Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.     

Molecular cloning and functional analysis of duck Toll-like receptor 5

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the single-exon TLR5 gene of the Maya breed of Common Shelduck (Tadorna tadorna). The TLR5 open reading frame is 2580 bp in length and encodes an 859-amino acid protein. The putative amino acid sequence of duck TLR5 consisted of a signal peptide sequence, 11 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain, a transmembrane domain, and an intracellular Toll-interleukin-1 receptor domain. The duck TLR5 gene was highly expressed in the lung, bone marrow, spleen, and liver; moderately expressed in kidney, small intestine, large intestine, and brain. A plasmid expressing duck TLR5 was constructed and transfected into HEK293T cells, and expression was confirmed by indirect immunofluorescence assay. HEK293T cells transfected with duck TLR5- and NF-κB-luciferase-containing plasmids significantly responded to flagellin from Salmonella typhimurium, indicating that it is a functional TLR5 homolog.     

金定鸭不同组织和血液中TLR1、TLR2、TLR4、TLR5的表达差异分析

试验旨在研究不同Toll样受体（Toll-like receptor,TLR）在鸭不同组织中的表达情况,选取300日龄雄性金定鸭10只,解剖后采集其血液及脾脏、肝脏、睾丸、肺脏、下丘脑、垂体、皮肤、腿肌、心脏、肾脏、胸肌、盲肠、小肠、胸腺,采用Primer Premier 5.0软件设计特异性引物,并用实时荧光定量PCR法检测TLR1、TLR2、TLR4、TLR5在不同组织中的相对表达情况。结果显示,各基因扩增产物的熔解曲线均有一特异性的单峰,无其他杂峰,说明引物的特异性较强,可以准确定量。4种目的基因与内参基因的扩增效率在101.4%~105.0%之间,均接近100%,相关系数（R²）为0.98~1.000。TLR1、TLR2、TLR4、TLR5 4种TLRs在金定鸭不同组织和血液中均有表达,但每种TLR受体在各组织中表达水平略有差异,其中TLR1在下丘脑中表达量最低,在胸肌中最高;TLR2在小肠中的表达量最低,在肺脏中最高;TLR4、TLR5在睾丸中的表达量最低,在皮肤中最高。以上结果说明,鸭TLRs在多种组织中能够广泛表达,本试验结果可为TLRs在鸭源感染过程中的作用机理研究提供科学依据。     

Analysis of TLR1,TLR2,TLR4 and TLR5 Expression in Different Tissues of Jinding Duck

The aim of this study was to explore the expression profiles of Toll-like receptor (TLR) in different tissues,and to lay the foundation of TLRs research for ducks.Ten male Jinding ducks of 300 days of age were slaughtered,and the blood sample and 14 different kinds of tissues (spleen, liver, testise, lung,hypothalamus,pituitary gland,skin,leg muscle,heart,kidney, chest muscle,caecum,small intestine and thymus) were collected. The Primer Premier 5.0 software was used to design specific primers and the quantitative Real-time PCR method was used to detect the relative expression levels of TLR1,TLR2,TLR4 and TLR5 mRNA. The results showed that there were only one specific peak on the melting curve of each gene amplification products,indicating that the specificity of the primers were strong. The amplification efficiency of the target genes was 101.4% to 105.0% and the correlation coefficient (R²) was 0.98 to 1.000. The four kinds of TLRs expressed in all of the 14 tissues and blood with varying abundance in different tissues for each of the 4 kinds of TLRs.The expression levels of TLR1 was lowest in hypothalamus,and highest in chest muscle. The expression levels of TLR2 was lowest in small intestine,and highest in lung. The lowest expressed tissues for TLR4 and TLR5 were testis, and the highest expressed tissues were skin.The study indicated that TLRs were widely expressed in various tissues of duck,which laid the foundation for further study on the role mechanism of duck TLRs in the process of pathogen infection.     

Cloning, expression and bioinformatics analysis of the duck TLR 4 gene

1. Toll-like receptors (TLRs) are type I transmembrane proteins that play an essential role in the innate immune system. Studies on the structure and function of TLRs can be applied to the development of new approaches to control diseases of humans and animals.

2. A 3432-bp cDNA encoding duck toll-like receptor 4 (duTLR4) was cloned from duck splenic lymphocytes using RT-PCR and rapid amplification of cDNA ends.

3. The encoded protein, which was predicted to contain 843 amino acids, had a molecular weight of 96·01?kDa and included an archetypal toll/interleukin-1 receptor domain, a transmembrane domain, and a distinctive arrangement of extracellular leucine-rich repeat regions similar to chicken TLR4, human TLR4, and mouse TLR4. The duTLR4 showed 82·1% amino acid sequence identity with previously described chicken TLR4, and 43·2–45·2% sequence identity with mammalian homologs.

4. RT-qPCR analysis indicated that the duTLR4 gene was strongly expressed in the liver, kidney, spleen, intestine, and brain.     

鸭β-防御素-2基因的克隆测序与组织表达分析

利用RT-PCR技术,从肝脏组织中扩增到鸭AvBD2基因.经测序表明,扩增到的鸭AvBD2大小为350 bp,含有1个大小为195 bp的开放阅读框,编码64个氨基酸残基.组织表达分析表明,鸭AvBD2基因在鸭脾脏和肾脏中大量表达;心脏和肝脏中有少量表达;肺脏和骨髓有中等水平表达;在胸腺、腔上囊、小肠、胰腺、腺胃、食管、气管、舌头、胸肌、卵巢、皮肤中未见表达.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Molecular cloning, polymorphism and tissue distribution of the MHC class IIB gene in the Chinese goose (Anser cygnoides)

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5'-RACE sequences (Ancy-MHC II5'-1, Ancy-MHC II5'-2, Ancy-MHC II5'-3), one 3'-RACE sequence (Ancy-MHC II-3') and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.     

蒙古马不同组织器官TLR1、TLR2、TLR4和TLR6 mRNA转录水平研究

根据GenBank中马Toll样受体基因序列设计特异性引物,建立检测马Toll样受体（TLRs）mRNA转录水平的SYBR GreenⅠ实时荧光定量PCR方法,检测TLR4、TLR2、TLR1和TLR6在蒙古马不同组织器官中的转录水平。4种TLRs在心脏、肝脏、脾脏、肺脏、肾脏、胃、十二指肠、空肠、盲肠和骨髓中均有转录。其中,TLR4mRNA除在空肠和肝脏外,在其他组织器官中的表达水平均高于TLR2、TLR1、TLR6。免疫器官中,TLR4、TLR1mRNA在骨髓中表达量高于脾脏,而TLR2、TLR6mRNA在脾脏中表达量高于骨髓。各肠段,TLR4、TLR2、TLR1、TLR6mRNA表达水平之间在空肠的差异不是很大,而在十二指肠和盲肠中差异很大。结果表明,TLRs mRNA在马各组织器官转录水平差异较大,可能与其对病原体的识别和抵抗能力有关。     

PK15细胞α干扰素效应因子qPCR检测方法的建立及其初步应用

 
为建立一种用SYBR Green I荧光染料检测PK 15细胞α干扰素(α IFN)效应因子Mx1、OAS的mRNA表达水平的qPCR检测方法，通过在猪圆环病毒2型（Porcine Circovirus Type 2, PCV2）抑制α IFN发挥效应的信号通路中进行初步应用。根据GenBank中目的基因的序列，利用分子生物学软件Premier 5.0在其保守区设计并合成相应的特异性引物。利用TRIzol法提取总RNA，经Oligo d(T)15进行反转录，利用PCR扩增各段目的基因，并克隆至pMD 18 T载体，转化大肠杆菌DH 5α，经鉴定为阳性的重组质粒作为标准品模板建立SYBR Green I qPCR标准曲线和溶解曲线，并进行灵敏性、特异性和重复性试验。根据建立的实时qPCR方法，检测PCV2对α IFN效应因子的抑制效果。对建立的PK 15细胞α IFN效应因子SYBR Green I qPCR方法进行分析，结果表明Mx1、OAS和内参β actin基因的Ct值与标准品稀释度在1×101～1×108 copies/μL的范围分别呈良好的线性关系。PK 15细胞在接种PCV2，并受到α IFN刺激后Mx1、OAS的相对表达量较未接种PCV2明显降低。本试验建立了PK 15细胞α IFN效应因子的qPCR检测方法，为在mRNA水平上对PK 15细胞α IFN效应因子的定量分析奠定了基础，并成功地初步应用于PCV2抑制α IFN发挥效应的信号通路研究中。     

五指山猪不同组织中Myf5与MyoD1基因的表达研究

 MRFs家族成员包括Myf5、MyoD1、Myf4和Mfy6,利用Real time PCR技术,检测Myf5、MyoD1基因在从出生到体成熟（30 d、210 d、360 d）五指山猪背部肌肉组织中的表达变化趋势,以及Myf5、MyoD1基因在体成熟五指山猪心脏、肝脏、肺脏、脾脏、肾脏、肌、胃和小肠以上组织中的mRNA表达水平。结果表明：Myf5和MyoD1基因在出生后五指山猪背部肌肉组织中的mRNA表达水平与五指山猪的生长年龄成正比（P<0.05）;Myf5及MyoD1基因在成年五指山猪以上8种组织中均有表达,其中在肌肉组织中相对表达量最高。     

牦牛IGFBP4和IGFBP5基因的克隆及其在各组织和不同生长阶段肝中的差异表达分析

旨在克隆牦牛胰岛素样生长因子结合蛋白4和5（insulin-like growth factor binding protein 4 and 5,IGFBP4 and IGFBP5）基因,并检测其在牦牛不同组织及不同生长阶段肝中的表达水平。本研究选取1日龄、15月龄和5岁龄健康雌性麦洼牦牛共9头（每组各3头）,体重分别约为（12.35±1.85）、（98.88±2.50）和（268.55±27.82） kg,采集5岁龄牦牛心、肝、脾、肺、肾、小肠组织及3个不同生长阶段牦牛肝组织。本试验克隆IGFBP4和IGFBP5基因,并对其序列进行生物信息学分析,采用qRT-PCR方法、Western blot技术与免疫组化技术检测其在5岁龄牦牛6种组织及1日龄、15月龄和5岁龄3个不同生长阶段肝中的表达差异。结果表明,获得牦牛IGFBP4和IGFBP5基因的CDS序列分别为777和816 bp,分别编码258和271个氨基酸,GenBank序列号分别为MT012934和MT003005。在同源性比较中,牦牛IGFBP4和IGFBP5序列均与黄牛的同源性最高,分别为99.6%、99.5%。组织表达谱分析显示,IGFBP4和IGFBP5基因均在5岁龄牦牛肝中表达量最高,与肾、心、小肠、脾、肺相比差异极显著（P<0.01）,且6种组织中IGFBP4基因的表达量均极显著高于IGFBP5基因（P<0.01）。不同生长阶段肝中差异表达结果显示,随着年龄的增长,IGFBP4和IGFBP5的mRNA与蛋白水平在牦牛肝脏组织中的表达量均呈现上升趋势,即5岁龄>15月龄>1日龄,在5岁龄时表达量最高,与1日龄和15月龄均存在极显著差异（P<0.01）,且IGFBP4的表达量极显著高于IGFBP5（P<0.01）。结果提示,IGFBP4和IGFBP5可能协同调控牦牛肝的生长发育,这为深入研究IGFBP4和IGFBP5在牦牛肝生长发育过程中的作用以及表达调控提供了一定的基础数据。     

鼠源重组UBC13蛋白对脂多糖诱导的小鼠急性炎症的影响

试验旨在探讨鼠源重组UBC13蛋白对脂多糖(lipopolysaccharide,LPS)诱导的小鼠急性炎症的影响。将24只SPF雌性小鼠随机分成4组:PBS组,LPS模型组,重组UBC13蛋白高、低剂量组(分别为100和25μg/只),每组6只。LPS模型组与各蛋白剂量组腹腔注射20 mg/kg LPS,PBS组腹腔注射等体积PBS;注射结束1 h后,各蛋白组按相应剂量背部皮下多点注射重组UBC13蛋白,PBS组与LPS模型组注射等体积PBS。给予蛋白24 h后处死小鼠。收集小鼠肺脏、脾脏、胸腺及肝脏组织,计算脏器指数,HE染色观察组织病理学变化,实时荧光定量PCR检测肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA的相对表达量,以及肺脏中iNOS mRNA的相对表达量,综合评价鼠源重组UBC13蛋白对LPS诱导小鼠急性炎症的影响。结果显示,与PBS组相比,LPS模型组小鼠肺脏、脾脏及肝脏指数均显著或极显著升高(P<0.05;P<0.01),且肺脏、脾脏和肝脏组织均出现病理变化。实时荧光定量PCR结果显示,与PBS组相比,LPS模型组肺脏、脾脏、胸腺和肝脏中IL-1β、TNF-α、IL-6 mRNA相对表达量均极显著升高(P<0.01),肺脏中iNOS mRNA相对表达量也极显著升高(P<0.01);与LPS模型组相比,UBC13蛋白高剂量组肺脏、脾脏和肝脏中病理变化明显改善,肺脏、肝脏、脾脏中IL-1β、TNF-α、IL-6及肺脏中iNOS mRNA表达量均极显著降低(P<0.01);胸腺中TNF-αmRNA表达量显著降低(P<0.05),IL-6 mRNA和IL-1β表达量极显著降低(P<0.01)。表明鼠源重组UBC13蛋白可下调炎性因子的表达,从而改善LPS诱导的小鼠急性炎症反应。     

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.     

HeLa细胞Ⅰ型干扰素效应因子实时荧光定量RT-PCR检测方法的建立及初步应用

本试验旨在建立一种用SYBR GreenⅠ荧光染料检测HeLa细胞Ⅰ型干扰素效应因子ISG15、ISG56、Mx1、OAS和PKR mRNA表达水平的实时荧光定量RT-PCR检测方法,并在口蹄疫病毒(FMDV)L蛋白抑制Ⅰ型IFN发挥效应的信号通路中进行初步应用。利用TRIzol法提取总RNA,经Oligo d(T)15进行反转录,利用PCR扩增各段目的基因,并克隆至pMD18-T载体,转化大肠杆菌DH5α,经鉴定为阳性的重组质粒作为标准品模板建立SYBR GreenⅠ荧光定量RT-PCR标准曲线和熔解曲线,并进行灵敏性、特异性和重复性试验。根据建立的实时荧光定量RT-PCR方法,检测FMDV L蛋白对Ⅰ型IFN效应因子的抑制效果。HeLa细胞在转染FMDV L蛋白真核表达质粒,并受到Ⅰ型IFN刺激后ISG15、ISG56、Mx1、OAS和PKR的相对表达量较转染空载体或表达GST的真核表达质粒明显降低。本试验建立了HeLa细胞Ⅰ型IFN效应因子的实时荧光定量RT-PCR检测方法,为在mRNA水平上对HeLa细胞Ⅰ型IFN效应因子的定量分析奠定了基础,并成功地初步应用于FMDV L蛋白抑制Ⅰ型IFN发挥效应的信号通路的研究中。     

冷应激对湖羊血清因子及热休克蛋白70 mRNA表达的影响

为研究冷应激对湖羊免疫系统及热休克蛋白70(heat stress proteins 70,Hsp70)的影响,试验分别采用实时荧光定量PCR和ELISA方法检测冷应激前后湖羊肝脏、肺脏、脾脏、淋巴结组织中Hsp70mRNA表达量及血清中白细胞介素-2(IL-2)、白细胞介素-4(IL-4)浓度。结果显示,与冷应激前相比,冷应激后湖羊肝脏、肺脏及脾脏组织中Hsp70mRNA表达量均极显著增加(P0.01),其中肝脏的表达量尤为显著;冷应激后湖羊血清中IL-2、IL-4的浓度均呈下降趋势,其中IL-4浓度下降尤为显著(P0.01)。结果表明,冷应激条件下湖羊各组织中Hsp70mRNA表达增强,可提高动物机体的自我保护机能,增强对外界不良刺激的抵抗力,但细胞因子IL-2和IL-4的浓度下降,表明冷应激抑制机体免疫系统。