Toll-like receptor expression in feline lymphoid tissues

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that activate the innate immune system. While it is clear that TLRs are important in the immune response against pathogens, they may also be exploited by some pathogens. Our objective is to determine whether feline immunodeficiency virus (FIV) infection affects TLR expression or function thereby resulting in innate immune dysfunction. To this end, we cloned partial sequences for feline TLRs 1--3, 5--8, and developed real-time PCR assays to quantify feline TLRs 1--9. TLR expression was quantified in normal cat lymphoid tissues, purified lymphocyte subsets, and FIV-infected cell lines. Different expression patterns of TLRs were found in spleen, mesenteric lymph node, retropharyngeal lymph node, thymus, intestinal intraepithelial lymphocytes, and lamina propria lymphocytes. B lymphocytes, CD4+ T cells, and CD8+ T cells all expressed TLRs 2--5, 7--9; however, the relative levels of expression varied among lymphocyte phenotypes. Infection of cell lines with FIV resulted in altered TLR expression levels that differed depending on cell type. These results demonstrate that tissue distribution of TLRs is associated with the immunological role of a particular tissue, that lymphocytes may also express these 'innate immune' receptors, and that FIV infection can alter TLR expression.     

猪Toll样受体2基因的克隆和序列分析

本研究从猪肠系膜淋巴细胞中，克隆了猪Toll样受体2基因（pTLR2）。基因序列分析表明，克隆的pTLR2基因ORF为2358bp，编码785个氨基酸，合15．01％的亮氨酸，合有一段21个氨基酸的信号肽序列；与GenBank中登录的pTLR2序列（NM213761）的同源性为99．2％；与牛、马、绵羊和人的同源性较高，与家鼠、中国仓鼠相比次之．而与鱼类的最低。蛋白分子结构预测表明，该分子由胞外区（588个氨基酸）、跨膜区（23个氨基酸）和胞内区（174个氨基酸）组成，其胞外结构域由多个串联重复的LRR基序（XLXXLXLXX）组成，而胞内区合有与人白细胞介素1受体（IL-1R）高度同源的区域即TOLL／IL-1受体同源区（TIR），说明pTLR2分子具有病原分子模式识别和信号传导的作用，这为其结构与功能的进一步研究奠定了基础。     

梅花鹿天然Toll样受体9基因克隆与序列分析

Trizol法提取梅花鹿肝脏总RNA,采用ORF两端兼并引物,RT-PCR方法克隆梅花鹿天然Toll样受体9基因（TLR9）并进行系统的生物信息学分析。RT-PCR结果显示,梅花鹿TLR9基因的mRNA长4 043bp,含有800bp左右的5’UTR,完整ORF编码1 081个氨基酸（GenBank序列号为：HQ260632）。同源性分析显示梅花鹿TLR9基因编码区与其他物种高度同源,但5’UTR区存在基因结构的变异。功能结构域分析显示不同物种间TLR9结构域相对保守但也存在细微差别,结构域的差别导致梅花鹿TLR9蛋白胞外区马蹄形弧顶内侧空间结构由人类TLR9蛋白的弧形改变为梅花鹿TLR9蛋白的三角形。进化分析显示TLR9基因分子进化关系与物种间真实进化相一致。     

猪Toll样受体9基因的克隆、序列分析及其结构预测

本研究应用反转录-聚合酶链式反应(RT-PCR)扩增技术,从猪脾脏淋巴细胞中,克隆了猪Toll样受体9基因(pTLR9).基因序列分析表明,克隆的pTLR9基因ORF为3 093 bp,编码1 030个氨基酸,含18.5%的亮氨酸,含有24个氨基酸的信号肤序列,属于Ⅰ型跨膜受体,具有富含亮氨酸的重复序列(LRR)和Toll/IL-1R同源区结构域;与GenBank上登载的pTLR9参考序列(AY859728)的同源性为99.3%,与牛、马、羊和人的同源性较高,与家鼠、褐鼠的次之,TLR9的演化关系与亲缘关系密切.     

犬瘟热病毒疫苗株H蛋白基因片段的克隆与表达及表达产物抗原性的初步鉴定

根据发表的犬瘟热病毒（CDV）参考株Ondetstepoort的序列设计一对引物,以CDV感染的Vero细胞总RNA为模板,经RT-PCR扩增得到549bpDNA片段,将该片段以正确的阅读框架定向克隆于pGEX-4T-1中,然后将重组质粒转化宿主菌Rosetta^TM,在37℃、1．0mmol／LIPTG诱导下外源基因获得良好表达。经SDS．PAGE鉴定,表达的融合蛋白约46ku,与预期值一致。Westernblot试验显示,CDV免疫的小鼠血清可特异的识别该重组蛋白,表明该重组蛋白具有一定的免疫原性。     

Characterization of canine filaggrin: gene structure and protein expression in dog skin

Background – Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. Hypothesis/Objectives – The aim of this study was to determine the structure of the canine FLG gene and to raise anti‐dog FLG antibodies, which will be useful to detect FLG protein in dog skin. Methods – The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti‐dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. Results – Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti‐dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. Conclusions and clinical importance – This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti‐dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG‐deficient dogs with canine atopic dermatitis or ichthyosis.     

Molecular cloning of canine thymus and activation-regulated chemokine (TARC) gene and its expression in various tissues

Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.     

Molecular cloning of canine interleukin-31 and its expression in various tissues

The newly discovered cytokine, interleukin-31 (IL-31), belongs to the short-chain cytokine group. It was reported that transgenic expression of IL-31-induced pruritus, similar to atopic dermatitis, in mice, further, excessive amounts of IL-31 was also expressed in the skin from human patients with atopic dermatitis as compared to that from normal people. In this study, canine IL-31 was molecularly cloned from concanavalin A-stimulated canine peripheral blood mononuclear cells (PBMCs), and its nucleotide sequence was determined. Canine IL-31 contains 4 alpha-helix structures characteristic of the IL-31 family, and the amino acid identity of canine IL-31 with those of human or mouse is 54% and 28%, respectively. Furthermore, we detected low levels of canine IL-31 in the thymus, testis, spleen, and kidneys, but not in the skin of atopic dogs.     

犬细小病毒结构蛋白VP2基因的克隆和在毕赤酵母中表达

表达犬细小病毒VP2蛋白（CPV—VP2）,用于重组蛋白免疫小鼠制备单克隆抗体,并为犬细小病毒病的诊断奠定基础。采用PCR方法对CPVVP2基因进行扩增,将CPVVP2基因克隆到毕赤酵母分泌表达载体pPICZAA中,构建真核重组表达载体pPICZAA-VP2,将该重组质粒线性化后,转化巴斯德毕赤酵母（Pichiapastoris）X-33中,甲醇诱导表达CPVVP2,SDs_PAGE和Westernblotting鉴定表达蛋白。结果,成功扩增了CPV—VP2基因,构建了真核重组表达载体pPICZAA—VP2,在毕赤酵母菌中表达出约68000蛋白。Western—blotting鉴定表明,表达蛋白为目的蛋白VP2。表达菌株扩大表达体系于培养基中培养,上清液用70％过硫酸铵4℃沉淀浓缩,采用His选择镍-亲和层析柱分离纯化获得重组的酵母表达的带组氨酸标签的VP2蛋白,表达量约3mg／L。结果表明,在毕赤酵母中成功地表达了CPV—VP2蛋白,且能被犬细小病毒VP2单克隆抗体特异识别。     

Molecular cloning and tissue-specific expression of Toll-like receptor 5 gene from turkeys

Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1beta, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.     

猪GDF11基因部分序列的克隆及组织表达分析

为了探讨生长分化因子11(Growth differentiation factor11,GDF11,又名BMP11)在胸腰椎数变异中的作用,本试验克隆了该基因包含外显子2在内的部分编码区,并进一步采用RT-PCR技术对其在猪胚胎和初生仔猪中的表达进行了分析。结果表明,在35d的猪胚胎中,后肢、牙龈、脑、肝脏、肾脏、胸椎、腰椎各组织均有明显的表达,而在前肢、眼、心脏、肺脏中的表达较弱,在颈椎和荐尾椎中没有观察到GDF11的表达。在45d猪胚胎的后肢、脑、眼、胸椎组织中GDF11的表达较强,而在前肢、牙龈、肺脏、肾脏、腰椎和荐尾椎的表达相对较弱,在肝脏中的表达极其微弱。在心脏和颈椎中没有检测到GDF11的表达。在55d的猪胚胎中,前肢、后肢、脑、眼、肝脏、颈椎、胸椎、腰椎组织中有明显的表达,肺脏和肾脏组织中的表达较强,牙龈和荐尾椎中的表达较弱,而在心脏中没有检测到GDF11的表达。3d仔猪的后肢、牙龈、脑、肾脏和腰椎组织中GDF11有明显的表达,脾脏组织的表达量较高,前肢、肝脏、心脏、背腰最长肌和肺脏中的表达相对较弱,在眼、颈椎和荐尾椎中的表达极弱,在胸椎中没有检测到表达。在所检测的不同时期的所有组织中,脑和肾脏组织表达明显地高于其他组织。     

中国新吉细毛羊天然Toll样受体8基因(TLR8)克隆与序列分析

为了解绵羊Toll样受体8(TLR8)蛋白的结构特征和进化关系。本试验采用Trizol法提取新吉细毛羊肝脏组织总RNA,设计ORF区两端兼并引物,RT-PCR方法克隆新吉细毛羊TLR8并进行了系统的生物信息学分析。RT-PCR结果显示,试验获得了大小为3 102bp的片段,包括完整的ORF区2 982bp,编码993个氨基酸(GenBank序列号为:GU936186和GU936187),含15.7%的亮氨酸。同源分析结果显示,TLR8在进化过程中具有高度保守性,与人、家牛、山羊、猪、梅花鹿和小鼠的氨基酸序列同源性分别为79.9%、96.3%、97.6%、84.7%、96.5%和74.2%。蛋白分子功能结构域预测显示,该分子由胞外区(781个氨基酸)、跨膜区(52个氨基酸)和胞内区(160个氨基酸)组成,其中胞外区具有LRR结构域,胞内区具有TIR结构域,且绵羊较人类多了LRR3和LRR12结构域。进化分析显示TLR8分子与不同物种间的进化关系符合真实物种间的进化顺序。试验结果表明新吉细毛羊TLR8分子具有病原分子模式识别作用,为其结构和功能的研究奠定了基础。     

Expression and function of Toll-like receptor 2 in canine blood phagocytes

Toll-like receptors (TLRs) are a family of highly conserved pattern recognition receptors (PRR) of mammals that participate in the activation of innate immune responses against microbial infections. Among these receptors, TLR2 is essential for the recognition of conserved structural components of bacteria, protozoa and fungi. Until now, expression of TLR2 in dogs has not been investigated. In this work we describe a partial sequence of the gene coding for canine TLR2 and show that TLR2 mRNA is constitutively expressed in canine blood PMNs. We also show that stimulation of purified PMNs with lipoteichoic acid (LTA), a ligand of TLR2, leads to the release of proinflammatory chemokine IL-8. Furthermore, TLR2 protein is easily detectable by flow cytometry on the canine peripheral blood granulocyte and monocyte cell surface, and slightly on lymphocytes. These findings suggest that, also in dogs as in humans the initial antibacterial response of PMNs could be elicited through engagement of TLR2.     

Influence of age,sex and rearing systems on Toll-like receptor 7 (TLR7) expression pattern in gut,lung and lymphoid tissues of indigenous ducks

1. The objective of the experiment was to determine the influence of age, sex and rearing system on Toll-like receptor 7 (TLR7) gene expression in gut, lung and lymphoid tissues and physiological responses to stress in male and female indigenous ducks of Tamil Nadu, India.

2. A total of 36 ducks (12 males and 24 females) were obtained from local farmers and tissue samples of gut tissues (duodenum, jejunum, ileum and caecum), lymphoid organs (spleen and bursa) and lungs were collected in RNAlater solution followed by RNA extraction.

3. After normalisation to β-actin (endogenous control) qPCR analysis identified a significant effect of age, sex and rearing system on TLR7 expression in the ducks.

4. A significant up-regulation of TLR7 expression was observed in lungs, duodenum, jejunum, ileum and caecum of sexually mature (45 wk) compared with that of immature ducks (16 wk). Among sexes, male ducks had significantly higher TLR7 expression than female ducks.

5. Age and sex interactions were significant in lungs, duodenum, jejunum and caecum. Ducks reared in an extensive housing system showed significantly higher TLR7 expression in bursa, lungs, duodenum, ileum and caecum compared to intensively reared ducks. There were no effects of age, sex and rearing systems on TLR7 expression in the spleen.

6. The heterophil-to-lymphocyte ratio and serum corticosterone were higher in ducks reared on an intensive system compared with ducks from an extensive rearing system.     

犬瘟热病毒水貂分离株F基因的克隆及原核表达

根据GenBank中登录的犬瘟热病毒F基因序列设计了1对引物，以从水貂犬瘟热病毒中提取的RNA为模板，扩增出一约1000bp的F基因片段。将PCR产物按相应的阅读框架克隆到原核表达载体pET-32a中，并将重组质粒转化E．coli BL21（DE3），用1．0mmol／L IPTG在30℃下诱导表达。结果显示，F基因的表达量约占细菌总蛋白的35％。SDS-PAGE电泳显示，表达产物的分子质量约为55ku，与预计大小相符；经Western-blotting试验进一步证实，该基因获得了正确表达。     

猪繁殖与呼吸综合征病毒ORF7基因的克隆及其在毕赤酵母中表达

用RT-PCR扩增猪繁殖与呼吸综合征病毒(PRRSV)重庆分离株C14-2的ORF7基因(384 bp),构建克隆质粒pMD19-T-ORF7,经EcoR Ⅰ /Not Ⅰ双酶切回收ORF7基因插入酵母表达载体pPIC9K,构建了重组表达质粒pPIC9K-ORF7,进行PCR鉴定和双酶切鉴定.鉴定的pPIC9K-ORF7经Sac Ⅰ线性化后电转化毕赤酵母宿主菌GS115,筛选获得阳性重组菌GS115(pPIC9K-ORF7),再经G-418/YPD筛选获得高拷贝重组菌,重组子经表型鉴定为Mut.重组菌GS115(pPIC9K-ORF7)经甲醇诱导表达,在96 h表达的N蛋白量最大,N蛋白经SDS-PAGE鉴定大小约为15 000;Western blot表明N蛋白能与美洲型PRRSV阳性血清发生特异性反应,具有良好的反应活性.本研究为开展PRRSV ORF7基因在毕赤酵母中表达及应用奠定基础.