【作者】 金兴坤；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 动物学， 2014， 博士

【摘要】 中华绒螯蟹是我国重要的养殖经济动物品种,是长江流域居民的重要传统食物。随着对其集约化养殖的大规模发展,由细菌,病毒或立克次氏体引起的各种疾病随之大规模爆发,造成了巨大的经济损失。因此针对该物种先天免疫系统以及生物防御机制的深入研究刻不容缓。先天免疫和适应性免疫(获得性免疫)是动物宿主(Host)防御病毒、细菌和寄生虫等病原体(Pathogen)的两种免疫防御形式,两者间本质的区别在于：适应性免疫包括抗原特异性识别和免疫记忆,而先天免疫则没有。目前认为,先天免疫存在于所有后生动物中,而适应性免疫则仅存在于高等脊椎动物。无脊椎动物先天免疫“非己”识别的基础是其体内存在某些模式识别受体(PRRs),能够特异性地识别并结合病原体表面保守的病原相关分子模式(PAMPs),如脂多糖(LPS)、肽聚糖(PGN)和葡聚糖(GLU)等。通过识别PAMPs,这些受体可以激活免疫系统中的蛋白酶以及通过免疫细胞内的信号转导途径引发免疫反应。本研究通过RNA-Seq高通量二代测序技术,构建了中华绒螯蟹血细胞经病原相关模式分子差异诱导后的转录组文库,从中筛选得到了无脊椎动物重要的模式识别受体分子。其中C型凝集素蛋白(C-type lectin domain,CTLD containing proteins)家族基因具有庞大的基因组分子多样性(至少存在40种以上的亚型分子),从而能够在蛋白质水平产生丰富的功能多样性。同时,本研究还发现单个免疫基因能够通过转录水平的外显子可变剪切调控,从而使得单个免疫分子产生数以万级的分子多样性,例如免疫球蛋白超家族基因Dscam,并针对其在中华绒螯蟹先天免疫系统中的重要功能进行了深入研究。针对无脊椎动物模式识别受体C型凝集素在甲壳动物中的分子多样性和特异性免疫功能,我们基于E.sinensis进行了以下研究。通过本实验室成功构建的一系列基于RNA-Seq的表达序列标签cDNA文库以及血细胞转录组文库,我们克隆得到四条C型凝集素基因,分别是EsLecA、EsLecF和EsLecG。通过序列比对和聚类分析发现上述基因序列均具有一个N端信号肽和一个C端糖类识别功能域(Carbohydrate Recognition Domain)。转录水平检测结果表明(1)EsLecA和EsLecG在所有检测组织中均表达；(2)经脂多糖(LPS)诱导后,EsLecA和EsLecG在肝胰腺、鳃和血细胞等免疫器官中均有不同程度的诱导表达；而EsLecF仅在肝胰腺中特异性表达,并且经LPS诱导后能够大量表达。原核表达重组rEsLecA、rEsLecF和rEsLecG蛋白能够广泛结合不同的病原微生物,包括革兰氏阳性菌、革兰氏阴性菌和真菌,并且其微生物结合活性是钙离子不依赖的。病原微生物生长抑制检测和抗菌活性检测结果表明rEsLecA、rEsLecF和rEsLecG蛋白均能够不同程度地抑制细菌生长甚至直接杀死细菌。体外细胞包裹实验验证了rEsLecA、rEsLecF和rEsLecG蛋白均具有不同程度的细胞调理活性,并能够在体外刺激血细胞的包裹活性。通过上述研究,初步阐明了中华绒螯蟹C型凝集素具有核酸水平的分子多样性和蛋白水平的功能多样性,为甲壳动物乃至其他无脊椎动物先天免疫特异性分子机制的研究提供了重要参考。近年来,无脊椎动物先天免疫的特异性水平及其免疫记忆受到了极大关注,“替代适应性免疫”(Alternative adaptive immunity)在节肢动物、软体动物以及棘皮动物等无脊椎动物中的发现,使得“适应性免疫是脊椎动物免疫的一种专有特征”这一传统观点受到了严峻挑战。节肢动物Dscam基因可通过外显子选择性剪切方式产生惊人的分子多态性从而特异性地识别不同的病原体。我们从大量转录组信息的分析中发现并成功克隆得到了中华绒螯蟹Dscam基因(EsDscam),通过生物信息学比对分析发现其由典型功能域组成,包括1个信号肽(Signal-peptide),10个免疫球蛋白功能域(Immunoglobulin,Ig domains),6个三型纤连蛋白功能域(Fibronectin type Ⅲ domains,FNⅢ),一个跨膜功能域(Transmembrane domain, TM)和一个细胞质尾(Cytoplasmic tail)。在EsDscam中我们通过TA克隆测序技术发现了4个可变剪切区：Ig2功能域的N端部分(25个亚型),Ig3功能域的N端部分(30个亚型),Ig7功能域(18个亚型)和跨膜功能域(2个亚型),至少可以产生27000种不同的亚型。EsDscam在E.sinensis的所有组织中均广泛表达,尤其是在免疫系统、消化系统和神经系统中表达最高。使用脂多糖(LPS)、肽聚糖(PGN)和葡聚糖(GLU)分别对E.sinensis进行体内免疫刺激后,EsDscam基因在血细胞中表现出了差异诱导表达模式。通过RACE-PCR技术,我们发现EsDscam具有两种表达类型,分别是跨模型(Transmembrane或Membrane-bound)和分泌型(Secreted或Cytosolic).且与跨模型相比,分泌型在不同模式分子(PAMPs)刺激后显著表达。免疫荧光定位结果证明EsDscam蛋白定位在中华绒螫蟹血细胞表面。本研究初步阐明中华绒螯蟹Dscam基因能够通过外显子可变剪切产生数以万级的分子多样性及其经病原刺激后的差异诱导剪切现象,为后续针对其免疫识别的功能多样性研究提供数据支持,为中华绒螯蟹乃至甲壳动物疫苗的研发提供了一定的理论基础。更多还原

【Abstract】 The freshwater Chinese mitten crab, Eriocheir sinensis (Henri Milne Edwards1854), is a traditional savory food especially in the Yangtze River Area, and comprises one of the most economically important freshwater aquatic species of China. With the development of intensive aquaculture, various diseases caused by bacteria, viruses or rickettsia-like organisms have also begun to emerge and have resulted in enormous economic loss. Therefore, an improved understanding of the innate immunity of this particular crab and its bio-defense mechanisms has become a research priority. In the absence of an adaptive immune system, which in the higher vertebrates is characterized by the vast diversity of somatically rearranged immunological receptors like antibodies, invertebrates including E. sinensis rely solely on innate immune mechanism to alleviate the long lasting selective pressure from various kinds of parasites. The most straightforward basis for immune specificity in these organisms relies on the genetic diversity of pattern recognition receptors (PRRs) and/or immune effectors. In my PhD project, I firstly constructed comparative transcriptomic libraries using Next Generation Sequencing approach (e.g. via Roche454and Illumina), subsequently carried out sequence alignments and annotations. Aiming to dissect the molecular mechanisms of invertebrate specific immune responses, I focused on the function of some important PRRs in crustaceans such as the C-type lectin domain-(CTLD) containing proteins which were identified to be abundant in immune organs or cells like the hepatopancreas or hemocytes with highly molecular diversity (40isoforms at least). I also studied other pattern recognition molecules such as Dscam, which was detected to have surprisingly molecular diversity using exons alternative splicing, acted as important role in the innate immune system of E.sinensis.As an important member of PRRs, CTLD containing proteins exist as either transmembrane receptors or soluble proteins in circulating fluids. They play crucial roles in innate immunity such as non-self-recognition and clearance of invading microorganisms, via recognizing and non-covalently binding to specific sugar moieties and agglutinating pathogens by binding to cell surface glycoproteins and glycoconjugates. Based on our previously performed transcriptomic analysis using large-scale RNA-Seq (plus unpublished data from differentially expression transcriptomes from hemocytes), we successively obtained numerous sequences with identical CTLDs inclusions (approximately40unique sequences). Subsequently, we performed a set of in-vivo immuno-challenge tests of these CTLDs (to be continued) using Lipopolysaccharide (LPS:an endotoxin from Gram-negative bacterial Escherichia coli) as a stimulus. Real-time quantitative PCR results revealed that:i) two of these CTLDs (EsLecA and EsLecG) were immune-inducible in all tested immune organs/cells such as hepatopancreas, gills and hemocytes; and ii) an additional CTLD (EsLecF) was immune-inducible in hepatopancreas only. To dissect their exact roles in innate immunity, we performed a series of in-vitro tests with the respective recombinant CTLD proteins, which were generated using prokaryotic expression systems combined with affinity chromatography. The in-vitro protein-level analysis demonstrated that, these selected three CTLD proteins consistently expressed wide spectrum microbial binding activities and could all differentially induced microbe aggregation. Surprisingly, their microbial-binding and microbial-aggregation activities were calcium-independent. Moreover, microbial growth inhibitory and also antibacterial activity assays revealed the proteins’abilities of suppressing microbial growth and directly killing microbes respectively. Furthermore, an encapsulation assay demonstrated an opsonic function of these CTLD proteins.As discussed above, invertebrates lack adaptive immunity which can mediate both immune specificity and memory. At the same time, invertebrate taxa are known from phenotypic analysis to express high levels of immune specificity which possibly depends on some diversified protein family involved in pathogen recognition or elimination. The Dscam proteins (Down syndrome cell adhesion molecule) belong to the immunoglobulin super family (IgSF) and are of particular interest in this context because of their diversified domain architecture. We were able to successfully isolate and characterize the first crab Dscam from E. sinensis. EsDscam shows the typical Dscam domain architecture, including one signal-peptide,10immunoglobulin (Ig) domains,6fibronectin type III domains (FNIII), one transmembrane domain (TM) and one cytoplasmic tail. It was detected to have four hypervariable regions in the N-terminal halves of Ig2(25) and Ig3domain (30), the complete Ig7(18) and also the transmembrane domain (2), thus potentially generating at least27,000unique isoforms through exons alternative splicing mechanisms. Transcription of EsDscam was both i) detected in all tissues, especially those of the immune system, digestive system and nerve system; and ii) significantly induced in hemocytes upon exposure to pathogen surface molecules, e.g. LPS, peptidoglycans (PGN) and β-1,3-glucans (Glu) injection. Importantly, both membrane-bound and secreted Dscam isoforms were expressed in E. sinensis. Moreover, the secreted isoforms were extensively transcribed upon challenge with different pathogen molecules. Results from immuno-localization assay revealed that EsDscam evenly distributed on the cell surface of hemocytes. These findings strongly suggest that EsDscam is a hypervariable PRR in the innate immune system of the E. sinensis.更多还原