新型重组人IFN-λ2的高效表达、纯化与抗病毒活性研究

目的在大肠埃希菌中高效表达重组人干扰素λ2(rhIFNλ2),并对其抗病毒活性进行初步研究。方法根据大肠埃希菌的偏爱密码子人工设计合成人干扰素λ2(hIFNλ2)的高表达基因,将其克隆到原核表达载体pBV220,在大肠埃希菌中进行表达,表达产物经变性、复性、阳离子交换层析及分子筛层析纯化,测定其抗病毒活性、种属特异性及抗HBV活性。结果在大肠埃希菌表达的目的蛋白占细胞总蛋白量的15%左右,纯化后的纯度达到90%以上,在WISH细胞上抗VSV活性约为1.5×106IU/mg。与rhIFNα2b比较,表达产物在非灵长类细胞上的抗病毒活性较在灵长类细胞上弱,目的蛋白在HepG2.2.15细胞上表现有与rhIFNα2b相似的抗HBV活性。结论hIFNλ2可以在大肠埃希菌内实现高效表达,表达产物具有抗病毒活性,有与rhIFNα2b相似的抗HBV活性,本型IFN有较严格的种属特异性。     

重组酵母鸡α干扰素的研制及其抗病毒活性测定

目的通过酵母真核表达系统获得鸡α干扰素,并鉴定其抗病毒生物活性.方法以Con A刺激4～10小时后的鸡全血淋巴细胞提取的总RNA为模板,通过RT-PCR方法克隆出鸡α干扰素成熟蛋白基因,通过EcoR I 和Xba I两个酶切位点使该基因插入酵母表达载体(pPICZa-A),并把线性化该重组酵母鸡α干扰素载体,通过电转化使鸡α干扰素基因整和到酵母(X33)基因组上,利用微量病变抑制法测定表达的鸡α干扰素的抗病毒活性.结果实验结果表明重组酵母鸡α干扰素活性单位为10×48U/ml,具有较好的抗病毒效果.结论实验研究结果表明重组酵母鸡α干扰素具有较好的抗病毒能力.     

人新型干扰素K的基因克隆、表达、纯化及其抗病毒活性的初步研究

目的制备人干扰素κ(hIFN-κ)并初步研究其生物学活性.方法克隆hIFN-κ的cDNA并根据大肠埃希菌的密码子偏好性人工合成了hIFN-κ基因,在大肠埃希菌DH5α中高效表达、纯化后测定了rhIFN-κ的多种生物学活性,包括抗病毒活性、抗细胞增殖活性、种属特异性以及NK细胞调节活性.结果成功地获得了高纯度的rhIFN-κ,纯度在90%以上.利用WISH-VSV系统检测rhIFN-κ的抗病毒活力为2.0×106 IU/mg.与rhIFN-α2b的比较发现,rhIFN-κ无论在抗病毒活性、细胞生长抑制,还是在促NK细胞活性方面均弱于前者.结论rhIFN-κ在不同种属来源细胞上的抗病毒活性因种属来源而异,不同的病毒对rhIFN-κ的敏感性是不同的.     

干扰素λ1在CHO细胞中的表达

目的 构建干扰素λ1表达载体PCI-dhfr-λ1和连接增强子SP163的表达载体PCI-dhfr-SP163-λ1,并在CHO(dhfr-)细胞中表达.方法 在合成的干扰素λ1基因中引入相应酶切位点,并通过融合PCR获得连接增强子SP163-λ1基因,测序正确后将该基因插入表达载体PCI-dhfr,构建重组表达载体PCI-dhfr-λ1和PCI-dhfr-SP163-λ1.将所构建的重组表达载体用脂质体法转入CHO(dhfr-)细胞,通过间接免疫荧光法和Western Blot鉴定了λ1蛋白的表达,应用细胞病变抑制法初步鉴定了λ1蛋白的抗病毒活性.结果 成功构建了干扰素λ1真核表达载体PCI-dhff-λ1和PCI-dhfr-SP163-λ1.免疫荧光结果显示分别转染了两种表达载体的CHO(dhfr-)细胞均能表达干扰素λ1蛋白,SP163增强子明显增强了干扰素λ1蛋白的表达,Western Blot结果显示转染SP163的CHO(dhfr-)细胞表达干扰素λ1蛋白.通过细胞病变抑制实验,在转染细胞48h后的细胞培养液中检测到了干扰素λ1的抗病毒活性.结论 本研究成功的在CHO(dhfr-)细胞中表达了干扰素λ1,表达的蛋白具有抗病毒活性,为进一步建立稳定表达干扰素λ1的细胞系提供了条件.     

免疫细胞参与的干扰素抗病毒活性在细胞间转移的研究

干扰素与细胞膜上的干扰素受体相互作用而诱导细胞建立抗病毒状态。Blalock等最近报道,淋巴细胞经干扰素活化后能够将抗病毒活性传递给与其共同培养密切接触     

干扰素及其最新研究进展

20世纪初,病毒学家发现,当两种病毒感染同一宿主细胞的时候,两种病毒间普遍存在相互拮抗的现象,于是提出了病毒间存在着干扰（interference）的概念.1957年Issacs和Lindermann用灭活的流感病毒和鸡胚绒毛尿囊膜一起培养,产生了一种可溶性物质,该物质作用于其他鸡胚绒毛尿囊膜后,可以抑制随后攻击的活病毒繁殖,于是把这种物质命名为干扰素（Interferon,IFN）,干扰素是产生干扰现象的主要原因.后来发现除两栖类外的大多数脊椎动物受病毒感染后均可产生这类抗病毒物质.干扰素是一类具有多种生物学活性的低分子量蛋白质.1980年,国际干扰素命名委员会指出：干扰素是一类在同种细胞上具有广谱抗病毒功能的活性蛋白,其活性的发挥又受细胞基因的调节和控制,涉及RNA和蛋白质的合成.     

Effects of RNA silenced HLA-A2 in hBMSCs on secretory function of human allogeneic T lymphocytes and osteogenic ability of hBMSCs

目的:探讨应用RNAi技术沉默HLA-A2表达的人骨髓间充质干细胞(hBMSCs)对人异体T淋巴细胞分泌功能的调节作用及诱导成骨能力的影响.方法:用siRNA转染至第3代hBMSCs将转染前、后各组的hBMSCs与PHA刺激的人异体T淋巴细胞共同培养,用ELISA检测各培养组上清液γ干扰素(IFN-γ)、白细胞介素2(IL-2)的水平;对2组细胞进行成骨诱导分化培养,观察细胞形态变化;碱性磷酸酶染色、计数和Vor- Kossa染色、计数检测成骨能力.结果:hBMSCs表面抗原CD44、CD166阳性.转染前hBMSCs的HLA-A2表达为阳性,转染后hBMSCs的HLA-A2表达为弱阳性;转染前hBMSCs的HLA-A2蛋白表达量高于转染后的HLA-A2蛋白表达量.转染前、后的hBMBCs均能抑制人T淋巴细胞分泌IFN-γ、IL-2,转染前、后比较有差异.ALP染色显示转染前、后的hBMSCs均出现胞质呈棕黑色的阳性反应,ALP活性检测结果显示2组之间无差异.Vor-Kossa染色显示转染前、后的hBMSCs均有钙结节形成,统计2组之间无差异.结论:应用RNAi技术沉默HLA-A2表达的hBMSCs可以抑制PHA刺激人T淋巴细胞分泌因子IFN-γ、IL-2的水平,转染后的hBMSCs比未转染的hBMSCs抑制作用更强.应用RNAi技术沉默HLA-A2表达的hBMSCs,不影响其成骨能力.     

无血清培养对人绒毛膜来源间充质干细胞免疫活性的影响

目的 评价人绒毛膜来源间充质干细胞(MSCs)在无血清培养体系中的免疫活性,探索干细胞安全的应用于临床的条件.方法 应用无血清与含10％胎牛血清培养基培养人绒毛膜来源MSCs,比较两种培养条件下培养细胞的形态、免疫表型、分化潜能及对混合淋巴细胞反应的抑制作用等生物学特性.结果 两种方法培养的细胞形态相似,表达CD73、CD90、CD105,不表达CD34、人类白细胞抗原(HLA)-DR;具有多向分化潜能,同样能明显抑制混合淋巴细胞反应体系中淋巴细胞的增殖,抑制率与加入的干细胞数量成正比,可以减少混合淋巴细胞反应中白细胞介素2、γ-干扰素的分泌.结论 人绒毛膜来源间充质干细胞经无血清培养后仍保持原有的细胞形态、免疫表型、多向分化等生物学特性,没有改变免疫学活性.无血清培养可取代有血清培养用于细胞培养,避免经有血清培养的干细胞移植等临床应用研究引起的免疫原性反应及人畜共患病.     

干扰素与病毒相互作用的研究进展

干扰素（interferon,IFN）,是由英国科学家Isaacs于1957年通过鸡胚绒毛尿囊膜研究流感病毒干扰现象时首先发现的[1].干扰素是病毒感染机体时,宿主细胞通过抗病毒应答产生的一组结构类似、功能相近的低分子糖蛋白,是机体抗病毒感染机制中最重要的一种免疫因子,IFN具有抗病毒、抗肿瘤及免疫调节等功能.干扰素抗病毒效应往往早于特异的机体免疫反应,可有效地限制病毒复制,并具有抑制肿瘤细胞增殖和调节免疫功能的活性,是世界范围内许可的治疗多种病毒性疾病、肿瘤和免疫紊乱的药物.一方面,干扰素可有效抑制病毒在细胞中的增殖,在宿主抗病毒效应中发挥重要作用;另一方面,病毒也进化出相应的机制以对抗干扰素的抗病毒效应,从而成功侵染宿主并维持长期感染状态.鉴于此,本研究就干扰素的抗病毒作用及病毒对干扰素效应的逃逸机制等领域的研究进展加以综述.     

不同型别重组人干扰素在细胞培养上抗SARS病毒的研究

目的　研究重组人干扰素α2b、α1b、β1b和ω1b对严重急性呼吸综合征 (SARS)病毒在细胞培养上的抑制作用。方法　采用细胞病变抑制法在人横纹肌瘤 (Rda)细胞系培养上测定干扰素的抗SARS病毒活性。结果　高度纯化的重组人干扰素α2b、α1b、β1b和ω1b在Rda细胞培养上抑制SARS病毒产生 5 0 %细胞病变 (CPE)的最小活性单位各为 (16 0 5± 12 9 5 )IU ml、(14 9 0± 71 7)IU ml、(6 9 5± 6 1 5 )IU ml、(87 3± 4 7 1)IU ml或各为 (0 6± 0 5 )ng ml、(10 6± 5 1)ng ml、(3 5± 3 1)ng ml、(0 9± 0 5 )ng ml。结论　本研究中所用的重组人干扰素在细胞培养上均具有较好的抑制SARS病毒的活性。本研究就各型干扰素抗病毒敏感性的差异及干扰素预防SARS病毒感染潜在的临床应用价值进行了讨论     

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.     

Herpes simplex virus type 1 suppresses the interferon signaling pathway by inhibiting phosphorylation of STATs and janus kinases during an early infection stage.

We examined the influence on the interferon (IFN) signaling pathway of infection with herpes simplex virus type 1 (HSV-1) strain VR3. Data from reporter gene assays showed that expression of both type I and type II IFN-inducible genes was dramatically suppressed during the early stage of HSV-1 infection (2 to 3 h postinfection). During these periods, phosphorylation levels of janus kinases (JAKs) and STATs did not increase after treatment of HSV-1-infected FL cells with IFN-alpha or IFN-gamma, although cellular protein levels of the JAKs and the STATs were not significantly changed. In contrast, the inhibitory effect of HSV-1 on phosphorylation of STAT1 was not observed in U937 cells, which show resistance to steady-state accumulation of RNA for HSV-1 immediate-early genes. The phosphorylation of STAT1 in FL cells was not inhibited by infection with a UV-inactivated virus. These results indicate that viral gene expression or viral protein production is necessary for the inhibition of phosphorylation by HSV-1.     

Gamma interferon production by combinations of human peripheral blood lymphocytes, monocytes, and cultured macrophages.

Mitogen-induced interferon (IFN) production was studied using human peripheral blood mononuclear cells and subpopulations of lymphocytes, monocytes, and cultured macrophages. Cell populations were prepared in suspension to permit quantitative analysis of the interactions among different cell types. After stimulation by staphylococcal enterotoxin A, nylon column-purified lymphocytes produced only 5% as much IFN as the peripheral blood mononuclear cells from which they were prepared. When lymphocytes were supplemented with as little as 2% monocytes, IFN production increased two- to eightfold; with the addition of up to 20% monocytes, IFN production increased further, to levels approximating those of peripheral blood mononuclear cells. Monocytes alone produced no or very little IFN. Macrophages were derived from monocytes by culturing in vitro for 7 days. The addition of 2 to 5% autologous macrophages augmented IFN production to the same extent as 2 to 5% monocytes. However, more macrophages consistently resulted in less, rather than more, IFN, so that lymphocytes with 20% monocytes produced three- to eightfold more IFN than did lymphocytes with 20% macrophages. Thus, whereas the addition of monocytes over a broad dose-response range (2 to 20%) progressively augmented IFN production, macrophages showed an optimal effect at 2 to 5%, with higher percentages being inhibitory. The IFN induced by stimulation with staphylococcal enterotoxin A was characterized as IFN-gamma by its resistance to neutralization by antibody to IFN- alpha and its inability to induce antiviral protection in embryonic bovine trachea cells.     

A sensitive antiviral neutralization bioassay for measuring antibodies to interferons.

An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low OD signals. This fall in OD can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the OD signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte IFN although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

Lack of interferon-induced transfer of viral resistance against murine leukemia virus or poliovirus in cocultivated cells

It has recently been reported that there can be a transfer of the interferon (IFN)-induced viral resistance between cocultivated animal cells. However, in those systems the challenge virus was capable of replicating in both cell types. A more convincing conclusion could be obtained if the infecting virus could not replicate in the cell type which is homologous to the species of IFN that is added to the cultures. An inhibition of the yield of this virus would indicate that there was a transfer of the antiviral state from one cell species to another. In cocultivated mouse and human cells, a murine ecotropic type-C virus can only multiply in the mouse cells whereas poliovirus will only replicate efficiently in the human cells. It is clearly evident from this study that only homologous but not heterologous fibroblast IFN is capable of inhibiting the replication of either of these viruses in various combinations of cocultivated cells.     

Platelet serotonin release by human polymorphonuclear leucocytes stimulated by cotton dust bacteria

Spontaneous cytotoxicity of human lymphocytes for tumours is increased by interferon (IFN) without change in the overall fraction of cells binding to targets. We developed an indirect immunofluorescent technique to stain lymphocytes conjugated to K-562 tumour cells in agarose with monoclonal antibodies. This allowed assessment of lymphocyte subpopulations binding to tumour cells without disruption of conjugates. Overall binding of non-adherent (NA) lymphocytes to tumour targets following incubation at 37 degrees C for 6 h was 13.3 +/- 0.3% compared to 12.5 +/- 0.7% with inclusion of IFN at 100 u/ml. When NA lymphocytes were incubated with K-562 tumour cells without IFN, OKM1 and OKT3 staining lymphocytes comprised 16.8 +/- 3.5% and 83.0 +/- 1.3% of the total lymphocyte population and 32.5 +/- 1.3% and 70.2 +/- 2.6% of lymphocytes conjugated to tumours. Incubation with IFN significantly increased OKM1 staining cells in the total NA population to 57.2 +/- 5.6% (P less than 0.01) and within tumour conjugates to 59.2 +/- 2.7% (P less than 0.01) while OKT3 staining cells decreased to 58.3 +/- 5.2% (P less than 0.02) and 45.3 +/- 1.2% (P less than 0.01), respectively. IFN increased cytotoxicity of NA cells for 51Cr-labelled K-562 by 66% at an effector to target ratio of 30:1 (P less than 0.001). These results demonstrate that OKM1 staining cells bind more avidly to tumour targets in the absence of IFN. IFN selectively increases the proportion of OKM1 staining lymphocytes with a concomitant increase in their binding to tumour cells. Therefore, enhancement of cytotoxicity by IFN in the NK system may result, in part, from conversion of OKT3 to OKM1 staining cells which are more efficient killers.     

The long-term but not the short-term antiviral effect of IFN-alpha depends on Flt3 ligand and pDC

The cooperation between IFN-alpha/beta and FL, the ligand of Fms-like tyrosine kinase 3 (Flt3), plays an important role in the defense against herpes simplex virus type 1 (HSV-1) in neonates. Treatment of neonatal mice with recombinant IFN-alpha has a short-term, FL-independent and a long-term, FL-dependent protective effect against HSV-1. In mice lacking FL, neonatal resistance against HSV-1 is very low and DC numbers in the spleen are reduced. The treatment of these mice with rIFN-alpha at day 6 resulted in an increased resistance against infection with HSV-1 at day 7. In C57BL/6 mice, treatment with rIFN-alpha at birth induced both FL and plasmacytoid DC (pDC), resulting in enhanced resistance against HSV-1 at day 7. In contrast, in mice lacking FL, IFN-alpha treatment at birth did not influence the splenic cell composition and had no effect on viral protection. The transfer of pDC to mice lacking FL enhanced viral resistance. Therefore, the induction and function of pDC, normally controlled by IFN-alpha/beta and FL, are decisive for viral resistance in neonatal mice.     

Selective expression of nonsecreted interferon by an adenoviral vector confers antiproliferative and antiviral properties and causes reduction of tumor growth in nude mice.

Replication-deficient adenoviruses expressing human interferon-alpha2b (HuIFN-alpha2b) or the hybrid IFN-alpha2alpha1 or those with the secretory signal deleted, whose express is driven by the alpha-fetoprotein (AFP) promoter, were constructed and characterized. Synthesis of IFN protein and secretion or intracellular retention were tested by Western blotting and immunoassay. Expression of IFN by the recombinant adenoviruses was restricted to cells that constitutively express AFP. In these cells, expression of both secreted and nonsecreted recombinant IFN resulted in inhibition of cell proliferation, resistance to viral infection, induction of major histocompatibility complex (MHC) class I expression, increased apoptosis, and activation of an IFN-stimulated response element (ISRE)-containing promoter. Also, the induction of protein kinase R (PKR), increased phosphorylation of Stat1, and accumulation of hypophosphorylated pRb were observed for both the secreted and nonsecreted IFN, suggesting that the nonsecreted IFN may act through a similar pathway. Hep3B cells, an AFP-positive line derived from a patient with hepatocellular carcinoma (HCC), were injected subcutaneously (s.c.) into athymic nude mice to generate established tumors. Intratumoral injection of recombinant adenoviruses expressing secreted as well as the nonsecreted IFN caused suppression of tumor growth. As the AFP promoter is activated in many HCC cells but is silent in normal cells, these constructs may be useful in restricting IFN effects to the tumor cells while reducing toxicity to the neighboring tissues.     

Axl and MerTK receptor tyrosine kinases maintain human macrophage efferocytic capacity in the presence of viral triggers

The requirement to remove apoptotic cells is equally important in homeostasis and inflammatory disease. In particular, during viral infections large quantities of infected cells undergo apoptosis and need to be efficiently cleared by phagocytes to prevent secondary necrosis. Although specific roles of several apoptotic cell sensors, such as the TAM (Tyro3, Axl, MerTK) receptor family, have been characterized in mouse models, little is known about their regulation and involvement in apoptotic cell uptake (efferocytosis) by human macrophages under inflammatory conditions. We show that whereas pro‐inflammatory stimuli consistently downregulated MerTK expression in human monocyte‐derived macrophages (MDMs), stimuli indicative of a viral infection, interferon‐α (IFN‐α) and the TLR3 ligand poly(I:C), specifically induced Axl expression and promoted binding of the bridging molecule Gas6. Axl induction by IFN‐α and poly(I:C) was associated with higher MDM efferocytic capacity compared to cells treated with other pro‐inflammatory stimuli, such as LPS and IFN‐γ. While MerTK blocking antibody uniformly suppressed apoptotic cell uptake by MDMs, Axl blocking antibody significantly reduced efferocytosis by poly(I:C)‐stimulated MDMs, but not by resting MDMs. Our observations demonstrate that Axl induction during viral infections contributes to maintaining macrophage capacity to engulf apoptotic cells, which may have important consequences for resolution of anti‐viral immune responses.