Ⅲ型干扰素——新型抗病毒细胞因子

干扰素（IFN）是抗病毒感染的第一道防线,Ⅰ型和Ⅱ型干扰素不仅可抑制病毒,而且还能参与天然免疫反应和获得性免疫反应。最近干扰素家族增添一位新成员：Ⅲ型干扰素,即IFN-λ,因其具有类似干扰素的抗病毒活性且能诱导干扰素相关基因的表达而命名。IFN-λ受体与Ⅰ型干扰素的受体不同,但具有与Ⅰ型干扰素类似的诱导表达方式和信号转导通路,并能激活一系列相似的干扰素刺激基因。就IFN-λ家族及其受体、基因表达和信号转导机制、抗病毒作用等进行综述。     

树鼩进化分类地位的分子证据

树鼩隶属攀鼩目,广泛分布于东南亚、南亚和中国南部等地区。由于其独特的特点,如体型小、脑-体重比例高、生殖周期短、寿命短和饲养成本低等,在生物医学研究中被认为是可望替代灵长类动物的新型实验动物。然而,关于树鼩与灵长类动物的亲缘关系一直存在争议。明确树鼩的分类地位是创建实验动物的重要研究基础。该文介绍了近年来关于树鼩分类地位探讨的分子证据。在现有的研究中,大部分核DNA序列研究,包括近期树鼩全基因组序列分析,都支持树鼩是灵长动物的近缘旁系群,然而绝大部分基于线粒体DNA序列的研究却显示树鼩与啮齿动物的亲缘关系更为接近。这样的分歧主要是由于线粒体序列和核基因数据的差异以及不同的算法导致。综合现有不同DNA数据的研究结果,作者认为树鼩作为灵长类的近亲这一结论应该成为共识。     

干扰素-λ的生物学作用

Ⅲ型干扰素（Interferon,IFN）,即IFN-λ,是一种新型干扰素,其家族包括IFN-λ1,IFN-λ2和IFN-λ3（也可分别称作IL-29,IL-28α和IL-28β）.IFN-λ的功能性受体复合物是由IL-28Rα（又称IFN-λR1或CRF2-12）和IL-10Rβ（又称CRF2-4）链组成的异二聚体,IFN-λ结合到受体上诱导受体异二聚体化,导致Jak-STAT信号转导途径的激活,从而发挥与Ⅰ型IFN相似的生物学效应.IFN-λ的生物学效应包括抗病毒、抗肿瘤和调节免疫活性等方面.IFN-λ很多生物学活性与临床上应用广泛的 IFN-α/β十分相似,但其受体表达局限,毒副作用相对较小,因此在抗病毒和抗肿瘤方面具有广阔的应用前景.     

树鼩神经肽Y的分子克隆及其灵长类类似物的同源性比较

树鼩由于与灵长类动物有较密切的亲缘关系和其个体小,以及繁殖周期短等特性而倍受关注,尤其是作为医用实验动物的研究,近年来已受到越来越多的重视,但树鼩的分类地位还一直有所争论。该研究从树鼩脑cDNA文库中克隆得到编码树鼩神经肽Y(neuropeptide Y,NPY)前体序列,序列比对发现该序列与灵长类NPY序列同源性高达96.9%。将该序列与GenBank数据库中其他物种的NPY序列构建系统进化树,发现树鼩与灵长类处于同一分支。该研究结果揭示了树鼩与灵长类较近的亲缘关系。     

非洲爪蟾干扰素-λ1基因的原核表达

干扰素(Interferon,IFN)λ包括IFNλ-1、-2和-3,分别又称为白细胞介素(Interleukin,IL)-29、IL-28A和IL-28B,在基因结构上与IL-10相似,含有多个外显子,IFN-λ1基因包含5个外显子和4个内含子,IFN-λ2和IFN-λ3则有6个外显子和5个内含子,它们作为一类新型干扰素统归为III型干扰素。哺乳类IFN-λ在功能上与I型IFN更为接近,具有抗病毒、抗肿瘤、抗细胞增殖及免疫调节功能。     

树鼩γ干扰素在大肠杆菌中的表达、纯化及免疫原性鉴定

目的:克隆树鼩γ干扰素(IFNγ)基因,在大肠杆菌中高效表达纯化并鉴定其免疫原性。方法:提取树鼩肺组织总RNA,经RT-PCR扩增出树鼩IFNγ基因,再克隆到原核表达载体p ET30a(+),构建重组表达质粒p ET-30a(+)-IFNγ,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达树鼩IFNγ;经金属螯合柱复性及纯化,Western印迹和ELISA检测其免疫原性,并检测不同组织中IFNγ的分布情况。结果:构建了重组表达质粒p ET-30a(+)-IFNγ,重组蛋白在30℃、1 mmol/L IPTG诱导4 h获得较高表达量,镍柱复性纯化后得到较高纯度的树鼩IFNγ。Western印迹显示IFNγ可与兔抗人IFNγ单克隆抗体特异性结合,应用Western印迹和ELISA分别检测树鼩IFNγ的免疫原性和抗体滴度,在树鼩的鼻、心、肝、肺、肾、气管中检测到IFNγ。结论:在大肠杆菌中高效表达了重组树鼩IFNγ,复性纯化后具有良好的免疫原性。     

IFN-λ家族基因遗传多态性与HBV感染、病毒复制及清除的关系

乙型肝炎病毒(hepatitisBvirus,HBV)感染是一个严重的全球性公共卫生问题。HBV感染后会引发肝炎,可进一步发展为肝硬化甚至恶化为肝癌。干扰素λ(interferonλ,IFN-λ)是干扰素家族的成员之一,是抗病毒防御的重要细胞因子。IFN-λ家族包括4个成员,分别为IFN-λ1、IFN-λ2、IFN-λ3和IFN-λ4,已有研究表明该家族基因的遗传多态性与HBV的复制和患者的治疗效果具有相关性。文中从遗传易感的角度总结了IFN-λ家族基因多态性在HBV的感染、患者的病程进展和治疗效果中扮演的角色,从而进一步了解其遗传多态性在HBV感染及其病程中的作用,为HBV感染的预防与患者的治疗提供理论基础,为该类细胞因子作为抗HBV药物的研发提供参考。     

树鼩IL-2全长编码序列的克隆及分子特征分析

树鼩作为多种人类疾病模型已受到广泛关注,而免疫因子对于树鼩模型评价至关重要,但目前对其白细胞介素-2(IL-2)的研究鲜有报道。该实验以经ConA(concanavalin)诱导培养的树鼩淋巴细胞总RNA为模板,RT-PCR克隆出465bp的树鼩IL-2全长编码序列,并采用ClustalW软件分析其序列和分子特征。结果表明树鼩IL-2cDNA编码一个由154个氨基酸组成的蛋白质,其cDNA及氨基酸序列与人的同源性分别为93%及80%,且其整体结构与人IL-2相似。MEGA5.0软件构建的进化树表明,树鼩与人及恒河猴的亲缘关系较近。Pymol软件对树鼩和人IL-2氨基酸序列进行的三维结构模建表明,两者的IL-2分子三维空间结构基本相似,表面大部分区域所带电荷相同,但在某些区域差异较大,且树鼩多出一个糖基化位点,这些差异对抗体的结合可能存在影响。该研究为今后树鼩IL-2单克隆抗体的制备及功能研究奠定了基础。     

树鼩CD4全长编码序列的克隆及分子特征分析

树鼩作为多种人类疾病研究模型的可能性已受到广泛关注,但尚缺乏研究其免疫功能的基本标志以及单克隆抗体。该实验首先以树鼩外周血总RNA为材料,通过RT-PCR扩增得到长度为1365bp的树鼩CD4全长编码序列,并确定了数据库中缺失的两个片段,进而通过ClustalW等软件对其序列和分子特征进行分析,发现树鼩CD4氨基酸序列胞外和胞内域保守性较好,且与人类和猴的亲缘关系较近。虽然树鼩和人CD4分子表面大部分区域均带正电荷,但与人CD4胞外域D1相比,树鼩CD4D1结构区域表面带负电荷较多,且多出两个N-糖基化位点。这些差异对抗体的结合可能存在影响。该研究为今后树鼩CD4单克隆抗体制备及功能研究奠定了基础。     

新型的干扰素λ家族

干扰素λ为一类新型的干扰素家族,分IFN-λ1、IFN-λ2和IFN一λ3等3种,也分别称为IL-29,IL-28A和IL-28B;其功能受体复合物是由新鉴定的CRF2—12和IL-10R2组成的异二聚体。配体与受体相互作用能活化Jak—STAT通路,并起抗病毒或其他防御功能作用。     

Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates

Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFN?1 and IFN?2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-α] and beta interferon [IFN-β]) and type III IFNs (lambda interferon [IFN-λ]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFN?1 and IFN?2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.     

IL-28 and IL-29: newcomers to the interferon family

IL-28 and IL-29 were recently described as members of a new cytokine family that shares with type I interferon (IFN) the same Jak/Stat signalling pathway driving expression of a common set of genes. Accordingly, they have been named IFN lambda. IFNs lambda exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumour activity. Importantly, however, IFNs lambda bind to a distinct membrane receptor, composed of IFNLR1 and IL10R2. This specific receptor usage suggests that this cytokine family does not merely replicate the type I IFN system and justifies its designation as type III IFN by the nomenclature committee of the International Society of Interferon and Cytokine Research.     

Induction of an Antiviral State and Attenuated Coxsackievirus Replication in Type III Interferon-Treated Primary Human Pancreatic Islets

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets'' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Herpesviruses and the Type III Interferon System

Type III interferons (IFNs) represent the most recently discovered group of IFNs. Together with type I IFNs (e.g. IFN-α/β), type III IFNs (IFN-λ) are produced as part of the innate immune response to virus infection, and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs). It was initially thought that type I IFNs and type III IFNs perform largely redundant functions. However, it has become evident that type III IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers, thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world, versus the generally more broad, potent and systemic antiviral effects of type I IFNs. Herpesviruseses are large DNA viruses, which enter their host via mucosal surfaces and establish lifelong, latent infections. Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses, our current knowledge on the interaction of herpesviruses with type III IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV). This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.     

High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli

Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physiological functions of these compounds. We describe a protein expression system for the production of IFNs alpha2, alpha8, and their hybrids in insoluble form in Escherichia coli, coupled to an efficient two-step optimized refolding and histidine-tag purification protocol. The expressed IFNs were of high biological value, as shown in antiviral and antiproliferative assays and some had specific activities higher than those of the commercially available interferon preparations and exhibited novel properties. This time-efficient, optimized protein expression method allows for the production of not just a single interferon subtype but several native and hybrid IFNs with relatively high yield and low cost that can be used in functional and potentially clinical assays.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.