【作者】 罗展；

【导师】 相建海；

【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】 C-型凝集素几乎存在于所有生物体内。作为一种模式识别受体,C-型凝集素参与“非己”识别和对入侵病原的清除过程,在无脊椎动物先天性免疫防御中发挥了重要的作用。对于甲壳动物C-型凝集素的研究在近几年逐渐开展起来。本研究采用Real-time PCR、原核重组表达、RNA干扰等分子生物学技术,对凡纳滨对虾的两种C-型凝集素（Lvlectin1和Lvlectin2）的生物学功能进行了研究。本课题组对NCBI中公布的16万余条凡纳滨对虾ESTs进行了拼接、注释和聚类分析。在此基础上,本文作者重点对其中注释为lectin的序列进行了分析,并从中选取了注释为远海梭子蟹（Portunus pelagicus）C-type lectin receptor和中国明对虾（Fenneropenaeus chinensis） C-type lectin 3的2个contig（分别命名为Lvlectin1和Lvlectin2）进行了深入研究。Lvlectin1基因开放阅读框（ORF）为510 bp,编码169个氨基酸,其中N末端有19个氨基酸的信号肽,在C末端有一个CRD结构域。Lvlectin2基因包含一个474 bp的开放阅读框,编码157个氨基酸,N末端有17个氨基酸的信号肽,C末端有一个CRD结构域,该结构域中具有决定糖结合特异性的基序为“EPS”（Glu120-Pro121-Ser122）序列。在健康凡纳滨对虾中,Lvlectin1和Lvlectin2基因都主要在肝胰脏中表达。Real-time PCR结果显示,分别利用LPS、灭活溶壁微球菌和WSSV免疫刺激对虾后,对虾肝胰脏中Lvlectin1和Lvlectin2基因在转录水平都有明显的变化但表达模式不同。利用生物信息学对Lvlectin1基因编码的氨基酸序列进行分析,发现在Lvlectin1的CRD结构域中不具有与半乳糖或甘露糖结合相关的特征序列“QPD”或“EPN”,为了验证其是否具有凝集活性,对Lvlectin1成熟肽进行了体外原核重组表达,并对纯化的重组蛋白进行了活性分析,结果发现rLvlectin1蛋白对溶壁微球菌和鳗弧菌表现出较强的凝集效应。rLvlectin1蛋白的凝菌活性具有Ca2+依赖性,并且其凝集活性可被D-半乳糖、D-葡萄糖、D-甘露糖和N-乙酰甘露糖胺所抑制。结果充分证明了Lvlectin1是一种Ca²⁺依赖型的C-型凝集素。为进一步了解Lvlectin1基因在对虾防御白斑综合症病毒（WSSV）侵染中的作用,利用RNA干扰技术沉默对虾Lvlectin1基因的表达,并用WSSV病毒提取液进行了对虾感染病毒的实验。结果显示,Lvlectin1基因的沉默显著降低了凡纳滨对虾对WSSV的防御能力,半数死亡时间明显比对照组提前,同一时间点的死亡率较对照组偏高。利用PCR筛选法在L. vannamei基因组BAC文库中筛选到4个含有Lvlectin1基因的BAC克隆,通过进行BAC克隆测序将有助于获得Lvlectin1基因相关的基因组结构、启动子和调控元件等信息。本研究结果表明,Lvlectin1可能作为凡纳滨对虾先天性免疫中重要的模式识别受体,参与了凡纳滨对虾对细菌和WSSV的防御。更多还原

【Abstract】 C-type lectins exist in almost all the animals. As a pattern recognition receptor(PRR), C-type lectin is believed to mediate pathogen recognition and play an important role in the clearance of pathogens in innate immunity. A number of C-type lectins in crustaceans have been isolated and characterized in recent years. In this study, two C-type lectin genes (named Lvlectin1 and Lvlectin2) cloned from shirmp Litopenaeus vannamei were studied by molecular biotechnologies including Real-time PCR,recombinant protein expression and RNA interference(RNAi).The 161, 075 expressed sequence tags of Litopenaeus vannamei published at the NCBI have been jointed, noted and cluster analyzed(data not published) in our lab. The contigs and singletons noted as lectin were analyzed and two C-type lectin fragments (named Lvlectin1 and Lvlectin2) were selected frrom the ESTs assembled library of L. vannamei. Lvlectin1 and Lvlectin2 share high identity with C-type lectin receptor from Portunus pelagicus and C-type lectin 3 from Fenneropenaeus chinensis respectively. The open reading frame of Lvlectin1 is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain (CRD) at the C-terminal. Lvlectin2 gene contains a ORF which is 474 bp. The deduced amino acid sequence of Lvlectin2 is 157 amino acids, containing a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain (CRD) at the C-terminal. There is a potential carbohydrate-bingding motif“EPS”(Glu120-Pro121-Ser122) presented in the CRD may support its ability to bind mannose-type sugars. In healthy shrimp L. vannamei, Lvlectin1 and Lvlectin2 were both mainly expressed in hepatopancreas. Real-time PCR analysis indicated that Lvlectin1 and Lvlectin2 transcripts level show significant change in hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus(WSSV).According to the deduced amino acid sequence analysis of Lvlectin1, there was no key motif EPN or QPD which is predicted to bind to mannose or galactose in its CRD. The function of Lvlectin1 was investigated by recombinant expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21(DE3). The recombinant Lvlectin1 could agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-galactose, D-Glucose, D-mannose and N-Acetyl-D-mannose.In order to study the antiviral function of Lvlectin1, RNAi-based silencing of Lvlectin1 gene resulted in significantly increasing mortality rate when the shirmp were challenged with WSSV, and the median lethal time was much earlier than the control.Four BAC clones containing Lvlectin1 gene were screened with special primers for Lvlectin1 from the Bacterial Artificial Chromosome (BAC) Library of Litopenaeus vannamei, which will be utilized to research genomic characters, transcriptional promoter and regulating element of Lvlectin1.These results suggested that Lvlectin1 was involved in the immune response against bacterial and WSSV infections and contributed to nonself recognition as a pattern recognition receptor in the innate immune system of shrimp L.vannamei.更多还原