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1.
《中国兽医杂志》2021,(1)
为研究牛分枝杆菌N和C68004菌株间蛋白水平的差异表达对其致病性的影响,本试验采用串联质谱标记(TMT)定量蛋白组学技术,对2个菌株的毒力及其致病性进行鉴定与分析。结果分析得到2 174个共有蛋白,其中有479个差异表达蛋白(P<0.05)。GO分析结果显示,表达差异显著蛋白的功能包括催化、结合、运输、转录调控以及分子结构相关作用,特别是对内部或外部刺激产生的反应。KEGG分析结果显示,表达差异显著蛋白主要参与代谢和生物过程的调节。导致N菌致病性增强的毒力基因可能是mmaA4、ecce1、IpqY、hspX、Mpb63和mmpl4。并且N菌的耐药基因rpoA、rpoB和rpoC表达量显著高于C68004,可以推断N菌对抗结核一线药物利福平的耐药性可能比C68004更强。随着临床用药频繁和免疫应答的改变,N菌在适应环境过程中,与代谢、DNA复制以及耐药相关的蛋白表达出现了差异。 相似文献
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《畜牧兽医学报》2016,(5)
为了寻找不同品种马骨髓之间的差异表达基因,本研究构建了蒙古马-骨髓和纯血马-骨髓基因表达谱文库,经Illumina Miseq高通量测序,分别获得了7 219 956和7 116 170个有效reads;两个文库映射到11、8和1号染色体的reads最多,而映射到29、30和31号染色体的reads最少;两个文库共筛出318个差异表达基因,其中与免疫相关的基因有21个。GO功能富集分析结果表明差异表达基因与转运、细胞间信号传导、生物学过程和细胞质等有关;KEGG通路富集分析结果表明,差异最大的显著富集KEGG条目为神经系统。这些结果为研究马匹抗病力的遗传和免疫机理等提供基础数据。 相似文献
3.
以猪传染性胸膜肺炎放线杆菌（APP）血清7型25-4株基因组DNA为模板，用PCR扩增外膜蛋白（OMP）基因特异片段，并克隆于pMD18-T中，经酶切及核苷酸序列分析鉴定后，亚克隆于原核表达栽体pGEX-6P-1，成功构建了重组表达载体pGEX-omp；以此转化大肠埃希氏菌BL21（DE3），经SDS-PAGE鉴定，表达的可溶性融合蛋白分子质量约为61 ku，命名为GST-OMP。以GST亲和层析柱纯化并利用Xa因子酶解，获得切掉标签的OMP。经ELISA检测，该OMP蛋白能够与兔抗APP的阳性血清反应，具有很好的免疫活性。GST-OMP蛋白的成功表达为APP OMP相关分子生物学功能的研制奠定了基础。 相似文献
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为了进行抗蜱和蜱传病疫苗的研究,本实验对亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中获取的一个全长编码基因P18进行了研究。该基因全长519bp,共编码170个氨基酸,分子量为18.36Ku,等电点为4.28。BLAST分析表明,该基因预测的氨基酸与肩突硬蜱、篦子硬蜱唾液腺的抗凝血小肽有30%～40%低度同源性。将该基因亚克隆到pET-32a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,重组融合蛋白以可溶性形式高效表达。将可溶性重组蛋白免疫小鼠后获得的抗血清。经免疫印迹分析表明,该重组蛋白抗体可特异性的识别半饱雌蜱唾液腺中的天然蛋白抗原,而未吸血雌蜱唾液腺中则不显现特异条带。RT-PCR结果进一步证实,该基因在蜱吸血后的唾液腺中差异表达。 相似文献
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家蚕中肠cDNA T7噬菌体展示文库的构建和免疫相关基因的淘选 总被引：1，自引：0，他引：1
中肠是昆虫重要的免疫防御器官之一。为探究家蚕中肠的分子免疫机制,尤其是针对不同病原体的模式识别分子或结合蛋白,以家蚕5龄第3天幼虫中肠为材料,构建家蚕中肠cDNA T7噬菌体展示文库,并分别以几丁质和脂多糖为配体,通过不同方法进行文库的生物淘选。结果共获得10个基因片段,其中在以脂多糖为配体的淘选中获得了β-1,3葡聚糖识别蛋白4,该分子是已知的模式识别受体。另外还获得了多个免疫相关候选基因,如分别编码几丁质结合蛋白、翻译控制的肿瘤蛋白、氨肽酶N、脂肪酰辅酶A结合蛋白等的几个基因。研究结果为深入开展家蚕中肠免疫功能的研究提供了重要线索。 相似文献
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旨在利用抑制性削减杂交(SSH)技术筛选西农萨能奶山羊泌乳中期和后期的差异表达基因,并用实时定量PCR(Q-PCR)分析差异基因的表达丰度,探讨奶山羊不同泌乳阶段乳腺组织的基因表达规律。本研究以西农萨能奶山羊泌乳中期和后期乳腺组织的mRNA互为检测子(Tester)和驱动子(Driver)构建cDNA消减文库(M-L和L-M),随机挑选克隆测序,进行序列比对分析,并检测部分差异基因在乳腺组织中的表达丰度。结果,成功构建了泌乳中期和后期的cDNA文库,随机挑选30个克隆测序,得到M-L和L-M文库中与细胞凋亡、抗氧化、脂类代谢、能量代谢等生理过程相关的差异基因,对其中的6个基因进行实时定量分析,发现5个均为阳性克隆,表达水平增加了1.3~5.5倍不等。结果表明,利用SSH技术成功构建了泌乳中期和后期乳腺组织正反向消减cDNA文库,筛选出24个差异基因,为进一步研究奶山羊乳腺组织基因调控机理奠定了基础。 相似文献
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《中国兽医学报》2017,(5)
为筛选北京油鸡和来航鸡脾脏组织中与抗病力差异相关的候选基因和信号通路,本研究采用高通量测序技术,构建了42日龄北京油鸡(抗病力较强)和来航鸡(抗病力相对较弱)脾脏组织的数字基因表达谱,对差异表达基因进行了GO功能分类、KEGG信号通路分析和STRING互作网络构建,并利用荧光定量PCR验证了部分差异表达基因。结果表明,以来航鸡为对照组,北京油鸡与之相比,差异倍数在2倍以上的共有1335个基因,其中693个上调表达,643个下调表达,包括多个与淋巴细胞的激活、分化和增殖、免疫器官发育等相关的基因,主要涉及到结合功能、细胞组成、细胞加工和生物学调节等功能。对通路显著性分析发现,与免疫相关生物学通路共有4个,其中BCR信号和TLR信号涉及脾脏B细胞介导的体液免疫应答反应,两条通路上的差异表达基因构成1个相互连接的互作网络,其中D79B、CD44、IL1B、SOCS1及TLR4等位于重要节点位置。为今后挖掘新基因和研究影响鸡抗病力的遗传因素等提供理论依据。 相似文献
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家蚕微孢子虫感染BmN细胞的差异表达基因筛选及Akirin的原核表达 总被引：1，自引：0，他引：1
为探究家蚕微孢子虫(Nosema bombycis,Nb)感染宿主细胞的分子机制,以感染Nb的家蚕卵巢上皮细胞(BmN)为材料,通过GeneFishing技术进行基因差异表达分析,发现感染Nb的BmN细胞有10个基因被诱导或抑制表达,这些差异表达基因功能涉及蛋白质合成、细胞信号转导、线粒体呼吸链电子传递、细胞能量代谢、细胞免疫及一些未知功能。运用RT-PCR方法克隆在感染Nb的BmN细胞中差异表达的免疫相关基因Akirin,获得一个长588 bp的完整ORF,编码195个氨基酸,预测蛋白质分子质量21.98 kD,等电点为8.97,属于碱性蛋白,该蛋白质经SignalP在线预测未见信号肽。通过原核表达获得家蚕Akirin的外源融合表达蛋白,为进一步研究家蚕Akirin在家蚕先天免疫系统中的功能奠定了基础。 相似文献
10.
为挖掘重要的抗盐碱相关基因,进而阐述羊草(Leymus chinensis)耐盐碱胁迫的分子机制,采用荧光双向差异凝胶电泳(2D-DIGE)结合串联飞行时间质谱(MALDI TOF/TOF)方法,利用非盐碱条件下及Na2CO3处理下生长的羊草建立蛋白质表达谱,并进行差异蛋白质的分离鉴定。结果表明:2D-DIGE获得的74个差异蛋白点,其中蛋白质表达丰度上调的蛋白点44个,丰度下调点为30个,质谱鉴定显示其中33个蛋白质信息已知,参与了能量转换、代谢、光合作用、植物抗性和转录的过程。 相似文献
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通过鲎素抗菌肽和超高静水压联合作用,制备出一种胸膜肺炎放线杆菌菌影。利用胸膜肺炎放线杆菌血清5型（CVCC263）制备菌影并检测其灭活率。CVCC263菌影疫苗接种免疫仔猪,二免14d后攻毒,每天测量体温,并观察精神状态,呼吸,食欲等。攻毒第8天对存活猪进行剖杀,测量肺部病变面积,进行病理检测。结果显示,免疫组抗体效价及血清中的IgG、IgM、IgA、IL-2、IL-4含量均较对照组显著增加。免疫组临床症状和肺部病变面积均小于对照组。CVCC263菌影疫苗免疫仔猪抗APP感染的保护效果是明显的,并且APP5型的菌影疫苗可对APP7型感染有交叉保护。 相似文献
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广西巴马小型猪IL-4和IL-10基因的克隆和序列分析 总被引：6，自引：0，他引：6
为研究猪白细胞介素4（IL-4）和白细胞介素10（IL-10）在机体免疫应答中的作用,从ConA刺激的巴马小型猪的外周血淋巴细胞提取总RNA,用RT-PCR方法扩增出IL-4和IL-10基因,将其分别克隆到PMD18-T 载体上,进行序列分析.结果表明,克隆的IL-4和IL-10基因的核苷酸和氨基酸序列与GenBank上登录的其它猪种的IL-4和IL-10基因的核苷酸和氨基酸序列同源性分别为98.8%～100%、97.0%～99.3%和99.2%、97.7%. 相似文献
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【目的】丰富非洲猪瘟病毒（African swine fever virus,ASFV）感染后猪外周血淋巴细胞长链非编码RNA（long non-coding RNA,lncRNA）表达谱,并进一步挖掘影响Toll样受体信号通路的调控网络。【方法】试验动物感染ASFV,于第7天采集外周血并分离得到外周血淋巴细胞,运用Illumina高通量组学测序对外周血淋巴细胞中lncRNA进行测序,原始数据经处理后筛选获得差异表达的lncRNA,并进行靶基因预测,利用生物信息学方法对靶基因进行GO功能和KEGG信号通路富集分析,初步绘制与Toll样受体信号通路相关的lncRNA-mRNA调控网络,并对lncRNA-ENSSSCG00000041959在内的4个lncRNAs进行实时荧光定量RT-PCR验证。【结果】共筛选到73个差异表达的lncRNAs,其中上调表达lncRNAs 38个,下调表达lncRNAs 35个。GO功能分析结果显示,靶基因显著富集在调节免疫系统过程、防御反应、生物刺激、病毒反应和先天性免疫;KEGG信号通路富集分析显示,大部分靶基因与细胞循环、疾病及免疫应答有关,其中免疫相关信号通路有Toll样受体信号通路、TNF信号通路、产生IgA的肠道免疫网络等。进一步挖掘出lncRNA-ENSSSCG00000041959-RIPK1和lncRNA-ENSSSCG00000041959-IRAK1可能是影响Toll样受体信号通路的重要调控网络,实时荧光定量RT-PCR与测序结果一致。【结论】本研究初步鉴定出lncRNA-ENSSSCG00000041959-RIPK1和lncRNA-ENSSSCG00000041959-IRAK1可能是影响Toll样受体信号通路的lncRNA-mRNA调控网络,为进一步探索lncRNA调控ASFV感染机体免疫反应奠定了理论基础。 相似文献
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为了评价猪传染性胸膜肺炎与猪肺疫二联亚单位疫苗(由多杀性巴氏杆菌外膜蛋白H(OmpH)质粒导入胸膜肺炎放线杆菌(APP)菌影制备)的免疫效果,于某规模化猪场选取30头无上述病原菌和抗体的4周龄(w)仔猪,随机分为安全评估组和试验组进行免疫,试验组包括APP组、OmpH组、APP+OmpH联苗组,分别于4、6 w肌肉注射2 mL/头相应疫苗,采取4、6、9 w外周血,用流式细胞术、ELISA、血常规、血生化试验分析疫苗免疫后,相关免疫因子、免疫细胞及免疫球蛋白的变化。结果显示:APP+OmpH组外周血中CD3+、CD8+细胞增加数量比APP、OmpH组显著增多(P<0.05);血清抗体结果显示,APP+OmpH组结果优于APP和OmpH组;APP+OmpH组的外周血白细胞数、淋巴细胞数、中性粒细胞数、单核细胞数在二免后增多,白蛋白、球蛋白亦明显增加,且APP+OmpH组增加数量多于APP和OmpH组。结论:APP+OmpH组能更好地刺激机体细胞免疫和体液免疫反应,进而发挥免疫保护效果。 相似文献
16.
Nabin Rayamajhi Sung Jae Shin Sang Gyun Kang Deog Yong Lee Jeong Min Ahn Han Sang Yoo 《Journal of veterinary diagnostic investigation》2005,17(4):359-362
Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae. 相似文献
17.
Actinobacillus pleuropneumoniae is a gram-negative bacterium and is the causative agent of swine pleuropneumonia, a highly contagious respiratory disease. Biofilm formation is an important ability possessed by numerous bacterial pathogens. The purpose of this study was to identify and characterize biofilm mutants of A. pleuropneumoniae serotype 1 strain S4074 created using a mini Tn-10 transposon. The transposon library was screened to identify mutants with a modified ability to form biofilms in polystyrene microtiter plates. A total of 1200 mutants were screened and the analysis identified 24 mutants that exhibited abnormal biofilm formation, at least 16 unique genes were identified. Most genes identified in the enhanced-biofilm mutants encoded proteins with unknown functions, whereas most genes identified in the biofilm-reduced mutants encoded proteins related to transport, protein synthesis and nucleic acid synthesis. Approximately 50% of genes, including hns, potD2, ptsI, tig and rpmF, identified in our screen have been previously associated with biofilm formation in A. pleuropneumoniae and other bacterial species, and thus validated the screening method. The rest of genes identified, such as APL_0049, APL_0637 and APL_1572, have not been previously associated with biofilm formation. Interestingly, gene APL_0049 was previously seen among the genes differentially expressed during a natural infection of pig lungs. Preliminary characterization of the mutants was also initiated by assessing their hydrophobicity, their biofilm matrix composition and their ability to adhere to a polystyrene surface or NPTr cells. Based on the preliminary characterization, some of the mutants identified appear to have deficiencies during the initial attachment or growth of the biofilm. In conclusion, transposon mutagenesis analysis allowed the identification of new genes associated with biofilm formation in A. pleuropneumoniae. 相似文献
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Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon. 相似文献
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Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species? 总被引：3，自引：0，他引：3
Gottschalk M Broes A Mittal KR Kobisch M Kuhnert P Lebrun A Frey J 《Veterinary microbiology》2003,92(1-2):87-101
Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae. 相似文献
20.
Actinobacillus (A.) pleuropneumoniae is the causative agent of a porcine pleuropneumonia occurring worldwide. In order to identify novel non-cytoplasmic putative virulence-associated proteins, we prepared fractions enriched in surface-associated proteins for differential proteome analysis by two-dimensional (2D) gel electrophoresis and quadrupole time-of-flight mass spectrometry (Q-Tof MS). Bacteria grown under standard culture conditions were compared to an ex vivo model based on the addition of bronchoalveolar lavage fluid (BALF) to the culture media. Twelve proteins were found to be upregulated upon induction with BALF, among them a superoxide dismutase, a parvulin-like peptidy-prolyl isomerase, a polynucleotide phosphorylase and the highly immunogenic lipoprotein OmlA. Four of the proteins upregulated by BALF were additionally constitutively expressed by an isogenic A. pleuropneumoniae fur deletion mutant and could be identified by Q-Tof MS as the heat shock protein GroES, a putative dipeptide transporter, a putative metal ion transporter and a conserved protein of unknown function. In silico analysis of the putative promoter regions of the encoding genes revealed putative Fur boxes upstream of two genes, one of which encodes part of a putative metal ion transporter. An isogenic mutant with a deletion in this protein was constructed and designated as A. pleuropneumoniae Deltafui. Analysis of the mutant in an aerosol infection model revealed symptoms indistinguishable from those seen upon infection with wild type A. pleuropneumoniae. This result implies that not all proteins upregulated by BALF are directly involved in A. pleuropneumoniae virulence. 相似文献