝固有免疫中重要的模式识别受体参与了扇贝对外源微生物刺激的响应。它们在分子结构和功能上与高等动物补体系统中的C3、C1q、甘露糖结合凝集素和ficolin等成分有一定相似性,有可能作为它们的一种原始形式存在于低等软体动物中。该研究结果为进一步描绘扇贝中存在的简单原始的补体系统及揭示补体系统的进化脉络提供了分子生物学证据,同时对深入了解扇贝免疫防御机制,丰富和发展海洋无脊椎动物免疫学内容也具有重要理论意义。更多还原

【Abstract】 Complement system, as the important part of innate immunity and one of the major effecter arms of immune responses, plays a critical role in innate immune defense of invertebrates. The study on invertebrate complement-like system is still on the beginning and the information about complement-like molecules in mollusks remains deficient. In the present study, Chlamys farreri thioester-containing protein (CfTEP), Clq-domain-containing protein (CfC1qDC), C-type lectins (Cflec-3, Cflec-4 and Cflec-5) and Argopecten irradians C-type lectin (AiCTL-6) and fibrinogen-related protein (AiFREP) were studied by molecular biotechnologies including RACE, RT-PCR and recombinant protein expression. The full-length cDNA sequences of CfTEP, CfC1qDC, Cflec-3, Cflec-4, Cflec-5, AiCTL-6 and AiFREP were of 4616,777,2256,2080,1412,1063 and 990 bp, respectively. The open reading frame (ORF) encoded a polypeptide consisted of a signal peptide, with anα2-macroglobulin domain in CfTEP, a Clq domain in CfClqDC, three or four carbohydrate recognition domain (CRD) in Cflec-3, Cflec-4, one CRD in Cflec-5 or AiCTL-6, and a fibrinogen-related domain in AiFREP. In healthy scallops, CfTEP and Cflec-4 mRNA was only expressed in hepatopancreas and gonad, while others could be found in all the tissues detected. All of their mRNA expression levels could be remarkably up-regulated by Listonella anguillarum challenge. The genomic DNA of CfTEP consisted of 40 exons and 39 introns, and seven different transcripts were produced by alternative splicing. These transcripts displayed different expression pattern in gonads in response to different bacterial challenges. The recombinant CfClqDC protein displayed a significantly strong activity to bind LPS. The recombinant Cflec-3 agglutinated Pseudomonas stustzeri in the calcium ions-dependent D-mannose-binding manner. The recombinant Cflec-5 agglutinated Pichia pastoris GS115 in the calcium ions-independent D-mannose-binding manner. The recombinant AiCTL-6 agglutinated bacteria Escherichia coli TOPI OF’, Micrococcus luteus and Staphylococcus aureus. And the recombinant AiFREP agglutinated not only bacteria L. anguillarum, E.coli JM109 and M. luteus, but also chicken and human erythrocytes. These activities were calcium ions-dependent and could be inhibited by acetyl group-containing carbohydrates.These results collectively suggested that these molecules were involved in the immune response against microbe infections and functioned as pattern recognition receptors in the innate immune system of scallops. They shared structural and functional similarity with the mammalian complement components, including C3, Clq, MBL and ficolin, and they might be the ancestor forms of these molecules contributing to nonself-recognition in invertebrates. It would provide clues for elucidating the evolution origin of the complement components and contribute to further study on the primary and simple complement-like system in scallops. Meanwhile, it would be helpful for understanding the scallops’immune defense mechanisms and provide new information for the enrichment and development of marine invertebrate immunology.更多还原