大黄鱼免疫相关基因的筛选与鉴定

Abstract

<DIV align=left>大黄鱼是我国特有的经济鱼种，由于人工养殖技术的成熟使其成为目前网箱养殖量最大的经济鱼类，但是随着养殖量的增加和养殖环境的破坏，，导致大面积细菌性疾病和病毒性疾病的爆发，给大黄鱼的养殖业造成了巨大的损失。为了更好了解研究大黄鱼的免疫系统为将来的大黄鱼养殖的疾病防治提供有效支持，我们进行如下的工作：</DIV> <DIV align=left>1. 构建了polyIC诱导的CDNA SMART文库，并进行了1039个EST随机测序，发现了252个基因，其中包括了46个免疫相关基因，这些免疫相关基因广泛地参与了免疫应答包括补体系统，干扰素系统，抗原加工提呈系统，免疫球蛋白等重要免疫亚系统，而且还首次报道了hepcidin的切割位点的多态性，采用半定量PCR分析几个免疫相关基因发现Mx protein, β2m, placenta-specific 8 genes, Cyba 和CDBP2在poly IC诱导之后会上调表达，初步表明这些基因参与了大黄鱼的免疫应答中。</DIV> <DIV align=left>2. 克隆分析γ-干扰素诱导的溶酶体硫还原酶（GILT）基因，GILT是目前在哺乳动物中唯一发现的可以在溶酶体的酸性环境中催化还原二硫键的硫还原酶，它催化还原二硫键从而帮助蛋白去折叠使之容易被溶酶体中的蛋白酶降解加工成抗原肽。大黄鱼的GILT CDNA全长1033bp，编码256个氨基酸，理论分子量为29.3KD，在NCBI上进行比对分析表明，它分别与斑马鱼、人、小鼠、大鼠、狗的GILT蛋白有着54.1%, 43.2%,40.0%, 39.4%, and 39.2%同源性。初步序列结构分析表明所克隆到的GILT基因含有一个保守的CXXC（74~77）motif，一段gilt特征序列CQHGX2ECX2NX4C（119~134），六个重要的半胱酸，两个N-糖基化位点。根据这些序列资料可以初步判断所克隆到的基因是哺乳动物GILT的同源蛋白。进一步的基因组结构分析表明大黄鱼的GILT基因组结构与哺乳动物的GILT基因组结构具有一样内含子/外显子结构。利用三联细菌疫苗刺激大黄鱼分析GILT的表达调控模式发现，GILT在三联细菌疫苗刺激之后在脾脏和肾脏上调表达，而在肝、脑、鳃、心脏只有组成型表达，这个结果进一步确定了GILT参与了大黄鱼的免疫应答。通过搜索NCBI数据库我们找到的pufferfish的BAC文库中一个克隆含有GILT的全基因，继续在这个序列分析发现在GILT转录起始位点上游有两个GAS位点（TTCNNNGAA 和TCCNNNGAA）。GAS位点是γ-干扰素通过JAK-STAT信号传导通道激活目标基因表达的重要启动子，这个发现更进一步说明了鱼类GILT基因具有与哺乳动物中的GILT基因相似作用，也说明了γ-干扰素的JAK-STAT信号传导通道在鱼类也是保守的。</DIV> <DIV align=left>3. 克隆分析了大黄鱼G-型溶菌酶基因，大黄鱼G-型溶菌酶的CDNA全长716bp，编码193个氨基酸，理论分子量21KD，在NCBI上进行比对分析表明它与其他物种的G-型溶菌酶相似度为40~85%。初步的序列结构分析表明大黄鱼溶菌酶具有保守的酶催化位点（E71,D84,D101）和酶结合位点（L97,L121,L128,G152），基因组结构分析它具有和其他鱼类的相似的内含子/外显子结构，表达调控分析表明大黄鱼G-型溶菌酶在小肠，心脏，肝脏，脾脏，肾脏，鳃当中都有组成形表达，在三联疫苗刺激之后溶菌酶在小肠，脾脏，肾脏中表达量上调8~10倍，而在其他的组织只有波动表达，这个结果说明了G-型溶菌酶在天然免疫应答中重要的作用。另外采用甲醇诱导的毕氏酵母表达系统体外表达溶菌酶，并检测体外表达的 G-溶菌酶的溶菌活力，发现它对革兰氏阳性菌溶壁微球菌(<I>M.lysodeikticus</I>)，以及革兰氏阴性菌温和气单胞菌(<I>A.sobria</I>)，溶藻弧菌(<I>V. alginolyticus</I>)，副溶血弧菌(<I>V. parahae molyticus </I>)，创伤弧菌(<I>V.vulnficus</I>)均有溶菌作用，这些革兰氏阴性菌都是主要的水产养殖病害菌，这些结果为将来水产养殖疾病防治奠定了良好的基础。</DIV>

<DIV>Large yellow croaker (<I>Pseudosciaena crocea</I>), is a species of jewfish and is found mainly in the coast in the temperate zone. Large yellow croaker is an economically important marine fish species in China, and also represents the largest yield for a single species in Chinese marine net-cage farming. In recent years, with the rapid development of large yellow croaker culture industry, the infectious diseases caused by viruses, bacteria, and parasites are becoming more and more severe, resulting in great economical losses. At present little is known about the genetic and immunological basis of this fish. This lack of knowledge may represent a major obstacle that hinders the establishment of effective measures in disease control and genetic improvement.</DIV> <DIV>To better understand the molecular mechanism of the immune system of large yellow croaker and increase genomic resources in cultured fish, we constructed a cDNA library from mRNA isolated from the spleens of large yellow croaker stimulated with a viral micmic，polyinosinic polycytidynic acid (poly I: C). 1039 ESTs from the library were sequenced and compared with sequences in GenBank, and 252 genes were identified by EST analysis, of which 46 genes may be implicated in the immune functions, including complement system components, immunoglobulins, antigen processing and presentation proteins, interferon system proteins, cytokines, and some innate defense molecules. The expression analysis of selected genes during polyI:C induction was performed by reverse transcription-PCR (RT-PCR), including Mx protein, beta2-microglobulin (b<SUB>2</SUB>m), CD2 binding protein 1(CD2BP1), placenta-specific 8 genes, MHC class II associated invariant chain (li) and Cytochrome b-245 alpha peptide (Cyba). The results revealed that expression levels of Mx protein, b<SUB>2</SUB>m, placenta-specific 8 genes, and Cyba were significantly upregulated at 30h after induction with poly I:C, and the CD2BP1expression was also induced by polyI:C, suggesting that these genes may be involved in an immune response induced by poly I:C in large yellow croaker. In this study we also report two novel immune-related genes (CD2BP and placenta-specific 8 gene) and the presence of polymorphism at the maturation site of hepcidin.</DIV> <DIV align=left>Based on the work of large yellow croaker EST project, we further clone and analyze interferon-γ-inducible-lysosomal thiol reductase (GILT) gene of large yellow croaker. In mammals, GILT has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. Here, we reported the cloning of a GILT gene homologue from the spleen of large yellow croaker(LycGILT). The full length cDNA of LycGILT gene is 1033 nucleotides (nt) encoding a protein of 256 amino acids (aa)，with a putative molecular weight of 28.9 kDa. The deduced protein is highly homologous to that of mammalian and zebrafish GILTs and shares 54.1% sequence identity to that of zebrafish and 43.2%-39.2% sequence identity to that of various mammals. The deduced LycGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX2ECX2NX4C, and other six cysteines responsible for the formation of disulfide bonds in the C-terminus. Genomic analysis revealed that LycGILT gene, spanning a 3159 nt fragment, contained seven exons interrupted by six introns and exhibited a similar exon-intron organization to human and mouse GILT genes except for a slightly more compact intron arrangement. The LycGILT expression is obviously up-regulated in spleen and kidney after immunization with inactivated trivalent bacterial vaccine consisting of Vibrio alginolyticus, Vibrio paraphaemolyticus, and Aeromonas hydrophila although it is also constitutively expressed in liver, gills, brain, and heart, suggesting that LycGILT may be involved in the immune response to bacterial challenge in large yellow croker. A search of NCBI sequence data with LycGILT cDNA identified a pufferfish (fugu rubrides) GILT homologue cDNA and its genomic DNA sequence, where two putative interferon-γ activation sites (GAS) were found within the promoter region. This provided evidence that a fish GILT homologue like mammalian GILT, may also be regulated by interferon-γ (IFN-γ ) through the JAK-STAT signal pathway. These results indicate that the bony fish GLIT is a functional homologue of mammalian GILT.</DIV> <DIV>Finally we clone and characterize G-type lysozyme from large yellow croaker (lyc-g-lys). the full length of lyc-g-lys is 716bp encoding a protein of 193 aa, with a putative molecular weight of 21kd.The deduced protein show 40~85% homologous to other speice. The deduced amino acid squence possesses the typical structural feature of other G-type lysozyme including catalytical residues (E71,D84,D101) and substrate binding site(L97,L121,L128,G152). Genomic analysis revealed that lyc-g-lys gene has similar exon-intron organization to other bony fish G-lysozyme gene. Expression analysis by relative quantification real time PCR shown that g-lys was unregulated about 8~10 fold in instine, spleen and kidney after the trivalent bacterial, vaccine while the expression of G-lys in other organ was not change obviously, these results indicating complexity of G-lysozyme regulation mechanism and the important role of G-lysozyme in the innate immunity. The recombinant large yellow croaker g-lysozyme expressed by yeast shown lytic activity against <I>M.lysodeikticus</I>，<I>A.sobria</I>，<I>V. alginolyticus</I>，<I>V. parahae molyticus</I>，<I>V.vulnficus</I>.</DIV>

学位：理学硕士

院系专业：生命科学学院生物学系_生物化学与分子生物学

学号：200326140