【作者】 牛丽莉；

【导师】 杜立新；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 奶牛乳房炎是奶牛乳腺的一种炎症,它的发生是病原微生物、环境因素、管理因素、奶牛自身及遗传因素等综合作用的结果。奶牛乳房炎是对奶业生产造成巨大损失的疾病之一,严重影响世界奶牛业发展,在畜牧、兽医、食品等领域一直受到广泛关注。随着生物技术的发展不断有新的技术被用来探索乳房炎的发病机理,本实验运用蛋白质组学的方法,鉴定健康与患乳房炎奶牛血浆中的差异蛋白,以及分娩前后血浆中的差异蛋白,旨在寻找奶牛乳房炎早期诊断的标志性蛋白,并为今后的抗病育种提供理论依据。主要试验结果如下:第一,建立了奶牛血浆蛋白质组双向电泳分离鉴定技术平台。(1)对不同的样品处理方法进行了比较。DEAE Affi-Gel兰胶柱,阴离子柱和超滤管被用于对血浆样品进行预处理,步骤增多,而且低丰度蛋白富集效果不明显。(2)使用经典的热SDS法处理血浆样品,采用pH4-7的胶条和7.5%-17.5%的梯度胶,经硝酸银染色后,建立了奶牛血浆蛋白质双向电泳图谱。此方法简单易行,稳定性好。第二,乳房炎奶牛血浆差异蛋白的分离与鉴定。分别收集健康和患病奶牛血浆,根据体细胞数将患病奶牛的血浆分为2组,一组为体细胞数大于40万个/ml小于200万个/ml,第二组体细胞数大于200万个/ml。采用双向电泳(2-DE)技术得到奶牛血浆蛋白质组图谱,PDQuest7.0软件进行分析,识别3倍差异表达蛋白斑点8个;将选取的差异蛋白质点胶内酶解后进行MALDI-TOF/TOF MS分析,经MASCOT搜索数据库鉴定为白蛋白、α-2-HS-糖蛋白、结合珠蛋白和转甲状腺素蛋白;其中结合珠蛋白在患病牛血浆中含量增加,白蛋白、α-2-HS-糖蛋白和转甲状腺素蛋白含量降低。并使用Western Blot方法在个体中验证,与双向电泳的分析结果一致。这4种差异蛋白均为急性期蛋白。α-2-HS-糖蛋白与转甲状腺素蛋白为首次在血浆中鉴定的乳房炎相关蛋白,有可能作为奶牛乳房炎发病前的检测蛋白。第三,比较奶牛分娩前后血浆差异蛋白。分别收集分娩前后一周内的奶牛血浆,进行双向电泳分析。PDQuest7.0识别3倍差异表达蛋白点12个,质谱鉴定为白蛋白和结合珠蛋白,白蛋白在产后奶牛的血浆中的含量降低,结合珠蛋白的含量增加。Western Blot检测结合珠蛋白与双向电泳的分析结果一致。产后乳房炎个体与健康个体间的结合珠蛋白的血浆含量没有差异。结合珠蛋白的产后的不敏感性与慢性疾病或急性炎症高度相关。因此,这种现象可能与奶牛围产期的免疫抑制有关。更多还原

【Abstract】 The mastitis is the inflammation of cow mammary gland, its occurrence is a combined action result including the pathogenic microorganism, the environmental factor, the management factor, the cow own and the hereditary factors and so on. The mastitis is one of diseases that causes heavy economic loss and influences the development of dairy industry in the worldwide.It has became the hotspot not only in the fieldof livestock and veterinarian but also in food security. Following the development of biotechnology, more new technologies are applied to investigate the mechanism of mastitis pathogenesis. In this study, plasma proteomics analysis was performed for analysis the protein differentiations between healthy and patient cows, as well as around the calving period. It will be help for detecting mastitis process on pathological levels, and seeking for the proteins related to mastitis to provide the reference for mastitis treatment ahead of time. The main results are as follows:First, different methods for the preparation of plasma sample were compared—DEAE Affi-Gel blue column, anion exchange column and ultrafiltration tube. More steps were needed, and the enrichment effect was not better. The heating with 10%SDS and 2.3% DTT to denature sample at 95℃for 5min, IEF on pH 4-7 IPG strips were better for effectively improving the resolution and intensity of low-abundant proteins.Second, analysis of different proteins between health and mastitis cow. Plasma was collected from health and mastitis cow respectively. According to somatic cell counts (SCC), plasma from mastitis cow was divided into two group: mastitis1 (2000x103cells/ml>SCC>400 x103cells/ml), mastitis2 (2000x103cells/ml<SCC). After sliver-staining, the images of the gels were acquired by the scanner and then analyzed by using PDQuest7.0 software to find the different protein spots. Further the different protein spots that showed more than 3 fold changes in abundance were dentified by MALDI TOF/TOF MS after enzymatical digestion. The identified proteins were searched in database and confirmed by Western Blot in individuals. The different proteins between health and mastitis cows were identified as albumin,α-HS-glycoprotein, transthyretin and haptoglobin. all of the different proteins were all acute phase proteins.α-HS-glycoprotein and transthyretin were novel proteins detected in the plasma of mastitis cow. It need further study to assess whether they can be look as the morbidity examination proteins for cow mastitis.Third, different protein analysis between pre-perinatal and post-perinatal. Haptoglobin and albumin were identified as the different proteins between pre-perinatal and post-perinatal.The haptoglobin was related to retained placenta and endometritis during post perinatal. Poor sensitivity in postpartum periods could be related to the higher incidence of chronic (vs acute) inflammation. Haptoglobin may be appropriate for routine screening, but further work needs to be done to assess its value as an indicator of herd health and relationship with immune depression.更多还原