猪Toll样受体4基因SNPs功能分析

Toll样受体4(Toll-like receptor 4,TLR4)在机体的免疫反应中发挥重要作用,该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上,进一步分析c.611 T>A(p.Leu204His)、c.1027C>A(p.Gln343Lys)和c.1605 G>T(p.Leu535Phe)3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变;利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms,SNPs)对猪TLR4配体识别和信号转导能力的影响;同时,利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果,成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体,构建了不同等位基因的真核表达载体,在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01),该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递,可能和机体的疾病抗性/易感性有关。     

Toll样受体4与病毒感染的研究进展

Toll样受体4（TLR4）是固有免疫系统中能够识别病原相关分子模式的受体家族成员,可识别革兰氏阴性菌的脂多糖（LPS）而在细菌感染性疾病的发生中起重要作用。近年来越来越多的研究发现,TLR4还广泛参与病毒感染性疾病的发生和病毒的免疫逃逸,由于其信号转导通路的独特性和细胞定位的可变性,再次引起人们极大的研究兴趣。该文将介绍TLR4的生物学特性、信号转导通路及TLR4与病毒感染的最新研究进展。     

Toll样受体与脑缺血

脑缺血是临床上的常见病,目前已成为导致人类死亡的主要原因之一,脑缺血后存在一个十分复杂的病理生理过程,涉及很多机制。包括离子稳态的破坏,自由基的损伤作用,兴奋性氨基酸的毒性作用,免疫炎症作用和细凋亡等机制。TOLL样受体即TLR（Toll-like receptor TLR）,是一类介导天然免疫的跨膜信号传递受体家族,在细胞活化信号传导中发挥着重要作用,已成为联系天然免疫和后天性免疫的桥梁。现在共发现了13中TOLL样体,最近发现Toll样受体能诱发机体的固有免疫应答,介导炎症因子细胞因子的释放,与全身多种重要器官的缺血再灌注损伤有关,研究表明部分TOLL样受体在脑缺血损伤中发挥十分重要的作用。本文就TLR的结构、分布、配体、信号转导通路及其脑缺血中的作用综述如下。     

Toll样受体与脑缺血

脑缺血是临床上的常见病,目前已成为导致人类死亡的主要原因之一,脑缺血后存在一个十分复杂的病理生理过程,涉及很多机制。包括离子稳态的破坏,自由基的损伤作用,兴奋性氨基酸的毒性作用,免疫炎症作用和细凋亡等机制。TOLL样受体即TLR(Toll-like receptor TLR),是一类介导天然免疫的跨膜信号传递受体家族,在细胞活化信号传导中发挥着重要作用,已成为联系天然免疫和后天性免疫的桥梁。现在共发现了13中TOLL样体,最近发现Toll样受体能诱发机体的固有免疫应答,介导炎症因子细胞因子的释放,与全身多种重要器官的缺血再灌注损伤有关,研究表明部分TOLL样受体在脑缺血损伤中发挥十分重要的作用。本文就TLR的结构、分布、配体、信号转导通路及其脑缺血中的作用综述如下。     

Toll样受体4表达的调节

Toll样受体4(TLR4)是最早发现的人类Toll样受体,主要表达于树突状细胞和巨噬细胞等免疫细胞表面,在免疫应答和炎症反应中起重要作用。本文综述了影响TLR4表达的包括上调因素和下调因素在内的一些主要调节因素。     

Toll样受体及其信号转导

Toll样受体(TLR)介导着绝大部分哺乳动物、昆虫及植物的宿主防御. TLR4与配体结合涉及膜抗原CD14和分泌蛋白MD-2的调节并一起形成受体复合物, 然后与接头分子MyD88结合, 使IRAK磷酸化, 再使TRAF6寡聚化, 随后激活控制着各种效应基因表达的转录因子NF-κB.     

选择性剪接在Toll样受体4信号转导通路中的作用

Toll样受体4(Toll-likereceptor4,TLR4)属于模式识别受体,可识别来自G-细菌细胞壁的脂多糖(lipopoly-saccharides,LPS),并通过MyD88依赖途径或MyD88非依赖途径进行信号转导,引发核因子-κB(NF-κB)和其他转录因子的表达,从而诱导细胞因子、化学趋化因子的产生,引起系统性炎症反应。选择性剪接是真核生物控制基因表达的一种重要机制,在TLR4通路中很多信号分子都存在着选择性剪接产生的异构体,且这些剪接异构体分子大都可负性调控TLR4信号转导通路。本文针对这方面的研究进展作一综述。     

Toll样受体4(TLR4)基因剔除小鼠构建及初步表型分析

目的 利用CRISPR/Cas9技术构建Toll样受体4(TLR4)基因敲除小鼠模型,并观察突变小鼠对革兰氏阴性细菌脂多糖(LPS)刺激响应的变化。方法 针对TLR4基因外显子2设计并合成1对sgRNA片段,与编码Cas9的mRNA混合后通过受精卵显微注射方法,建立TLR4基因敲除小鼠,通过繁育获得基因敲除纯合子小鼠(TLR4^-/-小鼠);通过LPS刺激,分析TLR4^-/-小鼠对炎症应激的反应情况,并在分子和病理水平上和野生型对照(WT)进行比较。结果 PCR及测序检测表明TLR4基因外显子2在小鼠基因中被成功敲除;给予LPS刺激后,IL1β、IL6、MyD88、iNOS及TNFa等炎症因子的表达在野生型小鼠的心、肝和肺组织中显著上调,而在TLR4^-/-小鼠中则几乎没有变化;血生化指标显示LPS刺激后WT小鼠血清中的尿素(Urea)和肌酐(Cre)水平显著升高,而TLR4^-/-小鼠刺激前后无显著变化,病理分析同样发现TLR4^-/-小鼠能够抵抗LPS对肾组织的损伤。结论 利用CRISPR/Cas9技术成功构建了TLR4基因剔除小鼠模型,TLR4的缺失能够降低IL1β、IL6、MyD88、iNOS及TNFa炎症因子对LPS刺激的响应,抑制LPS引起的炎症反应及对组织的损伤。     

赤眼鳟Toll样受体3基因cDNA全长克隆及表达分析

为探究赤眼鳟（Squaliobarbus curriculus）Toll样受体3（Toll-like receptor 3,ScTLR3）基因是否参与抗病毒免疫反应,实验运用RACE技术,克隆得到ScTLR3基因cDNA全长序列,并进行了生物信息学分析;通过Real-Time qPCR技术,检测了ScTLR3 mRNA在健康赤眼鳟10个组织中的分布以及感染草鱼呼肠孤病毒（GCRV）后肝脏、脾脏、体肾和头肾中的表达特征。结果表明:ScTLR3基因cDNA序列全长4043 bp,包括5-非编码区（UTR）216 bp,开放阅读框（ORF）2715 bp和3-UTR 1112 bp;ScTLR3共编码904个氨基酸残基,推导的蛋白分子量102.67 kD,理论等电点8.76;SMART结构域预测显示,ScTLR3由N端的信号肽（SP）、富亮氨酸结构域（LRRs）、跨膜结构域（TM）和C端的Toll/白介素-1受体结构域（TIR）组成。实时荧光定量结果显示,ScTLR3mRNA在检测的各组织中均有表达,肝脏中的相对表达量极显著高于其他组织（P0.01）;感染GCRV后,肝脏、脾脏、体肾和头肾组织中ScTLR3 mRNA均上调表达,肝脏、脾脏和体肾组织中的相对表达量在24h达到峰值,分别为对照组的5倍、7倍和6倍。研究表明,ScTLR3具有TLRs家族基因的典型结构特征,并能被GCRV诱导表达,推测其在赤眼鳟抗GCRV入侵免疫反应中发挥了重要作用。     

海洋动物Toll样受体的研究进展

Toll样受体(Toll-like receptor,TLR)是先天性免疫系统中古老的模式识别受体家族,一直都是免疫学家研究的焦点。从最古老的后生动物——海绵到高等的海洋脊椎动物——硬骨鱼中都有TLR存在。阐述处于不同进化地位海洋动物的TLR及其信号通路分子,TLR基因结构域混编和多态性机制,有助于揭示脊椎动物TLR的起源和预防海洋经济动物病害。     

Retrospective serological survey on selected viral pathogens in wild boar populations in Germany

The objective of this study was to retrospectively evaluate the occurrence of porcine parvovirus (PPV), Aujeszky’s disease virus (ADV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV) in selected wild boar populations in Germany (n  = 1,221). Commercial enzyme linked immunosorbent assay and hemagglutination inhibition tests were used for serological monitoring. The serosurvey revealed seroprevalence rates of 64.28%, 11.26%, 7.87%, 7.84%, 3.82% and 1.59% for PPV, ADV, PRCV, SIV, PRRSV and TGEV, respectively. The seroprevalence rates differed between populations and age classes with the highest number of antibody-positive wild boars in older animals (>1 year old). No antibodies to TGEV were found in Baden–Wuerttemberg and in Mecklenburg–Western Pomerania (investigation period 1997/1998). In addition, sera collected in Mecklenburg–Western Pomerania in 1997/1998 were negative for SIV. Even though the seroprevalence rates established for these viruses, except for PPV, were relatively low, wild boars may act as a reservoir for pathogens and a source of infection for domestic pigs and humans. Based on the epidemiological situation, no risk of a spread of these viruses should emanate from wild boars, neither for wildlife nor for livestock. However, effective and science-based disease monitoring programmes should continuously be carried out in wild boar populations.     

Udder preference in wild boar piglets

Competition to access the front teats in the domestic pig is strong during the first days of life, despite the fact that the number of piglets is fewer than the number of teats. Preference has been explained in terms of greater milk production (although it might also be the consequence rather than the cause), or reduced risk of crushing when the female lies down to begin a suckling session. Here, with the aim of determining whether one cause might be the body and reproductive modifications caused by artificial breeding, we analysed teat preferences of wild boar piglets in the wild. Fifty-one hunted females in lactation in the Western Iberian Peninsula were analysed. In contrast to domestic pigs, wild boar piglets showed a clear preference for rear udders. This preference was consistent for females of different ages and body characteristics, as well as for females hunted in different seasons. We suggest that the choice of rear udders in the wild boar may be related to the advantages of suckling at places offering higher protection from low temperatures and from predators, in contrast to domestic pigs, for which crushing is the main cause of mortality.     

野猪CAPN7基因的克隆、表达和变异分析

钙蛋白酶是细胞质中主要的蛋白水解酶,在肌肉生长、蛋白转化及嫩化过程中发挥复杂的作用。本研究利用RT-PCR、生物信息学方法克隆野猪（Sus scrofa ussuricus）CAPN7 cDNA并进行序列分析,利用相对定量RT-PCR方法研究其组织表达情况和利用PCR-SSCP方法对其进行变异分析。结果表明,野猪CAPN7基因编码区全长2 442 bp,预期编码813个氨基酸残基,多肽链中存在着calpain家族的催化结构域和催化活性中心;野猪CAPN7不同程度地表达于所检测的12种组织中,在6、9月龄野家杂交猪肌肉组织中的相对表达量高于同一时期的大白猪肌肉组织;所检测到的3种基因型在野猪、民猪和杜洛克中的分布存在着极显著差异。本研究为进一步揭示calpain7的功能提供了分子生物学基础。     

The use of faeces counts to estimate relative densities of wild boar in a Mediterranean area

The monitoring of population trends of wild ungulates is important to evaluate their population dynamics and to develop sound conservation/management plans. The wild boar Sus scrofa can impose heavy impacts on ecosytems and human activities, as well as be responsible for disease transmission. Estimating abundance of wild boars is a challenging issue, because of some peculiar biological and ecological traits of this ungulate. Indices of relative abundance could be used to evaluate its population trends. In a Mediterranean area we used faeces counts, through a two-stage stratified sampling, to estimate relative densities of wild boars, between 2007 and 2014. Faeces density estimates increased not significantly between 2007 (151.5 faeces/100 ha) and 2010 (203.8 faeces/100 ha) and decreased significantly from 2010 to 2014 (95.5 faeces/100 ha). The decrease in faeces density estimates was consistent with the increased harvest effort (number of harvest days), performed from 2010 to 2013 to limit impact on ecosystems and reduce damages to crops. The variation of faeces density estimates was also consistent with that of harvest indices (total harvest to harvest effort), with significantly positive values of Pearson and rank correlation coefficients. Results suggest that faeces density estimates achieved with the adopted sampling strategies can be effectively used as indices of relative abundance.     

The effect of season on the properties of wild boar (Sus scrofa L.) semen

The objective of the study was to determine the properties of wild boar semen and their changes in annual cycle. During a 14-month study period, 167 ejaculates were sampled from 3 mature boars. In each ejaculate the volume of liquid fraction, percentage of spermatozoa motility, spermatozoa concentration and the total number of spermatozoa were determined. The activity of acid and alkaline phosphatase, and aspartate aminotransferase in the fresh semen plasma was also measured. It was shown that wild boar ejaculates did not differ from those of domestic boars, and the semen of the highest volume, concentration and number of spermatozoa was produced in late autumn. The spermatozoa motility was the lowest in summer. The activity of aspartate aminotransferase and alkaline phosphatase in the semen plasma increased with shortening of the light period.     

First molecular identification of Sarcocystis miescheriana (Protozoa, Apicomplexa) from wild boar (Sus scrofa) in Iran

Sarcocystis isolate obtained from the thigh muscle of a wild boar (Sus scrofa), captured from Gilan Province, northern Iran, was subjected to molecular analysis. Genomic DNA was obtained using a DNA extraction tissue kit and Polymerase chain reaction (PCR) for amplification of the 18S ribosomal DNA region yielded an 842 bp DNA band on agarose gel. Analysis of DNA sequencing by BLAST confirmed the isolate as Sarcocystis miescheriana and the sequence was deposited in GenBank by Accession No. GU395554. This is the first molecular identification of an isolate of S. miescheriana in Iran.     

Bullet-derived lead in tissues of the wild boar (Sus scrofa) and red deer (Cervus elaphus)

Bullet-derived lead in game food products is an important source of human contamination. Careless removal of tissues from around the bullet pathway in the animal body results in elevated lead doses being ingested by humans. To assess bullet-derived lead contamination of soft game tissues, muscle tissue samples were collected from ten wild boars and ten red deer immediately after they had been shot. The samples were collected from around the entry and exit wounds, from around the bullet pathway at different sites along its length, and from a distance of about 5, 15, 25, and 30 cm from the bullet track. The individuals examined differed in the lead contents in their tissues surrounding the entry and exit wounds and at different sites along the bullet pathway. One of the animals showed as much lead as 1,095.9 mg kg⁻¹ wet weight in the tissue surrounding the bullet track near the entry wound, 736.0 mg kg⁻¹ being recorded around the exit wound.     

应用红外相机技术研究秦岭观音山自然保护区内野猪的行为和丰富度

2009年7月,在陕西观音山自然保护区凉风垭小区域（中高海拔）和西沟小区域（低海拔）安装18台红外相机,2009年8月至2013年4月共收集野猪照片1 195张。定义9种野猪行为,分别为站立、走动、跑动、采食、饮水、修饰、发情、拱土、坐着休息,并逐一比对照片中野猪的行为,统计各种行为所占的比例;引入月相对丰富度和时间段相对丰富度两个指数分别研究野猪的年活动规律和日活动规律;利用一个种群估测模型探讨野猪密度的年际变化。结果表明：（1）春季野猪以走动、采食和站立为主,分别占总行为次数的36%、25.6%和17.4%;夏季野猪以走动、站立、采食和跑动为主,分别占总行为次数的35.7%、23.6%、17%和16.5%;秋季野猪以采食、走动和发情为主,分别占总行为次数的50.3%、19.3%和17.8%;冬季野猪以采食、走动和站立为主,分别占总行为次数的53.7%、26.7%和11.9%。（2）野猪在8月、9月和12月活动较为频繁;全年日活动高峰出现在午后14:00-16:00,低谷出现在22:00-04:00,四季显示活动规律不同。（3）2009-2012年野猪密度呈逐年上升趋势。这些研究结果有助于了解野猪的行为活动和种群动态,并采取针对性的措施对野猪进行有效管理。     

湖北省神农架林区野猪分布与传播非洲猪瘟风险分析

野猪是非洲猪瘟(ASF)传播的重要媒介,神农架林区野猪种群数量高、分布广,调查野猪分布对ASF防控有着重要意义.本研究首先通过布设红外相机117台、样线287条调查神农架林区野猪实体和痕迹位点后,应用最大熵模型预测林区野猪分布,再根据野猪、居民区、交通要道的空间分布数据,应用核密度估计法分析神农架林区各地野猪传播ASF...