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题名: 非洲爪蟾Type II 和Type III 干扰素基因的克隆和表达分析
作者: 齐志涛
答辩日期: 2009-06-04
导师: 聂品
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 干扰素 ; IFN-γ ; IFN-λ ; 肽聚糖识别蛋白 ; 基因 ; 非洲爪蟾
摘要: 干扰素(interferon, IFN)作为重要的抗病毒免疫因子,自1957年首次报道以来,在哺乳类免疫反应中的作用得到阐明。哺乳类中的IFN主要分为三种类型:type I IFN(IFN-α/β)、type II IFN(IFN-γ)和type III IFN(IFN-λ)。近年来,低等脊椎动物如鱼类中的IFN的相关报道逐渐增多。两栖类作为水生脊椎动物向陆生脊椎动物、变温动物向恒温动物进化的过渡类群,在进化生物学上具有十分重要的意义。然而关于两栖类的IFN及其相关基因还知之甚少。因此,本文以两栖类的重要模式动物-非洲爪蟾(Xenopus tropicalis)为研究对象,分别对其type II IFN(xeIFN-γ)和type III IFN(xeIFN-λ)及其受体(xeIFNLR1和xeIL-10R2)进行了克隆、鉴定,同时对其表达模式进行了研究,期望能够阐明两栖类中IFN功能,揭示IFN系统的进化过程。 通过比较生物信息学方法,对非洲爪蟾的IFN-γ基因座中的细胞因子进行了比较分析。在该基因座中包含有三个细胞因子,分别为IFN-γ、IL-22和IL-26。基因同线性分析显示,该基因座中细胞因子的基因排列顺序及转录方向在进化过程中均保持一致。对该基因座中的细胞因子分别进行了克隆,得到的xeIFN-γ cDNA全长为929 bp,编码179个氨基酸。xeIFN-γ基因含有4个外显子和3个内含子。xeIL-22 cDNA全长为1375 bp,开放阅读框为579 bp,编码192个氨基酸。xeIL-26开放阅读框为525 bp,编码175个氨基酸。xeIL-22和xeIL-26均具有5个外显子和4个内含子的基因结构。RT-PCR分析显示,在正常的非洲爪蟾中,xeIL-22和xeIL-26在所选取的组织中均未检测到表达;LPS刺激24 h后,在脾脏和小肠中检测到了xeIL-22的表达而未检测xeIL-26表达;PolyI:C刺激后,只在脾脏中可以检测到xeIL-22和xeIL-26的表达。荧光定量PCR检测结果显示,在正常的非洲爪蟾中,xeIFN-γ只在脾脏和小肠中表达。在PolyI:C刺激后,IFN-γ在肝脏、脾脏、肾和小肠中转录水平明显上调表达,分别增加了5、23、4和11倍。而LPS刺激后xeIFN-γ则只在脾脏中明显上调表达,表达量增加了14倍。 xeIFN-λs 基因以聚簇的形式分布在同一染色体上,共有五个基因,xeIFN-λ1 ~ λ5,其中IFN-λ1、-λ2 和-λ5 ORF 均为540 bp,编码179个氨基酸(IFN-λ1与-λ5的核苷酸序列一致)。IFN-λ3 ORF为438 bp,编码145个氨基酸。在基因结构上,xeIFN-λ1,-λ2 和-λ3 均由5个外显子和4个内含子组成;xeIFN-λ3 则由4个外显子和3个内含子构成;IFN-λ4 缺失了第二和第三个外显子,为假基因。xeIFNLR1 ORF 为1572 bp,编码523个氨基酸。xeIFN-λ另外一个受体IL-10R2位于Scaffold_1108 基因组上,其 ORF 全长为927 bp,编码308个氨基酸。RT-PCR 及荧光定量PCR显示,在健康非洲爪蟾中xeIFN-λs在选取的非洲爪蟾组织(心、肝、脾、肺、肾、肠和胃)中均能检测到。在PolyI:C诱导后,xeIFN-λs在不同的组织中呈显著上调表达,其中脾脏中的xeIFN-λs表达量变化最大,xeIFN-λ1、-λ2和-λ3增加倍数分别为33.13、9.34和11.86。近一步分离了非洲爪蟾的脾脏细胞并用PolyI:C进行诱导,在体外对xeIFN-λ的表达情况进行了分析。结果显示,与对照组相比,xeIFN-λs在PolyI:C浓度为1 μg/ml时表达量就已出现上调表达;当PolyI:C浓度增加时,xeIFN-λs表达量也随之增加。xeIFNLR1在所检测的组织中呈组成型表达,其中在肺中表达量最高。在PolyI:C刺激后,所检测的组织中的xeIFNLR1表达量均呈上调表达,其中在脾脏和肺上调表达倍数最大,分别为14.8 和14.9。体外PolyI:C诱导后显示,在PolyI:C浓度为0.1 μg/ml时,xeIFNLR1表达量就已出现上调表达;当PolyI:C浓度增加时,xeIFNLR1表达量也呈增加的趋势。对xeIFN-λ1进行重组活性表达后,将重组xeIFN-λ1蛋白(RxeIFN-λ1)作用于非洲爪蟾脾脏细胞,结果显示RxeIFN-λ能够有效提高IFIT5的表达量。1 mg/ml RxeIFN-λ1作用6 h后,IFIT-5的表达量增加了10.1倍。 肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)作为一类重要的模式识别分子,在对细菌类病原体的固有免疫反应中起着关键性的作用。本文同时对非洲爪蟾中的一种长型PGRP基因,xePGRP-L进行了克隆、鉴定。xePGRP-L cDNA全长为1681 bp,开放阅读框为1491 bp,编码497个氨基酸。在xePGRP-L N-末端含有一个长约170个氨基酸的保守的PGRP结构域。在非洲爪蟾胚胎发育至3天时可以明显检测到xePGRP-L的表达,之后xePGRP-L呈持续性表达。荧光定量PCR和Western blotting分析显示,在正常非洲爪蟾中,xePGRP-L呈组成型表达,在肝脏中表达量最高,在肺、肠和胃中呈中度表达,在心和脾脏中表达量最低。在非洲爪蟾腹腔注射LPS诱导24 h后,在非洲爪蟾的心、肝、脾和肠中xePGRP-L表达量呈上调表达,表达量分别为正常组的3、26、7、和33倍。
英文摘要: Interferon (IFN) is an important cytokine in antiviral immunity. The functions of IFN have been clearly elucidated in mammalian since IFN was first identified in 1957. To data, the IFN in mammalian was divided into three types: type I IFN (IFN-α/β), type II IFN (IFN-γ), and type III IFN (IFN-λ). In recent years, more and more research on the IFN in lower vertebrates such as teleost has been reported. The amphibian, as the transitional species that vertebrates evolve from hydrophilous to terricolous, and from ectotherm to homeotherm, has an important role in the biology evolution. However, little is known concerning the amphibian IFN and IFN related genes. Based on these, the type II IFN (IFN-γ), and type III IFN (IFN-λ) in the amphibian model animal, xenopus tropicalis (xe), were cloned, characterized, and their expression pattern were analyzed, aiming to reveal the function of amphibian IFN and the evolution story of the IFN system. Using comparative bioinformatics approach, the xeIFN-γ gene locus was analyzed. In this locus, there are three cytokines: interlcukin (IL)-22, IL-26 and IFN-γ. The gene synteny analysis showed this locus is relatively conserved and the transcription orientation of the cytokine gens and the phase of the introns remain the same during evolution. The xeIFN-γ cDNA consisted of 793 bp encoding a 179 aa peptide, has a four-exon-three-intron genomic organization. The xeIL-22 cDNA is 1375 bp in length with a 579 bp open reading frame (ORF) encoding a 192-aa peptide. The xeIL-22 gene consisted of five exons and four introns. The xeIL-26 ORF is 525 bp in length, coding for a protein of 175 aa. Similar to xeIL-22, the xeIL-26 gene also possesses five exons and four introns. RT-PCR showed that xeIL-22 and xeIL-26 was not detected in all tissues tested, including heart, liver, spleen, lung, kidney, intestine, and stomach. Treatment of the LPS activated IL-22 expression in spleen and intestine 6 h after injection but had no effect on IL-26 expression. PolyI:C induced both IL-22 and IL-26 expression in spleen but not in other tissues. IFN-γ expression pattern was determined by real-time quantitative PCR. The result showed that constitutive expression of IFN-γ was detectable in spleen and intestine. Induction of the IFN-γ expression was apparent in liver, spleen, kidney, and intestine after PolyI:C treatment with increase of IFN-γ transcript level of 5-, 23-, 4-, 11-fold, respectively. In contrast, LPS significantly elevated IFN-γ transcript level by 14-folds in spleen but not in other tissues. xeIFN-λs (xeIFN-λ1~ -λ5) are clustered on the same chromosome and have a multiple-exons with xeIFN-λ1, -λ2 and -λ5 possessing five exons and xeIFN-λ3 four exons. xeIFN-λ4 is a pseudogene which losts the exon 2 and exon 3. The xeIFN-λ1, -λ2 and -λ5 ORF is 540 bp in length, encoding 179 aa (xeIFN-λ1 and -λ5 shared 100% identity in nucleotide and amino acid level); xeIFN-λ3 ORF is 438 bp and encodes 145 aa. The xeIFN-λ receptors (xeIFNLR1 and xeIL-10R2) were also cloned. The xeIFNLR1 ORF is 1572 bp which encod 523 aa. xeIL-10R2 was located on Scaffold_1108 and ORF is 927 bp in length, encoding 308 aa. RT-PCR and realtime PCR showed that the xeIFN-λ could be detected in the tested tissues, including heart, liver, spleen, lung, kidney, intestine, and stomach. After PolyI:C treated, the xeIFN-λ expression increased in some tissue and had apparent increasing in spleen with elevating 33.13- ,9.34- and 11.86- folds. Further, the xeIFN-λs were analyzed in vitro. After treated the freshly isolated splenocytes for 4 h with 0.1, 1, 10 and 50μg/ml PolyI:C, the results showed that contrast to the control group, xeIFN-λs were upregulated when stimulated with 1 μg/ml PolyI:C. Then the xeIFN-λs expression level increased with the PolyI:C dose. xeIFNLR1 had a constitutive expression in all tested tissues, and had a significantly high expression in lung. Afer treated with PolyI:C, the xeIFNLR1 expression level increased in all tested tissues with the highest increasing in spleen and lung with 14.8 and 14.9-folds. In vitro, the xeIFNLR1 was upregulated when stimulated with 0.1 μg/ml PolyI:C. Similar to xeIFN-λ, the xeIFNLR1 expression level also increased with the PolyI:C dose. To further analysis the function of xeIFN-λ, the recombinant xeIFN-λ1 (RxeIFN-λ1) was expressed and purified. Then, the freshly isolated splenocytes were treated for 6 h with the RxeIFN-λ1 at doses of 0.001, 0.01, 0.1, and 1 mg/ml, respectively. Then the IFIT-5 gene expression level was tested with realtime-PCR. The result showed that IFIT-5 gene was increased with dose of RxeIFN-λ1 and increased 10.1-folds after stimulation with 1 mg/ml RxeIFN-λ1. Peptidoglycan recognition proteins (PGRPs), as a family of pattern recognition receptors (PRRs), have an important role in the host innate immunity against bacteria pathogen. In this paper, a long type PGRP in xenopus tropicalis (xePGRP-L) was also cloned and characterized. The xePGRP-L cDNA is 1681 bp and encode 497 aa. At the N-terminal of xePGRP-L contained a conserved PGRP domain. X.ePGRP-L mRNA was clearly detected in the early development stages at 3 dpf. Realtime PCR and Western blotting results showed in the normal xenopus, the xePGRP-L was constitutive expressed with highest in liver, lung, intestine and stomach, lower in heart, spleen and kidney. After LPS stimulated, the x.ePGRP-L expression was apparently up-regulated in heart, liver, spleen and intestine after LPS treatment, and the transcript level increase 3-, 26-,7-, 33-fold, respectively. However, there is no significantly expression level change in lung, kidney and stomach.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12378
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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非洲爪蟾Type II 和Type III 干扰素基因的克隆和表达分析.齐志涛[d].中国科学院水生生物研究所,2009.20-25
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