健康奶牛与临床型乳腺炎奶牛乳腺线粒体蛋白组差异表达分析

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构再进行蛋白质组学研究,可提高低丰度蛋白在双向凝胶电泳中的检出率。通过对比分析乳腺炎奶牛乳腺与正常奶牛乳腺线粒体蛋白质组的表达变化,为奶牛乳腺炎的生物学治疗及抗病育种工作筛选出目基因和蛋白。超速离心法分离线粒体,双向凝胶电泳分离蛋白,PDQuest7．4软件分析差异蛋白斑点,高效液相色谱串联离子阱质谱鉴定差异蛋白。从奶牛乳腺线粒体蛋白2-DE图谱中筛选出17个差异表达的蛋白质斑点,质谱鉴定出17个差异表达蛋白（6个蛋白在奶牛乳腺炎发生过程中下调,8个上调,1个只在正常情况下表达,2个只在乳腺炎乳腺组织中表达）。筛选出的差异蛋白质涉及到细胞的能量代谢、蛋白质合成、mRNA的加工成熟及调亡调控等许多方面,表明奶牛乳腺炎发生时乳腺线粒体组织结构和代谢状态都发生了明显的变化。     

蛋氨酸对奶牛乳腺上皮细胞亚细胞蛋白质组的影响研究

为寻找奶牛乳腺上皮细胞亚细胞结构中与泌乳相关的重要蛋白质、从蛋白质水平揭示乳蛋白合成的调控机制,应用双向凝胶电泳技术分离体外培养的蛋氨酸处理组与正常组奶牛乳腺上皮细胞蛋白质,利用Image Maser 2D软件对图谱进行对比分析,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)和数据库检索鉴定,并采用实时荧光定量PCR技术在mRNA水平上验证2-DE结果。质谱鉴定出8个表达上调的差异蛋白质,大多数差异蛋白质的功能涉及细胞骨架构成、能量代谢等过程。这些差异点的发现为研究奶牛泌乳机理提供了有益的线索。     

果叶两用桑品种大 10 与 SG01 桑椹蛋白质组的比较分析简

为了探讨不同品种桑椹蛋白质营养及成熟软化机理方面的特性,以果桑品种大10和SG01为材料,采用二维电泳和质谱技术对成熟桑椹的蛋白质组分进行了分离和鉴定,结果显示：从大10和SG01成熟桑椹蛋白质的双向电泳图谱中分别检测到471个和545个斑点,表明不同果桑品种间成熟桑椹的蛋白质表达谱存在着显著差异;从中选取表达量丰富、分离良好的20个蛋白斑点进行质谱分析,成功鉴定了其中的5个蛋白质组分;这些蛋白组分主要涉及蛋白代谢、细胞构成、植物应答反应、核酸代谢、光合作用等生理过程.桑椹蛋白质组的表达差异,可能与不同品种桑椹的功能性蛋白及其营养价值有关.     

畜牧兽医科技文摘

<正>健康奶牛与临床型乳腺炎奶牛乳腺线粒体蛋白组差异表达分析/王建锋(甘肃农业大学动物医学院,甘肃兰州730070),陶金忠,张勇…//中国兽医学报.-2012,32(1).-63～68运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构再进行蛋白质组学研究,可提高低丰度蛋白在双向凝胶电泳中的     

奶牛乳腺组织亚细胞组分的双向凝胶电泳分析

利用超速离心结合梯度离心法分离亚细胞组分的技术路线,以提高奶牛乳腺组织蛋白双向凝胶电泳的分离效率。奶牛乳腺组织液氮研磨破碎后,差速离心分离成细胞核、线粒体、高尔基体、溶酶体4个亚细胞组分,Nyco-denz纯化,2-DE分离蛋白,PDQUest8.0软件分析凝胶图谱。结果显示,奶牛乳腺组织细胞经亚细胞分离后,电泳分辨率大大提高,特别是细胞核蛋白质检出效率明显提高。不同亚细胞蛋白质组电泳图谱的差异显示了其蛋白质组构成的不同。由此可见超速离心结合梯度离心是一种有效的奶牛乳腺亚细胞组分分离手段,亚细胞蛋白质组学克服了蛋白质组的一些缺陷并可将蛋白质组研究引向亚细胞水平。     

基于二维凝胶电泳分析健康泌乳奶牛血浆蛋白质组的个体差异

为了分析奶牛血浆蛋白质存在的个体差异,以合理选择牛血浆蛋白质组研究中试验奶牛的样本数。试验采集了临床健康的泌乳早期的初产中国荷斯坦奶牛的血液,分离血浆蛋白后用二维凝胶电泳技术分离,考马斯亮蓝G-250染色后,对分离形态好的56个蛋白点的表达丰度进行分析。结果发现,蛋白点的变异系数在8.7%～59.6%之间,且有5个蛋白点的丰度变异系数较大（35%～59.6%）,用液相色谱串联离子阱质谱分析后,3个蛋白点获得有效鉴定,为转甲状腺素蛋白、载脂蛋白A1和α1酸性糖蛋白。方差分析结果表明,当样本数≥4时可有效降低组内样本间的个体差异。因此,奶牛血浆蛋白的表达存在个体差异,本试验为奶牛血浆蛋白质组研究中合理选择样本数量提供了依据。     

雌激素处理的奶牛乳腺上皮细胞核磷酸化蛋白质组学分析

雌激素调节多种生理过程,包括乳腺生长发育、形态发生、增殖和分化及乳蛋白的表达。许多磷酸化蛋白调节乳蛋白合成,如磷酸化Stat5和mTOR,但在乳蛋白合成的转录和翻译后水平的调节鲜有报道。为了阐述雌激素在奶牛乳腺上皮细胞中的作用机制,本试验利用双向电泳和质谱分析,鉴定了雌激素调节的细胞核磷酸化蛋白质。在雌激素处理24h后,鉴定出7个表达上调的蛋白质,包括以前报道过的甘氨酰-tRNA合成酶,属于氨基酰-tRNA合成酶家族Ⅱ;这些蛋白质涉及翻译起始因子、GTP结合的核蛋白、热休克蛋白、泛素-蛋白酶体系统。这些筛选出来的蛋白质,揭露了雌激素影响奶牛乳腺上皮细胞核磷酸化蛋白质,为进一步研究乳合成的分子机制提供一个新的途径。     

奶牛乳腺上皮细胞核蛋白质组双向电泳方法的建立

蛋白质样品的双向电泳分离效果受各种试验条件的影响较大,针对不同来源的蛋白样品进行试验条件的优化可获得具有较高分辨率的双向电泳图谱。试验拟建立优化的奶牛乳腺上皮细胞核蛋白质双向电泳分离体系,按标准条件对蛋白质进行双向电泳分离,并对关键因素进行优化,采集电泳图谱并分析双向电泳图谱中蛋白斑点的数量、图像分辨率及背景条纹的变化。通过对试验条件的筛选和优化,成功建立了具有较高分辨率的奶牛乳腺上皮细胞核蛋白质组双向电泳分离体系。     

健康与患布鲁菌病奶牛血清蛋白2-DE的建立和初步分析

采用商业化试剂盒Albumin & IgG Depletion Spintrap处理奶牛血清样品,比较处理前后对2-DE图谱的影响,结果发现处理后奶牛血清2-DE图谱中高丰度蛋白明显减少,但低丰度蛋白也有损失.采用不去除高丰度蛋白的方法比较健康奶牛与感染布鲁杆菌病奶牛的血清2-DE图谱,发现了11个明显差异的蛋白点,其中3个在正常奶牛血清中高表达,1个只在正常奶牛血清中表达;6个在患布鲁菌病奶牛血清中高表达,1个只在患布鲁菌病奶牛血清中表达.这些差异蛋白点的发现为将来质谱分析奠定基础.     

奶牛不同乳腺健康状态下乳清蛋白的差异性表达研究

本试验旨在通过研究奶牛不同乳腺健康状态下乳清蛋白差异性表达,探究奶牛乳腺健康状况对乳清蛋白的影响以及病原微生物蛋白诱发炎症的分子机制,为诊断奶牛乳腺健康程度以及早期炎症的发生提供潜在生物标记物。选择102头荷斯坦奶牛[胎次2～3胎、泌乳天数(152±27) d、产奶量(27±3) kg/d]进行牛奶样本采集,根据乳体细胞数(SCC)及细菌学鉴定结果将牛奶样本分为6组,分别为细菌培养阳性组(传染型、环境型和机会型)和细菌培养阴性组[培养阴性-低SCC组(SCC100 000个/mL)、培养阴性-中等SCC组(SCC为100 000～400 000个/mL)和培养阴性-高SCC组(SCC400 000个/mL)]。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)与非标记定量蛋白质组学等技术对6组乳清样本中的蛋白质功能及表达水平进行鉴定。结果表明:1)奶牛健康乳腺与炎症乳腺之间的差异主要是由蛋白质的表达水平造成的。2)试验总共鉴定出272种蛋白质,其中有58种蛋白质显示出显著调节变化。与细菌培养阴性乳清样本相比,细菌培养阳性乳清样本中有20种蛋白质表达显著上调(P0.05),其中导管素-4(CATHL4)、导管素-3(CATHL3)、导管素-2(CATHL2)、α-球蛋白抑制因子H4(ITIH4)、丝氨酸蛋白酶抑制剂A3-1(SERPINA3-1)、前列腺素H-2 D异构酶(PTGDS)、血清淀粉样蛋白A3(SAA3)和免疫球蛋白κv3-20(IGKV3-20)在培养阳性乳清样本中的表达量超过培养阴性乳清样本的约8倍,且上调蛋白质的功能多数与特异性免疫反应有关。显著或极显著下调的蛋白质有38种(P0.05或P0.01),其中血小板糖蛋白4(CD36)、补体蛋白CD59(CD59)、κ酪蛋白(CSN3)、嗜乳脂蛋白亚家族1成员A1(BTN1A1)、ATP结合盒转运蛋白G2(ABCG2)、围脂滴蛋白2(PLIN2)、包膜糖蛋白2(GP2)、聚泛素-C(UBC)和异柠檬酸脱氢酶1(IDH1)呈现极显著下调(P0.01),BTN1A1与脂质合成和分泌有关,PLIN2和GP2与转运有关,ABCG2具有酶活性,CD59与免疫系统有关。3)在细菌培养阳性乳清样本中鉴定到21种菌体蛋白,这些菌体蛋白的存在既验证了细菌培养鉴定结果,又从菌体蛋白特性和功能角度阐明了病原微生物诱发炎症的分子机制。综上所述,通过对奶牛不同乳腺炎症状态下乳蛋白差异表达研究,根据不同炎症类型奶样中乳蛋白质及菌体蛋白的表达变化及功能揭示了不同乳腺健康状态下乳腺内的生物学现象,此外为诊断乳腺健康特别是早期乳腺炎症的发生提供潜在生物标记物。     

蛋白质组学及其在奶牛乳房炎研究中的应用

蛋白质组学是功能基因组学的重要组成部分,已被广泛应用于人类疾病等相关研究领域。二维凝胶电泳、质谱和生物信息学是蛋白质组学研究的三大关键技术。文章概述了蛋白质组学研究技术的最新进展和白细胞蛋白质组学、微生物蛋白质组学、差异蛋白质组学的研究进展,以及蛋白质组学在奶牛乳房炎发病机理、诊断和防治等相关研究中的应用与前景。     

奶牛隐性乳房炎研究进展

隐性乳房炎是奶牛高发病之一,对奶牛及奶业发展造成了重要影响。该文对奶牛隐性乳房炎的发病原因、发病规律、诊断方法、预防措施等进行了探讨,并介绍了基因疫苗和细胞因子在乳房炎预防中的作用,以及PCR与蛋白组学方法在奶牛乳房炎诊断方面的应用。     

奶牛乳腺炎的细菌学研究

在对山东7个地区14个奶牛场临床型和隐性乳腺炎调查的基础上采集234头临床乳腺炎病牛乳样、241个隐性乳腺炎乳样并分别做了细菌学检查,结果表明:泌乳期临床型乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌、链球菌、酵母菌和棒状杆菌为主;干奶期临床型乳腺炎病原菌以大肠杆菌、链球菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌和酵母菌为主;隐性乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌链球菌、酵母菌、假单胞菌和棒状杆菌为主;厌氧菌在隐性乳腺炎、干奶期乳腺炎和干奶期乳腺炎乳样的捡出率分别为 5．82％,4．17%,10．16％;隐性乳腺炎、干奶期乳腺炎细菌的共感染率较高,与泌乳期乳腺炎病原菌的差异极显著(P<0．01),隐性乳腺炎与干奶期乳腺炎病原菌共感染率差异不显著(P >0．05)。     

对奶牛隐性乳房炎发生规律的探究

[目的]进一步探讨奶牛隐性乳房炎发生与自然月份、胎次、乳区、牛舍、泌乳月等的关系,以便更好的掌握奶牛隐性乳房炎与外界环境及奶牛泌乳生理状态的关系,为隐性乳房炎的预防和治疗提供科学的依据。[方法]2008年11月至2009年4月通过对南方某牧场泌乳牛群进行隐性乳房炎的CMT逐月检测,分析了奶牛隐性乳房炎的发生规律。[结果]表明,该场奶牛隐性乳房炎二、三胎牛阳性率极显著高于其他胎次（P〈0.01）;第五、第六个泌乳月和第九个泌乳月以上牛新感染阳性率极显著高于其它牛（P〈0.01）,左乳区阳性率高于右乳区。[结论]隐性乳房炎的发生率有随着胎次和泌乳时间的增加而提高的趋势。     

奶牛乳腺炎无乳链球菌的分离鉴定

无乳链球菌是比较常见的导致奶牛乳腺炎的病原菌,该菌也是山羊、绵羊慢性乳房炎的病原菌之一,也能引起婴儿败血症、脑膜炎和肺炎等。人医临床上对该菌以B群链球菌相称。试验从内蒙古呼和浩特市周边5个牛场中患有乳房炎的病牛中采集120份乳样,通过分菌培养、形态学观察、生化试验,鉴定出14株无乳链球菌,同时对这14株菌进行了药物敏感试验、动物致病性试验等。试验结果发现,所分离到的无乳链球菌对青霉素类、氨基糖苷类药物高度敏感,对磺胺类、氟哌酸、喹诺酮类药物中度敏感。动物致病性试验对小鼠的致死率达到80%以上。     

Risk Indicators Associated with Subclinical Mastitis in Smallholder Dairy Cows in Tanzania

Smallholder dairy farmers in Tanzania appear to be unaware of the subclinical mastitis situation in their cows. A cross-sectional study was carried out between June and September 2002 on smallholder dairy herds in the Dar es Salaam region. The study objectives were to establish the prevalence of subclinical mastitis and related risk indicators, and to assess their contribution to the occurrence of subclinical mastitis. Three field procedures based on the principles of herd health and production management were followed: clinical, farm and data inspection. The California mastitis test (CMT) was carried out on quarter milk samples to determine the prevalence of subclinical mastitis. A total of 182 lactating cows from 62 herds were investigated. Clinical inspection indicated that 3.8% of the lactating cows had clinical mastitis. Subclinical mastitis was detected in 90.3% of lactating cows screened. Farm inspection revealed that water scarcity, barn size, residual suckling, single udder-towel and dairy labourers as the most substantial (p < 0.05) risk indicators. Although most of the risk indicators studied were not found to be statistically significantly associated with the occurrence of subclinical mastitis, possibly owing to sample size and the presence of confounders, the epidemiological need to address such risk indicators cannot be overemphasized.     

Prevalence and Aetiology of Mastitis in Cows from Two Major Ethiopian Dairies

The study was undertaken to determine the aetiology and prevalence of mastitis in hand-milked cows (n = 186) in two major Ethiopian dairies. The California Mastitis Test and culturing for bacteria revealed that 21.5% of the cows were clinically infected and 38.2% had subclinical mastitis. Most mastitis pathogens isolated from milk samples testing positive by the California Mastitis Test were Gram-positive cocci. Staphylococci constituted 57% of the isolates, of which the predominant cause of bovine mastitis was Staphylococcus aureus (40.5%). Other mastitis pathogens isolated include streptococci (16.5%), coliforms (9%) and corynebacteria (5%). Retrospective analysis of farm records indicated that mastitis was the second most important cause of culling and accounted for 27% of the cows removed from these two dairies.     

中兽药散剂治疗奶牛临床型乳房炎的研究

奶牛乳房炎严重影响奶业生产。本试验使用中兽药散剂替代抗生素对临床型奶牛乳房炎进行治疗,并对治疗过程中的奶牛乳房炎病程情况、奶牛免疫情况和牛奶品质进行观察,评估中兽药散剂的治疗效果。结果显示：中兽药散剂可有效通乳消痈;可降低牛奶中体细胞数量;可提高粒细胞含量（从2%以下提高到46.3%）;可抑制炎症,降低IL-1β、IL-6和TNF-α的含量（降低66.3%~71.0%）。得出结论：中药散剂可提高奶牛的免疫功能,治疗乳房炎。     

Mastitis in Post-Partum Dairy Cows

Transition from the dry period to lactation is a high risk period for the modern dairy cow. The biggest challenge at that time is mastitis. Environmental bacteria are the most problematic pathogens around parturition. Coliforms are able to cause severe infections in multiparous cows, and heifers are likely to be infected with coagulase-negative staphylococci. During the periparturient period, hormonal and other factors make the dairy cows more or less immunocompromised. A successful mastitis control programme is focused on the management of dry and calving cows and heifers. Clean and comfortable environment, proper feeding and adequate supplementation of the diet with vitamins and trace elements are essential for maintaining good udder health. Strategies which would enhance closure of the teat canal in the beginning of the dry period and would protect teat end from bacteria until the keratin plug has formed decrease the risk for mastitis after calving. Dry cow therapy has been used with considerable success. Yet, a selective approach could be recommended rather than blanket therapy. Non-antibiotic approaches can be useful tools to prevent new infections during the dry period, in herds where the risk for environmental mastitis is high. Vaccination has been suggested as a means to support the immune defence of the dairy cow around parturition. In some countries, implementation of Escherichia coli core antigen vaccine has reduced the incidence of severe coliform mastitis after calving.     

Effects of Clinical Mastitis on Ovarian Function in Post-partum Dairy Cows

Mastitis-induced ovarian abnormalities were studied in a field trial. At 1-3 day after calving, > or = 2 parity cows not affected with chronic recurrent mastitis and yielding < 400,000/ml somatic cell count (SCC) individual milk in the previous lactation, were enrolled in the study. Thereafter milk samples were collected three times weekly for 95-100 day for progesterone (P4) assay. Individual P4 profiles were used to monitor ovarian cyclicity. When mastitis was diagnosed in the first 80 day post-partum (pp), clinical signs were recorded and scored, and aseptic milk samples were taken to identify the mastitis pathogens. Depending on the isolated pathogens the cows were blocked into one of the three sub-groups affected by either Gram-positive (GP), or Gram-negative (GN) bacteria, or of those with no detected pathogens (NDP). Cows suffering from any type of mastitis between days 15 and 28 (n = 27) showed a delay in the onset of ovarian cyclicity, and estrus was postponed compared to cows affected during the first 14 day pp (n = 59) and controls (n = 175) (38.6 +/- 2.3 vs 33.4 +/- 2.1 and 32.0 +/- 1.0 day, respectively, for onset of ovarian cyclicity and 90.7 +/- 2.5 vs 80.2 +/- 2.8 and 83.9 +/- 2.1 day, respectively, for estrus; both p < 0.05). The percentage of cows ovulating by day 28 was lower in those affected by mastitis between days 14 and 28 compared to cows between days 1 and 14 and controls (22.2% vs 47.5 and 50.3%, respectively; p < 0.05). A significantly higher rate of premature luteolysis was observed in GN + NDP compared to GP mastitis and healthy cows (46.7% vs 8.3 and 2.0%, respectively; p < 0.001). If the mastitis outbreak occurred during the follicular phase, the duration of this cycle segment was lengthened in GN + NDP mastitis compared to GP mastitis and healthy cows (10.8 +/- 0.9 vs 7.9 +/- 0.1 and 7.2 +/- 0.1, respectively; p < 0.001). The results indicate that mastitis can affect the resumption of ovarian activity in pp dairy cows. Mastitis may also impair reproduction also in cyclic cows: this effect can be the consequence of premature luteolysis or a prolonged follicular phase.