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青鳉防御素基因的基因组结构、抗菌活性及免疫调控研究
Alternative TitleStudies on genomic organization, antimicrobial activity and immune modulation of a medaka β-defensin gene
赵久刚
Subtype博士
Thesis Advisor桂建芳
2008-11-24
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword青鳉 防御素 抗菌活性分析 Nf-κb Sp1
Abstract防御素(defensins)是一类在先天性免疫系统中起着重要作用的阳离子抗菌肽。因其在机体抵御病原入侵方面起着重要作用,固命名为防御素。防御素对许多病原微生物有着天然的抵抗活性,包括病毒和细菌。目前,几个跟防御素的转录调控相关的转录因子已经被鉴定,如NF-κB、NF-IL6等。在本论文中,我们主要就青鳉防御素的基因功能及转录调控进行研究。首先,我们运用3′, 5′末端快速扩增法 (Rapid Amplification of cDNA Ends techniques, RACE)在青鳉(Oryzias latipes)眼睛中克隆到一个新的β-防御素基因,命名为OlBD。该基因的cDNA全长480bp,其中5′端和3′端非翻译区(Untranslated region,UTR)分别为68bp和240bp,在3′ 末端还含有一个典型的加尾信号(ATTAA)和26bp的多聚腺嘌呤尾(poly A 尾)。在该cDNA中的189bp 的开放阅读框(open reading frame,ORF) 编码了63个氨基酸(aa)的多肽。Southern blot 结果表明青鳉防御素OlBD基因在青鳉HNI品系里是一个单拷贝基因。运用RT-PCR和Western blot,我们检测了该基因在不同组织中的RNA和蛋白表达谱。研究结果表明,在眼睛、肝脏、肾脏、脾脏、鳃、血细胞中均有OlBD基因的mRNA和蛋白表达。而且其mRNA和蛋白在眼睛中表达丰度最高。免疫组化的结果显示OlBD主要定位在机体跟免疫和抗病相关的组织和细胞中,这个结果表明OlBD在青鳉中的作用也主要是在机体免疫方面。进而,我们分析了该基因编码的蛋白的抗菌活性。首先我们将化学合成的多肽进行体外折叠,并用这种折叠好的多肽进行抗菌活性的测定。共用了六株实验菌株来进行抗菌实验(共包含两株革兰氏阳性菌和四株革兰氏阴性菌),结果表明OlBD的抗菌活性在革兰氏阳性菌和革兰氏阴性菌之间有一种典型的选择性。且革兰氏阴性菌对该基因的蛋白非常敏感。在我们所选择的革兰氏阴性菌之中,有三种是鱼类的强致病菌。接下来我们用活体刺激的方法去检测OlBD基因的免疫应答。在用脂多糖(LPS,Lipopolysacoharide)刺激成体活鱼12小时之后,我们在青鳉鱼的眼睛中检测到防御素mRNA的表达量增加了13倍。采用启动子分析的方法,我们发现该基因启动子的近端的100bp(从+26到-73)跟基因的核心启动活性相关,而相邻的1700bp(从-73 到-1755)则主要负责基因的转录调控。对启动子序列的突变分析我们在5端侧翼序列中发现三个核转录因子kappa B(NF-κB )的顺式元件和1个Sp1顺式元件跟防御素基因OlBD受细菌入侵时所诱导上调表达紧密相关,另外,我们发现转录因子Sp1也跟该基因的核心转录相关。同时我们还发现另外一个转录因子——TFIID在基因转录的起始过程中也起着重要的作用。
Other AbstractDefensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel β-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (ORF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of β-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the β-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka β-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13 fold upregulation of the β-defensin within first 12 hours. In addition, promoter and promoter mutagenesis analysis were performed in the medaka β-defensin. A proximal 100 base pair (bp) sequence (+26 to -73) and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-κB) cis-elements and a sp1 cis-element were revealed by mutagenesis analysis to exist in the 5′ flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka β-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka β-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-κB and Sp1.
Pages150
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12284
Collection学位论文
Recommended Citation
GB/T 7714
赵久刚. 青鳉防御素基因的基因组结构、抗菌活性及免疫调控研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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