Association of toll‐like receptor 2 polymorphisms with somatic cell score in Xinjiang Brown cattle

This study aims to identify single nucleotide polymorphisms (SNPs) and haplotypes in the TLR2 gene, and analyze the association of SNPs or haplotypes and somatic cell scores in 151 Xinjiang Brown cattle and 138 Holsteins to evaluate the role of TLR2 during intramammary infections. TLR2 coding region was amplified by PCR and screened for SNP sequencing. Genotypes and frequencies of SNPs were identified. Finally, the associations of genotypes or haplotypes and somatic cell scores (SCS) were analyzed. The results showed that: (i) 15 SNPs (E+653, E+945, E+978, E+1010, E+1250, E+1688, E+1707, E+1779, E+1782, E+1891, E+1995, E+2025, E+2055, E+2214 and E+2295) were observed and detected from 289 cows; (ii) distribution of the 14 SNPs were significantly different from Xinjiang Brown cattle and Holstein (P < 0.001) except for the E+945 (P > 0.05); (iii) in 11 SNPs (E+945, E+978, E+1010, E+1688, E+1707, E+1779, E+1782, E+1995, E+2025, E+2055 and E+2214), the SCS of AB genotype was lower than AA (P < 0.05) in Xinjiang Brown cattle; and (iv) haplotypes composed of the above‐mentioned 11 SNPs were constructed. The SCS of cattle with Hap5 was lower than that of Hap3 (P < 0.05). This suggests that Hap5 might play an important role in sub‐mastitis resistance in Xinjiang Brown cattle.     

The equine alveolar macrophage: Functional and phenotypic comparisons with peritoneal macrophages

Alveolar macrophages (AMs) constitute the first line of defence in the lung of all species, playing a crucial role in the regulation of immune responses to inhaled pathogens. A detailed understanding of the function and phenotype of AMs is a necessary pre-requisite to both elucidating their role in preventing opportunistic bacterial colonisation of the lower respiratory tract and developing appropriate preventative strategies. The purpose of the study was to characterise this important innate immune cell at the tissue level by making functional and phenotypic comparisons with peritoneal macrophages (PMs). We hypothesised that the tissue of origin determines a unique phenotype of AMs, which may constitute an appropriate therapeutic target for certain equine respiratory diseases. Macrophages isolated from the lung and the peritoneal cavity of 9 horses were stimulated with various toll like receptor (TLR) ligands and the production of nitrite, tumour necrosis factor alpha (TNFα), interleukin (IL) 10 and indoleamine 2,3-dioxygenase (IDO) were measured by the Griess reaction and enzyme linked immunosorbent assay (ELISA) and/or quantitative polymerase chain reaction, respectively. Cells were also compared on the basis of phagocytic-capacity and the expression of several cell surface markers. AMs, but not PMs, demonstrated increased TNFα release following stimulation with LPS, polyinosinic polycytidylic acid (Poly IC) and heat-killed Salmonella typhinurium and increased TNFα and IDO mRNA expression when stimulated with LPS. AMs showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and TLR4, whereas PMs showed high expression of TLR4 only. AMs, but not PMs, demonstrated efficient phagocytic activity. Our results demonstrate that AMs are more active than PMs when stimulated with various pro-inflammatory ligands, thus supporting the importance of the local microenvironment in the activation status of the macrophage. This information provides a valuable knowledge base on which to improve our understanding of the role of macrophages and their microenvironment in equine innate immunity.     

白羽番鸭TLR7基因多态性与部分生产性状的关联分析

以白羽番鸭为研究材料,根据鸭的TLR7基因序列(DQ888644)设计引物,扩增TLR7基因的第1外显子和部分内含子,并分析其多态性对生产性状的遗传效应。结果表明:PCR产物出现片段长度多态,产生AA和AB2种基因型,其中AA型为优势基因型,A为优势等位基因;测序结果显示,在内含子区209位碱基发生C→T突变,在280位碱基之后缺失/插入3个碱基AAT;χ2检验结果表明,该座位处于Hardy-Weinberg平衡状态;AA型个体的体重、半潜水长和胫围极显著高于AB型(P<0.01),胫长显著高于AB型(P<0.05),其他指标在各基因型间的差异均未达到显著水平(P>0.05),AA型是白羽番鸭生长性状较为有利的基因型。     

chTLR4及其信号通路在脂多糖致鸡淋巴细胞中的作用

Toll样受体4(Toll-like receptor 4,TLR4)是脂多糖(lipopolysaccharides,LPS)的跨膜受体,它介导了LPS诱导机体产生的多种损伤反应。本试验用LPS对鸡淋巴细胞进行6、12、24、48 h诱导,以得到IL-1β、NF-κB水平变化,为深入研究TLR4介导LPS胞内信号传递奠定了基础。结果显示,LPS诱导细胞后,IL-1β、NF-κB含量增多,均在24 h达到最大值,之后开始下降。且在12、24 h与对照组差异显著(P<0.05),在6、48 h与对照组无显著差异（P>0.05）。     

中国美利奴羊TLR2基因多态性及其与布鲁氏菌病的相关性分析

试验旨在研究Toll样受体2（Toll-like receptor,TLR2）基因的多态性及其与中国美利奴羊布鲁氏菌病易感性的相关性。利用生物信息学方法对NCBI上公布的绵羊TLR2基因序列进行比对,选出多态位点丰富的片段进行扩增,运用PCR-SSCP的方法对206个中国美利奴布鲁氏菌病阴性样本和80个中国美利奴羊布鲁氏菌病阳性样本进行TLR2基因的多态性检测,然后对不同等位基因的PCR产物进行测序,确定该基因的多态性位点,经卡方检验分析每个SNP位点的等位基因频率、基因型频率及其多态性与布鲁氏菌病易感性的相关性,利用生物信息学软件分析RNA二级结构及蛋白质的二级结构。结果表明,在279 bp的序列中共检测到3个SNPs,分别为：C1731T、G1737C和G1749T,均未引起对应氨基酸的改变,属于无义突变。这些位点在病例组和对照组之间的等位基因频率及基因型频率均不存在显著差异（P>0.05）。各突变位点均能引起RNA二级结构和最小自由能的改变,而蛋白质的二级结构均未改变。由此得出,中国美利奴羊TLR2基因的3个SNPs位点（C1731T、G1737C和G1749T）与中国美利奴羊布鲁氏菌病易感性无相关性。     

猪Toll样受体3、7和8基因的克隆与序列分析

为了对猪Toll样受体(TLR)3、7和8基因进行克隆与序列分析,本实验从猪肺泡巨噬细胞(PAM)中,利用RT-PCR方法分片段扩增猪TLR3、7、8基因,并克隆于pMD18-T载体中。根据测序结果分析,猪TLR3、7和8基因的ORF分别为2718bp、3153bp和3087bp,并分别编码906、1051和1029个氨基酸。同源性分析结果显示,它们与GenBank中登录的猪TLR的相应序列同源性达99%以上;与牛、马、羊和人的同源性较高,与鼠的同源性次之,与鸡的同源性最低,其蛋白分子结构预测表明猪TLR3、7、8均为跨膜蛋白。     

牛TLR4基因的遗传多态性与乳房炎的关联分析

TLR4通过识别病原体而激活免疫细胞，在先天免疫和适应性免疫防御中起着重要作用。以中国荷斯坦奶牛、三河牛和中国西门塔尔牛共397头为研究对象，利用创造酶切位点PCR法扩增243bp的目的片段，通过限制性内切酶HinfⅠ酶切来检测TLR4第3外显子的多态性，结果发现扩增产物的27bp处C到T的突变使得多态位点产生，编码的氨基酸由苏氨酸变为异亮氨酸。A、B2个等位基因在3个群体中均有分布，A等位基因占优势（大于78％），经χ^2适合性检验，三河牛在该位点未达到Hardy-Weinberg平衡状态（P〈0．05）。运用SAS8．2软件采用最小二乘法拟合线性模型，将该基因座不同基因型与奶牛乳房炎进行了关联分析，结果表明：AA基因型为乳房炎抗性基因型（P〈0．05），A等位基因为乳房炎抗性的有利基因。     

Characterization of an intravenous lipopolysaccharide inflammation model in calves with respect to the acute-phase response

Our objective was to develop a lipopolysaccharide (LPS) inflammation model in calves to evaluate the acute-phase response with respect to the release of pro-inflammatory cytokines and acute-phase proteins, fever development and sickness behaviour. Fourteen 4-week-old male Holstein Friesian calves were included and randomly assigned to a negative control group (n = 3) and an LPS-challenged group (n = 11). The latter received an intravenous bolus injection of 0.5 μg of LPS/kg body weight. Blood collection and clinical scoring were performed at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 18, 24, 28, 32, 48, 54 and 72 h post LPS administration (p.a.). In the LPS group, the following clinical signs were observed successively: tachypnoea (on average 18 min p.a.), decubitus (29 min p.a.), general depression (1.75 h p.a.), fever (5 h p.a.) and tachycardia (5 h p.a.). Subsequent to the recovery from respiratory distress, general depression was prominent, which deteriorated when fever increased. One animal did not survive LPS administration, whereas the other animals recovered on average within 6.1 h p.a. Moreover, the challenge significantly increased plasma concentrations of tumour necrosis factor-α, interleukin 6, serum amyloid A and haptoglobin, with peaking levels at 1, 3.5, 24 and 18 h p.a., respectively. The present LPS model was practical and reproducible, caused obvious clinical signs related to endotoxemia and a marked change in the studied inflammatory mediators, making it a suitable model to study the immunomodulatory properties of drugs in future research.     

利用脂质体法建立转Toll样受体4基因大尾寒羊胎儿成纤维细胞株的研究

利用脂质体转染法获得转Toll样受体4(Toll-like receptors 4,TLR4)基因的成纤维细胞,将其作为供体细胞,再通过体细胞核移植技术构建重构胚,最终生产出转TLR4基因且性别可控的大尾寒羊。本研究通过原代培养大尾寒羊胎儿皮肤成纤维细胞,并进行SRY基因性别鉴定,利用脂质体转染法转入TLR4基因,然后分别从形态学与分子水平检测TLR4基因的表达,将稳定表达转TLR4的成纤维细胞核移植入绵羊去核卵母细胞中构建重构胚。最终获得5个稳定表达TLR4的阳性细胞克隆,经SRY基因性别鉴定为雌性细胞株,通过实时荧光定量PCR分析,在第4代转染的细胞中TLR4表达量最高,比未转染细胞高出7.4816倍(P<0.01),卵裂的重构胚中有EGFP的表达。试验成功构建出含TLR4基因的重构胚,为今后生产出性别可控的抗病转基因绵羊地方新品种奠定基础。     

卵形鲳鲹TLR13基因的克隆、表达分析和亚细胞定位研究

Toll样受体(Toll-like receptor,TLR)是一种古老的先天性免疫受体,参与病原体相关分子模式识别,对维持免疫稳态和预防感染至关重要。本研究克隆和鉴定了卵形鲳鲹(Trachinotus ovatus)TLR13基因(命名为ToTLR13),其开放阅读框(ORF)为1 269 bp,编码422个氨基酸,等电点为8.13。保守结构域分析显示,ToTLR13含有跨膜结构域(TM)、LRR结构域和TIR结构域,符合TLR家族的典型特征。通过建立TLR13保守域三级结构发现,ToTLR13与小鼠(Mus musculus)和大黄鱼(Larimichthys crocea) TLR13功能结构域的蛋白三级结构具有较高重叠性。多序列比对显示,ToTLR13与其他硬骨鱼TLR13具有较高的相似性,与其他纲物种的序列相似性较低。系统进化树结果显示,To TLR13与硬骨鱼TLR13聚在一起,其中与鞍带石斑鱼(Epinephelus lanceolatus)最为接近,与哺乳动物、两栖类和贝类相分离。实时荧光定量PCR (Real-time fluorescence quantitativ...