干扰素调节因子新剪接异构体IRF7-e的结构及功能

干扰素调节因子7是诱导玉型干扰素表达的最主要的转录因子。寻找IRF7新的剪接异构体,研究其结构及功能,为探索IRF7参与调控玉型干扰素机制的多样性提供基础。通过PCR和Sanger测序获得了IRF7一种新的剪接形式IRF7-e,并通过RACE获取了IRF7-e 基因全长。IRF7-e全长为1994 bp,含5'-UTR 410 bp,3'-UTR 120 bp,开放阅读框1464 bp,编码487个氨基酸的蛋白,预测其等电点为6.659,蛋白分子量为52.8 kD。双荧光素酶报告分析表明过表达IRF7-e能够提高玉型干扰素IFNα和IFNβ启动子的活性,其中对IFNα启动子活性提高了12.18倍,对IFNβ的启动子活性提高了2.99倍。表明IRF7-e可能参与玉型干扰素的调控。     

斑马鱼IRF11基因的鉴定、亚细胞定位及表达特征

早期的研究表明IRF11是鱼类特有的IRF家族成员。查询最近解析的斑马鱼第九版基因组时,发现斑马鱼IRF1和IRF11命名出现了混乱。通过对脊椎动物IRF1和IRF11基因位点进行同线型分析表明,IRF11与IRF1是两个不同的基因,不宜命名为IRF1b和IRF1a。系统进化树分析发现,在脊椎动物中IRF11基因比IRF1起源更早;两栖类以后的脊椎动物基因组只有IRF1,没有IRF11,其中原因可能是因为基因丢失。斑马鱼IRF11与脊椎动物IRF1一样,其表达蛋白定位在细胞核中。缺失分析揭示斑马鱼IRF11的DBD有一个能引导蛋白定位进入细胞核的序列。表达分析发现poly（I:C）能诱导斑马鱼IRF11的表达,但其表达水平低于IRF1。     

斑马鱼TAK1正向调控IRF7介导的天然免疫反应

在鱼类中关于转化生长因子β-激活激酶1(TAK1)在天然免疫反应中的功能研究较少。为了探究TAK1在斑马鱼天然免疫中的功能,本文克隆并获得了一种斑马鱼tak1剪接异构体(Drtak1),其开放阅读框含有1737个核苷酸,编码578个氨基酸,其中包括N端的丝氨酸/苏氨酸蛋白激酶结构域和C端的卷曲螺旋结构部。通过免疫荧光试验,证实DrTAK1是一种胞质蛋白。双荧光素酶报告试验显现在EPC细胞中单转DrTAK1不能诱导IFN的产生,但与IRF7共转时能显著提高其诱导干扰素启动子表达的能力。本文研究结果首次在斑马鱼中发现TAK1能正向调控IRF7介导的天然免疫反应,为后续DrTAK1功能研究奠定了基础。     

斑马鱼fem-1c cDNA克隆与表达分析

为进一步探究鱼类性别决定的相关机理, 增加对鱼类性控基因表达和功能的认识, 克隆斑马鱼fem-1c 基因并对其进行表达分析。研究采用RACE-PCR方法从斑马鱼卵巢组织cDNA中克隆了fem-1c的cDNA全长序列, 其大小为2701 bp, 编码618个氨基酸。生物信息学分析显示, 斑马鱼FEM-1C蛋白包含9个ANK结构域、2个TPR结构域和2个低复杂性区域, 与其他脊椎动物的FEM-1C蛋白序列保守性较高。脊椎动物的fem-1c与tmed7、trim36等邻近的45个基因具有保守的同线性关系。半定量RT-PCR实验结果显示斑马鱼fem-1c在受精后17d开始表达, 并特异地表达于成体卵巢组织中。RNA原位杂交结果显示, fem-1c基因mRNA定位于卵巢组织的Ⅰ期和Ⅱ期卵母细胞胞质中。fem-1c的时空表达特征暗示其在斑马鱼卵巢分化中具有重要作用。       

鲫Nub1基因的克隆及特征分析

Nub1(NEDD8 Ultimate Buster-1)是近年来发现的一种干扰素诱导表达基因,过量表达该基因能抑制细胞生长。研究通过RACE-PCR方法从产生干扰素的鲫囊胚培养细胞(Carassius auratus blastulae embryonic cells,CAB)中克隆出鲫Nub1基因。鲫Nub1全长cDNA为2298bp,编码一个由589个氨基酸残基组成的蛋白。推导的鲫NUB1蛋白具有哺乳类同源蛋白保守的结构域,包括N端的UBL结构域和C端两个UBA结构域,与已知的哺乳类包括人和小鼠、鸟类和两栖类的同源蛋白具有41%-45%的相似性。诱导表达分析显示PolyI:C和LPS均能诱导CAB细胞Nub1mRNA的上调,表明Nub1基因在鱼类的抗病免疫反应中发挥某种重要作用。       

草鱼Noxa基因克隆及其应答GCRV入侵的表达响应

研究通过获得草鱼Noxa基因(Cinoxa)全长cDNA, 进行序列分析和进化树构建, 使用定量PCR (qPCR)的方法研究其在GCRV刺激下的表达模式。研究发现, 草鱼Noxa基因的蛋白序列与斑马鱼Noxa基因具有高度相似性。通过分析草鱼和斑马鱼的Noxa基因的蛋白三维结构(3D)模型, 研究发现两者具有高度一致的蛋白三维结构模式, 该模型与高等哺乳类中的Bcl-2家族蛋白中C端结构域同源。对Cinoxa在GCRV刺激下的表达模式的研究表明, Cinoxa在GCRV感染后中肾、脾脏和头肾中表达发生显著性变化, 攻毒后24h和120h出现显著上调表达。研究表明草鱼Noxa为斑马鱼Noxa的同源基因, 并且参与了草鱼对GCRV入侵的应答反应, 为深入研究鱼类Noxa应答病毒入侵的功能和转录调控机制奠定了基础。     

Ppp5c 和TTC16 基因的TPR 结构域与Hsp70、Hsp90 蛋白 的相互作用及对细胞周期的影响

目的:研究Ppp5c及TTC16基因的TPR(tetratricopeptide repeat)结构域短片段和Hsp70及Hsp90家族蛋白的相互做用,及其过表达对细胞周期的影响。方法:通过生物信息学的分析及PCR的方法,克隆Ppp5c及TTC16基因的TPR结构域以及HSPA1A、HSP90AA1的全长基因,并连入酵母双杂交载体,通过ClonTech的酵母双杂交实验体系研究蛋白和蛋白之间的相互作用。把Ppp5c及TTC16基因的TPR结构域克隆入真核表达载体,构架稳定表达Ppp5c及TTC16基因的TPR结构域的MCF-7细胞系,并通过流式细胞实验观察细胞周期。结果:Ppp5c及TTC16基因的TPR结构域能与HSPA1A或HSP90AA1发生相互作用。Ppp5c及TTC16基因的TPR结构域在MCF-7中的过表达能严重影响细胞周期,引起细胞凋亡和S期阻滞。结论:本实验初步揭示了不同蛋白的TPR结构域在与Hsp70及Hsp90蛋白的相互作用性质的异同点以及其过表达对细胞周期的影响,为全面理解TPR结构域的功能、Ppp5c以及TTC16蛋白在细胞内的功能奠定了前期实验基础。     

高粱LEA基因家族的鉴定及表达分析

有研究表明,干旱、低温和盐等环境胁迫能够诱导LEA基因的表达。为了探索LEA基因家族在高粱响应外界刺激过程中起到的作用,本研究通过生物信息学的方法对LEA基因家族在高粱全基因组水平进行鉴定和分析,于高粱全基因组中共鉴定出35个基因家族成员,不均匀地分布于高粱8条染色体上,结合系统进化树和保守结构域分析结果,将高粱LEA基因家族成员分为7组。亲水性分析和结构无序性预测表明高粱LEA蛋白绝大多数为亲水性且结构无序。基因结构分析显示了各分组基因结构上的保守性。高粱LEA基因的启动子分析发现了一些与激素和非生物胁迫响应相关的顺式作用元件。对激素和干旱胁迫下高粱LEA基因的表达分析发现外界胁迫能够诱导部分高粱LEA基因的表达。     

鲫鱼干扰素调节因子1功能初步研究

将鲫鱼IRF1基因的ORF与真核表达质粒pcDNA3．1（＋）相连,构建了真核重组表达载体,转染并通过G418筛选获得了稳定转染IRF1基因的细胞株。同时利用原核表达系统成功获得了包含鲫鱼IRF1C端165个氨基酸的多肽,制备了抗鲫鱼IRF1的多克隆抗体。进一步从RNA和蛋白水平证明,在稳定转染IRF1基因的细胞株中IRF1能够组成型的过量表达,并且IRF1的过量表达伴随IFN基因的表达水平增强。同时PolyI：C的诱导能使稳定表达IRF1的细胞中干扰素系统基因STAT1和IF158的表达显著增强。研究结果表明,过量表达IRF1能调控鲫鱼IFN以及ISG基因的表达。     

斑马鱼myd88和traf6真核表达载体的构建

髓样分化因子88(myeloid differentiation primary response gene 88,MYD88)和 TNF 受体相关因子6(TNF receptor associated factor 6,TRAF6)均属于 Toll 样受体(Toll-like receptors,TLR)家族成员中的关键衔接分子.斑马鱼是研究先天免疫应答的独特模式生物,为了构建myD88和traf6的真核表达载体,用于免疫应答机制的研究,克隆了斑马鱼myd88和traf6基因的编码区CDS全长序列,分别是855 bp和1 629 bp.结构分析表明,斑马鱼MYD88存在2个保守结构域为死亡结构域和TIR结构域,TRAF6存在4个保守结构域分别为RING结构域、2个锌指结构域、环-环(coiled-coil)结构域以及TRAF同源结构域(meprinand TR-AF-homology,MATH),并与其他物种氨基酸序列一致性较高.系统进化树分析发现斑马鱼myd88和traf6基因与硬骨鱼类亲缘关系较近,说明斑马鱼myd88和traf6基因的同源性符合其在物种进化上的地位并且具有很高的结构同源性.将斑马鱼myd88和traf6基因的CDS全长序列连接至pCMV-Tag2B表达载体.通过对重组质粒进行双酶切检测发现,成功克隆了斑马鱼pCMV-myd88和pCMV-traf6真核表达载体.为了验证这2个真核表达载体的生物学功能,本研究在HEK293T细胞中进行NF-κB的报告基因活性检测.结果表明,过表达myd88和traf6基因后,斑马鱼NF-κB家族nfκbl启动子的转录活性显著升高,分别约为空载对照组的2.5倍和8倍.鉴于MYD88和TRAF6在天然免疫功能等方面的重要作用,实验结果为以后研究斑马鱼的先天免疫信号传导过程提供了有力的研究工具.     

Molecular characterization and expression analysis of interferon- inducible protein 56 gene in large yellow croaker Pseudosciaena crocea

In mammals, interferon-inducible protein 56 (IFI56) has been considered to play a role in mediating inhibition of viral replication and cell growth, and possibly in mediating cell apoptosis. Here, we reported the cloning of an IFI56 homologue from the spleen of large yellow croaker, a marine fish (LycIFI56). The complete cDNA of LycIFI56 gene is 1628 nucleotides (nt) encoding a protein of 437 amino acids (aa), with a putative molecular weight of 50.8 kDa. The deduced LycIFI56 protein has a high-level homology with all members of IFIT (IFN-inducible proteins with TPR domain) family, and its 9 putative TPR motifs all locate the corresponding position of these IFIT proteins. Phylogenetic analysis showed that five fish IFIT members form a unique clad independent of mammalian homologues, reflecting a distant evolutionary relationship from mammals. LycIFI56 gene was constitutively expressed in various tissues examined, such as gills, intestine, liver, kidney, heart, spleen, muscle and blood. Upon induction with poly(I:C), LycIFI56 gene expression is obviously up-regulated in spleen, gills, intestine, liver and kidney at 24 h post-induction, suggesting that LycIFI56 may be involved in the immune response induced by poly(I:C). Analysis of the expression kinetics of LycIFI56 and IRF1 genes revealed that the up-regulation of LycIRF-1 expression by poly (I:C) was apparently earlier than that of LycIFI56. These results would facilitate a better understanding of the expression regulation of fish IFI56 gene, and of its roles in immunity of bony fish.     

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.     

CaPKR-like在鲫鱼与草鱼组织中的表达特性分析

摘要：PKR是由干扰素诱导的最重要的抗病毒蛋白之一,能抑制细胞和病毒蛋白的合成。鲫鱼PKR基因(CaPKR-like)是鱼类第一个,也是非哺乳类脊椎动物第一个报道的PKR全长cDNA序列。本研究制备了CaPKR-like N-端包含Za结构域的多克隆抗体,采用Western blots检验PKR-like在鲫鱼和草鱼组织中的表达特性。Western blots显示在未诱导的鲫鱼和草鱼中该基因表达水平非常低,但是经Poly I:C免疫一周后,在鲫鱼和草鱼等8种组织中有较强的PKR-like蛋白产生。结果暗示：CaPKR-like与哺乳类PKR有相同的组织表达特性。。     

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was upregulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly I:C and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection.     

IFN-induced TPR protein IFIT3 potentiates antiviral signaling by bridging MAVS and TBK1

Intracellular RNA viruses are sensed by receptors retinoic acid-inducible gene I/MDA5, which trigger formation of the mitochondrial antiviral signaling (MAVS) complex on mitochondria. Consequently, this leads to the activation of TNFR-associated factor family member-associated NF-κB activator-binding kinase 1 (TBK1) and phosphorylation of IFN regulatory factor 3 (IRF3). It remains to be elucidated how MAVS activates TBK1/IRF3. In this study, we report that IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) is significantly induced upon RNA virus infection. Ectopic expression or knockdown of IFIT3 could, respectively, enhance or impair IRF3-mediated gene expression. Mechanistically, the tetratrico-peptide repeat motif (E164/E165) of IFIT3 interacts with the N terminus (K38) of TBK1, thus bridging TBK1 to MAVS on the mitochondrion. Disruption of this interaction markedly attenuates the activation of TBK1 and IRF3. Furthermore, host antiviral responses are significantly boosted or crippled in the presence or absence of IFIT3. Collectively, our study characterizes IFIT3 as an important modulator in innate immunity, revealing a new function of the IFIT family proteins (IFN-induced protein with tetratricopeptide repeats).