甘草次酸脂质体体外对鸡IL-2、IL-4和IFN-γ mRNA表达的影响

为了研究甘草次酸脂质体(GAL)增强免疫的作用和分子机理.采用Primer Primier 5.0软件自行设计白细胞介素-2(IL-2)、白细胞介素-4(IL-4)和干扰素-γ(IFN-γ)3对引物.培养鸡外周血淋巴细胞,分别加入3种浓度的GAL、甘草次酸(GA)和空白脂质体(BL),另设LPS对照组,培养36 h后收集细胞,提取总RNA,然后对其进行反转录.用荧光定量PCR方法测定GAL对鸡外周血淋巴细胞IL-2、IL-4和IFN-γ的mRNA表达的影响.结果:在8.750 mg/L、2.188 mg/L、0.547 mg/L时,GAL促进外周血淋巴细胞3种细胞因子mRNA的转录能力显著强于GA和BL,且在8.750 mg/L时的作用最强.这可能是GAL增强机体免疫的分子机制之一.     

RHDV在多种属动物体内分布及细胞免疫水平

应用纯化兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV),接种小鼠、豚鼠、SPF鸡、仔猪4d后剖杀,分离组织,通过电镜观察、血凝试验(HA)、RT-PCR,进行体内分布检测;同时将上述动物进行3次免疫,三免后10d分离外周血淋巴细胞进行IFN-γ、IL-4、WST检测病毒刺激指数(SI)。结果显示,通过电镜观察未见到病毒粒子;HA检测结果为肝脏的HA价最高,而肺脏最低;RT-PCR未检测到目的基因;WST检测结果免疫组刺激指数高于对照组;免疫组的IFN-γ和IL-4检测数高对照组。结果表明,RHDV在多种属动物分布规律相同,均能产生细胞免疫反应,为下一步试验奠定基础。     

猪宿主蛋白G3BP1对猪圆环病毒2型在PK15细胞上复制的影响

猪宿主蛋白G3BP1在宿主抵抗病毒感染过程中起着重要作用,然而还未有相关报道G3BP1与猪圆环病毒2型(PCV2)感染过程中的联系。本文构建了G3BP1真核表达载体和干扰RNA,实现在PK15细胞上G3BP1的过表达及敲低。结果发现,PCV2感染细胞24 h后病毒复制出现显著差异,感染48 h相比于感染24 h病毒复制差异更显著,表明G3BP1能显著促进PCV2复制。过表达或敲低G3BP1后,用干扰素刺激DNA(ISD)刺激12 h, G3BP1能促进ISD诱导IFN-βmRNA的表达;感染PCV2 12 h后,G3BP1也能正调控IFN-βmRNA的转录。本文揭示了宿主蛋白G3BP1显著促进PCV2的复制,很可能是通过上调IFN-β表达引起的。     

猪体内筛选RHDV VP60 T细胞表位的优势区

应用戊二醛醛化的人＂O＂型红细胞纯化兔出血症病毒（RHDV）,对非免猪进行3次免疫后,分离外周血淋巴细胞经不同段重组杆状病毒（共分5段表达）、纯化的病毒、Con A刺激后进行IFN-γ、IL-2、IL-4、WST检测。结果显示,纯化病毒的血凝效价为28,与未纯化的RHDV毒价相差1个滴度;WST检测结果为免疫组经3段重组杆状病毒刺激的值最高;IFN-γ和IL-2检测结果也是3段重组杆状病毒刺激的值最高;IL-4检测结果为3段重组杆状病毒刺激的值最低。由此表明病毒经纯化后效果好,3段重组杆状病毒区域为T细胞表位优势区,这为RHDV T细胞表位的进一步研究奠定基础。     

重组虹鳟IFN-γ2的原核表达及抗IHNV活性分析

为获得虹鳟IFN-γ2(rt IFN-γ2)抗传染性造血器官坏死病毒(IHNV)活性的相关数据,实验根据NCBI已发表序列设计引物,提取经植物血凝素刺激后的虹鳟头肾细胞总RNA,采用RT-PCR方法扩增471 bp的该基因完整开放阅读框。将该基因重组至原核表达载体p ET32a中,并转化大肠杆菌Rosetta,进行诱导表达,SDS-PAGE结果显示,目的蛋白以包涵体形式表达,大小约为38.4 ku。重组蛋白经复性、纯化后在CHSE-214细胞上进行抗IHNV活性分析,结果显示,rt IFN-γ2在CHSE-214细胞上抗IHNV活性为6.63×106U/mg。Real-time PCR结果显示,rt IFN-γ2免疫后,虹鳟头肾、脾、肝中IRF-1、IRF-2、IFN-I、IFN-γ和Mx表达水平均显著提高,总体而言免疫后2天机体抗病毒状态弱于免疫后1天。攻毒保护实验结果显示,免疫后1天进行IHNV攻击时,鱼死亡率为40%,而免疫后2天进行IHNV攻击时,鱼死亡率达到80%。研究表明,原核表达系统制备的重组虹鳟IFN-γ2不仅具有体外抗IHNV活性,更能激发虹鳟的抗病毒状态,从而为虹鳟抵抗IHNV感染提供一定的保护力。     

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6 h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6 h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0⁺CD69⁺CD4⁺ memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.     

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.     

传染性造血器官坏死病毒微型基因组表达干扰素对病毒的抑制效应

为构建传染性造血器官坏死病毒(IHNV HLJ-09)微型基因组并表达虹鳟IFN,采用RT-PCR扩增IHNV HLJ-09株的N、P、L、G和NV蛋白基因并亚克隆入真核表达载体pCI中,构建辅助质粒pCI-N、pCI-P、pCI-L、pCI-G和pCI-NV;将扩增获得的IHNV基因组两末端序列、增强型绿色荧光蛋白(EGFP)报告基因、虹鳟I型干扰素(IFN)基因克隆到真核表达载体pCI中构建出表达EGFP的IHNV微型基因组pCI-LFGT和表达IFN的IHNV微型基因组pCI-LFIT;将pCI-LFIT质粒转染已接种IHNV HLJ-09毒株的EPC细胞,实时荧光定量PCR法测定细胞中IHNV G基因RNA。结果显示:构建的微型基因组不论与辅助病毒还是与5个辅助质粒共转染,外源基因均能正确表达;pCI-LFIT质粒转染已接种病毒的EPC细胞组与对照组相比其中的病毒核酸显著减少。     

发情期山羊腺垂体中IFN α和IFN γ 的免疫组化研究

为观察干扰素(IFN)的分布与山羊腺垂体内分泌细胞在发情免疫调节中的形态学依据。运用免疫组织化学SP法分别对发情前期、发情期、发情后期和间情期山羊腺垂体内IFN-α和IFN-γ阳性细胞的分布和变化特点进行研究。结果显示,2种干扰素阳性物质主要分布于腺垂体嗜酸性细胞、嗜碱性细胞和嫌色细胞的细胞质,远侧部和结节部较多;不同发情期IFN-α和IFN-γ的分布存在明显差异,IFN-α相对表达量均高于IFN-γ,但两者在发情周期中的变化趋势是一致的;发情前期和间情期相对表达量较高,后期最低,发情期呈中等强度表达。表明IFN-α和IFN-γ各自在发情的不同时期发挥作用或相互协同调节垂体激素的合成与分泌,进而参与动物的生殖调节。     

MDCK细胞中IFN-γ诱导的免疫相关GTP酶在弓形虫PVM表面的定位研究

为探究犬MDCK细胞中γ-干扰素(IFN-γ)诱导的免疫相关的GTP酶在刚地弓形虫(T.gondii)的纳虫空泡膜表面的定位情况,本研究采用慢病毒包装载体将免疫相关的GTP酶(Irgb11)与GFP的融合基因(Irgb11-GFP)引入犬MDCK细胞中,经荧光鉴定和western blot检测到Irgb11-GFP融合蛋白的表达,表明构建了过表达Irgb11的细胞系。采用IFN-γ诱导该细胞系24h后,接种弓形虫PLK株,以间接免疫荧光试验检测,并通过激光共聚焦扫描显微镜观察,结果显示Irgb11-GFP蛋白获得表达并能够与弓形虫PLK株的纳虫空泡膜(PVM)发生共定位,表明在IFN-γ存在的情况下,犬MDCK细胞中的Irgb11可以被募集到弓形虫PVM表面,从而发挥其破坏弓形虫PVM的功能。本实验为进一步研究犬细胞中抑制弓形虫生长的机制奠定基础。