法氏囊活性肽BP7和BP9调节T细胞的功能研究

旨在探索法氏囊活性肽BP7和BP9调节T细胞亚型的功能。采用禽流感灭活疫苗免疫小鼠,利用磁珠CD3阴选分选免疫小鼠T细胞,再用法氏囊多肽BP7和BP9刺激T细胞,检测T细胞活力及亚型变化情况。MTT结果表明,BP7和BP9均能提高活化T细胞的活力水平;流式分析图显示,法氏囊多肽BP7促进活化T细胞增殖分化成CD3~+CD4~+T细胞和CD3~+CD8~+ T细胞,而BP9在48 h促进CD3~+CD8~+ T细胞比例增加,但未达到差异水平;细胞凋亡结果显示,BP7和BP9均能抑制T淋巴细胞早期凋亡。此外,ELISA检测表明,BP7促进T细胞分泌IL-4和IFN-γ。以上结果表明:法氏囊活性肽BP7和BP9均参与T细胞分化成熟反应,但其调节功能略有差异。     

多糖作为兽用疫苗佐剂作用机制的研究进展

多糖可促进免疫调节,具有多靶点、多功能和多因子效应,因此可广泛用作兽用疫苗免疫佐剂。多糖作为疫苗佐剂可提高免疫动物脾脏、胸腺和法氏囊等免疫器官指数,增强巨噬细胞吞噬活性及抗原递呈能力,增强NK细胞杀伤能力,促进树突细胞的成熟和分化,增强机体对抗原的摄取能力,影响T细胞亚群的种类和数量,改变Th1、Th2型免疫应答反应的类型,促进B淋巴细胞增殖,刺激细胞因子及抗体的产生等。而多糖作为配体通过与Toll样受体(Toll-like receptor,TLR)、甘露糖受体(mannose receptor,MR)、C型凝集素受体(C-type lectins receptor,CLR)、清道夫受体(scavenger receptor,SR)等受体分子结合,激活下游信号通路,进而发挥上述免疫活性。文章主要从上述几方面对多糖作为兽用疫苗佐剂的作用机制进行阐述,以期为多糖的进一步开发和应用提供参考。     

gga-miR-142-5p负调控chMDA5抗传染性法氏囊病病毒感染的分子机制

传染性法氏囊病(infectious bursal disease,IBD)是由传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)引起的一种高度危害养鸡业的传染病。chMDA5在识别病原相关分子模式(pathogen associated molecular pattern,PAMPs)及激活宿主的天然免疫应答中具有重要的作用,但chMDA5识别IBDV的分子机制仍然不清楚。本研究用IBDV感染DT40细胞,在IBDV感染的DT40细胞中,chMDA5的表达水平显著升高。chMDA5过表达可以抑制IBDV的复制,并激活IFN-β promoter和Mx promoter活性,chMDA5抑制表达则得到相反的结果;chMDA5激活IFN-β promoter活性主要是通过IRF7通路。用生物信息学方法和双荧光素酶报告系统分析发现gga-miR-142-5p可作用于chMDA5的3'UTR;qRT-PCR和免疫印迹分析表明gga-miR-142-5p可以使chMDA5的蛋白水平表达降低,但mRNA水平没有变化;gga-miR-142-5p可依赖IRF7通路使IFN-β promoter和Mx promoter活性降低,IBDV复制水平升高。本研究表明,gga-miR-142-5p可以作用于chMDA5的3'UTR负调控chMDA5的表达从而影响其下游IFN-β promoter和Mx promoter活性来发挥抗IBDV感染的作用。     

鸡传染性法氏囊病免疫失败的原因分析及防制

传染性法氏囊病(Infectious Bursal Disease，IBD)是由双RNA病毒科的传染性法氏囊病病毒(IBDV)引起的、主要见于3-12周龄幼鸡的一种急性、高度接触性传染病。IBDV主要侵害鸡的中枢免疫器官——法氏囊和携带IgM的早期分化的B淋巴细胞，使鸡体免疫器官和免疫活性细胞功能受到严重影响，出现免疫抑制，导致其他疫苗接种后机体免疫应答功能低下或不发生免疫应答，因而常继发多种疫病，如鸡的大肠杆菌病、坏疽性皮炎、细菌性关节炎、禽传染性贫血、新城疫、马立克氏病、禽传染性支气管炎、鸡慢性呼吸道病等。     

传染性法氏囊病研究进展

一、IBD流行病学特性 传染性法氏囊病(IBD)是由双RNA病毒科的传染性法氏囊病病毒(IBDV)引起的、主要见于3～12周龄幼鸡的一种急性、高度接触性传染病.IBDV主要侵害鸡的中枢免疫器官一法氏囊和携带IgM的早期分化的B淋巴细胞,使鸡体免疫器官和免疫活性细胞功能受到严重影响,出现免疫抑制,导致其他疫苗接种后机体免疫应答功能低下或不发生免疫应答,因而常继发多种疫病,如鸡的大肠杆菌病、坏疽性皮炎、细菌性关节炎、禽传染性贫血、新城疫病、马立克氏病、禽传染性支气管炎、鸡慢性呼吸道病等.     

传染性法氏囊病研究进展

一、IBD流行病学特性传染性法氏囊病(IBD)是由双RNA病毒科的传染性法氏囊病病毒(IBDV)引起的、主要见于3～12周龄幼鸡的一种急性、高度接触性传染病。IBDV主要侵害鸡的中枢免疫器官—法氏囊和携带IgM的早期分化的B淋巴细胞,使鸡体免疫器官和免疫活性细胞功能受到严重影响,出现免疫抑制,导致其他疫苗接种后机体免疫应答功能低下或不发生免疫应答,因而常继发多种疫病,如鸡的大肠杆菌病、坏疽性皮炎、细菌性关节炎、禽传染性贫血、新城疫病、马立克氏病、禽传染性支气管炎、鸡慢性呼吸道病等。     

鞭毛蛋白诱导表达差异基因的分析

试验旨在分析鞭毛蛋白刺激机体基因表达谱的特征，揭示鞭毛蛋白佐剂的作用机制。从GEO数据库筛选目标微阵列GSE46421和GSE72016及其差异表达基因（DEGs），经DAVID分析DEGs参与的GO功能注释和KEGG信号通路、String构建DEGs的蛋白质-蛋白质相互作用（PPI）网络、Cytoscape可视化PPI网络并筛选高连通度的PPI子模块和hub基因。结果显示，共筛选出182个DEGs，包含上调基因161个、下调基因121个。DEGs参与GO生物过程主要有信号转导、免疫应答、炎症反应、细胞凋亡等，参与分子功能包含激酶激活、受体结合、细胞因子激活、膜信号转导。DEGs参与KEGG通路涵盖细胞因子-细胞因子受体相互作用、TNF信号通路、NF-κB信号通路、Toll样受体信号通路、NOD样受体信号通路等免疫相关途径。分析DEGs的PPI网络发现，2个重要的PPI子模块和10个关键基因（IL-10、IL-6、CCL2、CXCL2、NFKBIA、MyD88等）。研究表明，鞭毛蛋白通过识别TLR5和NOD样受体，触发MyD88依赖性和MyD88非依赖性途径发出信号，激活转录因子NF-...     

鸡传染性法氏囊病免疫失败的原因分析及防制

传染性法氏囊病(Infectious Bursal Disease,IBD)是由双RNA病毒科的传染性法氏囊病病毒(IBDV)引起的、主要见于3~12周龄幼鸡的一种急性、高度按触性传染病。IBDV主要侵害鸡的中枢免疫器官———法氏囊和携带IgM的早期分化的B淋巴细胞,使鸡体免疫器官和免疫活性细胞功能受到严重影响,出现免疫抑制,导致其他疫苗接种后机体免疫应答功能低下或不发生免疫应答,因而常继发多种疫病,如鸡的大肠杆菌病、坏疽性皮炎、细菌性关节炎、禽传染性贫血、新城疫、马立克氏病、禽传染性支气管炎、鸡慢性呼吸道病等。IBD疫苗的使用,曾使本病一度得…     

法氏囊多肽BSP-Ⅰ和BPP-Ⅰ的免疫调节活性及其B细胞结合蛋白的鉴定

为了研究法氏囊多肽的免疫调节作用,本试验用BSP-Ⅰ和BPP-Ⅰ分别与禽流感灭活疫苗或抗原混匀后免疫鸡。结果显示,BSP-Ⅰ和BPP-Ⅰ均明显提高免疫鸡体内禽流感抗体水平,且两者均促进Th1和Th2型细胞因子产生。采用鸡B淋巴细胞cDNA T7噬菌体文库筛选法氏囊活性肽相互作用的受体蛋白,PCR扩增富集噬菌体克隆的插入片段。序列分析结果表明,BSP-Ⅰ相互作用的蛋白受体有3种,分别为VSTM2、N-钙黏蛋白和v-myc。BPP-Ⅰ相互作用的蛋白分子主要是Ryanodine受体、MGC83917蛋白和BTB/POZ1。这些结果表明,法氏囊活性肽参与机体中多种生物学过程,为深入研究法氏囊活性肽的功能提供了理论基础。     

感染鸡贫血病毒雏鸡接种新城疫疫苗后免疫功能和免疫调节的变化

雏鸡1日龄感染鸡贫血病毒，8日龄接种Lasota疫苗，以未感染免疫雏鸡为对照，于免疫后7、14、28d检测血清免疫球蛋白IgG、IgM、IgA,在凝抑制抗体（HI）滴度；胸腺、法氏囊、脾脏T细胞、IgG^ 、IgM^ 、IgA^ ,抗体生成细胞数量及T、B细胞增殖反应；胸腺、脾脏细胞因子IL－2、IFN活性的变化。结果发现，感染CAV雏鸡Lasota疫苗免疫后，其血清IgG、IgM、IgA免疫球蛋白含量明显减少，HI抗体滴度降低；胸腺、法氏囊、脾脏T细胞、抗体生成细胞数量降低及T、B细胞增殖反应减弱，胸腺、脾脏IL－2及TNF诱生活性降低，表明其细胞免疫和体流免疫功能以及细胞因子免疫调节作用均未感染免疫雏鸡明显减弱。     

黄酮类化合物的免疫调节作用及机制

黄酮类化合物广泛分布于自然界中,具有抗炎、抗菌、抗病毒、抗肿瘤、抗氧化等多种生物活性。近年来的临床研究和试验表明,黄酮类化合物对机体免疫系统也具有重要的调节作用,主要通过影响免疫器官、细胞免疫、体液免疫和非特异性免疫以及免疫相关信号传导通路[核转录因子-κB(NF-κB)、Toll样受体(TLR)和丝裂原活化蛋白激酶(MAPK)信号通路]来发挥免疫调节作用。本文通过查阅国内外文献针对黄酮类化合物对动物机体的免疫调节作用及其分子作用机制相关研究进行了综述,以期为揭示黄酮类化合物免疫调节作用机制以及为兽医临床免疫调节中药的开发提供研究思路和方法。     

Bovine dendritic cells generated from monocytes and bone marrow progenitors regulate immunoglobulin production in peripheral blood B cells

We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.     

鸡毒支原体MG-HY株感染鸡气管的转录组学分析

旨在进一步探究鸡毒支原体（MG）感染SPF雏鸡后气管基因组转录水平的变化,筛选出MG感染后参与气管黏膜炎性损伤的差异表达基因和调控通路。使用MG-HY株菌液按0.2 mL·羽^-1经点眼滴鼻感染SPF雏鸡,感染后收集气管组织利用RNA-Seq技术进行测序分析。转录组分析结果显示,在MG感染期间RNA-seq共筛选出3 112个显著（P<0.01）差异表达基因（DEGs）,其中,1 646个上调基因,1 466个下调基因。GO功能分析显示,差异基因主要涉及生物调节、刺激的反应、多细胞生物过程、细胞成分组织或生物发生等生物过程。KEGG-Pathway分析发现差异基因参与黏膜免疫信号传导通路,例如：细胞因子-细胞因子受体相互作用,细胞黏附分子（CAMs）,细胞外基质（ECM）受体相互作用、紧密连接、PPAR信号通路和MAPK信号通路等,表明这些基因参与了MG诱导的雏鸡气管炎症反应和损伤。使用qRT-PCR验证与黏膜免疫相关的13个差异表达的基因,其结果与转录组分析一致。本研究为进一步阐明MG感染导致宿主气管黏膜上皮损伤和黏膜免疫机制提供了基础。     

新型鹅细小病毒感染鸭的肝、胸腺、回肠转录组差异表达分析

旨在探究新型鹅细小病毒（novel goose parvovirus,NGPV）与宿主细胞相互作用的分子致病机制。本研究采用转录组测序技术对感染NGPV SD株14 d雏鸭的肝、胸腺和回肠进行分析。结果显示：感染组回肠上调基因有78个,下调基因有31个;感染组胸腺上调基因有111个,下调基因有78个;感染组肝中上调基因有128个,下调基因有313个;NGPV感染雏鸭后,胸腺、回肠和肝中的差异表达基因参与免疫效应过程、淋巴细胞介导的免疫等多个免疫相关生物学过程,并在Toll样受体信号通路、JAK-STAT信号通路、NF-κB信号通路、PI3K-Akt信号等多条信号通路富集。综上表明：NGPV感染激活了机体免疫反应和宿主细胞的相关抗病毒信号通路。此研究为NGPV的致病机制研究提供了新的线索。     

TRAIL和HN基因融合表达重组腺病毒的构建及其对DT40细胞的抑瘤作用

分析携TRAIL和HN基因的重组腺病毒在DT40细胞中的表达规律及杀伤肿瘤细胞的生物学效应。通过RT-PCR分别从鸡外周血淋巴细胞和新城疫病毒(NDV)LaSota株中扩增出TRAIL和HN基因,通过定点突变和重叠延伸拼接法实现TRAIL和HN基因的2A连接,将获得的目的基因克隆到pShuttle-CMV穿梭载体中,与pAdeasy-1载体在BJ5183细菌内同源重组,构建了包含目的基因的重组腺病毒质粒pAd-TRAIL和pAd-TRAIL-2A-HN,将质粒线性化后转染AD293细胞,包装后的重组腺病毒,经RT-PCR检测、荧光显微镜观察和病毒滴度测定后,将获得的重组腺病毒体外转染禽淋巴白血病DT40细胞,通过RT-PCR和Western blotting检测重组腺病毒的感染性和外源基因的表达情况,采用流式细胞仪检测重组腺病毒对DT40细胞生长的影响以及Ad-TRAIL和Ad-TRAIL-2A-HN对DT40细胞的凋亡作用。PacⅠ酶切证实同源重组成功;RT-PCR、荧光显微镜检测证实重组腺病毒质粒成功导入AD293细胞中且具有良好的感染性,滴度为1.9×1010PFU/mL;Western blot检测结果显示,TRAIL-linker-HN基因能够在DT40细胞中稳定表达并对DT40细胞产生细胞毒作用,且诱导细胞的凋亡率为29.52%,表明TRAIL-linker-HN具有双基因抑瘤的协同效应。     

Limited phenotypic and functional maturation of bovine monocyte-derived dendritic cells following Mycobacterium avium subspecies paratuberculosis infection in vitro

After encountering antigen, dendritic cells (DC) must differentiate into a fully mature phenotype to induce a protective, lasting T cell immunity. Paratuberculosis is a disease caused by the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and is characterized by a transient cell mediated immune response, that when dissipates correlates to the onset of clinical disease. In order to study the mechanism of early cellular immunity associated with M. paratuberculosis infection, we tested the hypothesis that M. paratuberculosis infected bovine DC have impaired activation and maturation thus are defective in the initiation of a sustainable and protective Th1 immune response locally. Our results demonstrate that M. paratuberculosis infected DC showed decreased endocytosis of ovalbumin, indicating some functional maturation. Co-stimulatory molecules CD40 and CD80 mRNA expression from M. paratuberculosis infected DC was increased over untreated immature DC. M. paratuberculosis infection induced chemokine receptor CCR7 increase in DC, yet CCR5 remained high. MHC II surface expression remained low on M. paratuberculosis infected DC. M. paratuberculosis infection inhibited pro-inflammatory cytokine IL-12 production and promoted IL-10 secretion by bovine DC. Together, our findings showed evidence of phenotypic and functional maturation of DC. However, we did not see the expected antigen presentation via MHC II and cytokine responses as a fully mature DC. This may suggest semi-mature DC phenotype induced by M. paratuberculosis infection.     

Development-related expression of KGF and FGF-10 mRNA in the canine prostate gland

Keratinocyte growth factor (KGF) and fibroblast growth factor 10 (FGF-10) are stromal-derived growth factors that interact with their epithelial FGFR2 receptors to mediate stromal--epithelial cell interaction within the prostate gland. This study was conducted to compare the development-related mRNA expression of KGF, FGF-10 and their receptor FGFR2 in immature and mature canine prostate glands. In addition, their expression levels were correlated with the differentiation of stromal cells using vimentin as a mesenchymal cell marker. Quantitative mRNA expression was assessed by real-time polymerase chain reaction (PCR) and the results were expressed as relative mRNA expression of the target gene, which was normalized to the GAPDH reference gene. mRNA analysis revealed a differential expression of KGF, FGF-10 and FGFR2 receptor by the prostate glands of immature and mature dogs. The results showed a 7.3- and 9-fold decrease in mRNA expression of KGF and FGF-10 by mature and immature prostate glands respectively. However, there was no significant change in FGFR2 receptor mRNA expression by mature or immature prostate glands. This downregulation of KGF and FGF-10 expression was associated with a 15-fold decrease in vimentin expression. These results indicate that KGF and FGF-10 expression varied according to the differentiation status of stromal cells and might reflect differential developmental requirements of immature and mature canine prostate glands.     

Sheep erythrocyte and bluetongue virus antibody responses of spleen cell cultures from mice.

The optimum conditions for the culture of cells from dissociated spleens were determined. Routinely, 10(7) cells were seeded per ml of RPMI 1640 medium supplemented with 20% pre-tested foetal calf serum. For the assay of the immune response, cultures were supplemented with 30 muMolar mercaptoethanol. The immune responses to sheep erythrocyte and bluetongue virus antigens were determined by the haemolytic plaque-forming cell assays described by Oellermann (1974) and Oellermann, Carter & Marx (1976a). The optimum sheep erythrocyte antigen concentration was 2 X 10(6) erythrocytes per 10(7) spleen cells and maximum IgM plaque-forming cells were detected after 4 days in culture. Successful stimulation of the immune response to bluetongue virus was achieved in spleen cell cultures from mice previously primed with bluetongue virus. The optimum antigen concentration was 30-40 ng bluetongue virus per 10(7) spleen cells and the maximum plaque-forming cell response was observed after 4 days in culture.