牛源金黄色葡萄球菌分离鉴定及ClfA基因真核载体的构建
发布时间:2018-05-29 18:29
本文选题:奶牛乳腺炎 + 金黄色葡萄球菌 ; 参考:《甘肃农业大学》2015年硕士论文
【摘要】:奶牛乳腺炎是阻碍奶牛养殖业发展的主要原因之一,而金黄色葡萄球菌是主要的致病菌之一,且对于不同的地区,患病奶样所分离到金黄色葡萄球菌差异较大。为查明甘肃及周边地县由金黄色葡萄球菌引起的奶牛乳腺炎的情况,并研制相关的基因工程疫苗,分别从甘肃秦王川、青海民和、宁夏吴忠采集380份奶样。通过金黄色葡萄球菌耐高盐这一特点对这批奶样进行初步筛选,得到41株疑似金黄色葡萄球菌;再通过过氧化氢酶发酵、甘露醇发酵、三糖和Baird Parker平板等传统方法对金黄色葡萄球菌进行分离鉴定,获得37株疑似菌株;结合分子生物学方法鉴定Clf A基因,最后确定有35株为金黄色葡萄球菌的病原菌株。选取MD20菌株提取的DNA为模板,扩增ClfA基因扩增产物连接Trans T1感受态细胞精确测序,结果显示,扩增长度为1168bp,与已报道的Clf A因子的基因序列(Gene Bank:HQ_424292)同源性为98%。用HindⅢ、XhoⅠ两种酶对pGEMT-ClfA和pcDNA3.1-HisB双酶切,并回收目的片段。后将纯化后的目的片段用T4DNA连接酶于水浴锅中16℃连接过夜后,转化至感受态T1细胞,测序结果和菌液PCR鉴定显示,本实验已成功构建了真核表达载体pcDNA3.1B-Clf A。将载体pcDNA3.1B-ClfA稳定转染至人的乳腺癌细胞,传代筛选至细胞稳定,经过trizol法提取RNA,反转录进行检测,收集保存转染成功细胞。Western-blot检测外源蛋白的表达,分子量约为39KDa。本研究为后续的免疫动物实验做了基础工作,对金黄色葡萄球菌性奶牛乳腺炎的防控具有指导意义,同时也为动物之间病源互相传播的研究提供研究依据。
[Abstract]:Cow mastitis is one of the main reasons to hinder the development of dairy cattle breeding industry, and Staphylococcus aureus is one of the main pathogenic bacteria, and for different regions, there is a great difference in Staphylococcus aureus isolated from sick milk samples. In order to find out the situation of cow mastitis caused by Staphylococcus aureus in Gansu and its surrounding counties, and to develop the related genetic engineering vaccine, 380 milk samples were collected from Qinwangchuan, Qinghai Minhe and Ningxia Wu Zhong, respectively. According to the characteristics of high salt tolerance of Staphylococcus aureus, 41 strains of suspected Staphylococcus aureus were obtained by screening the milk samples, and then by catalase fermentation and mannitol fermentation. Staphylococcus aureus was isolated from Staphylococcus aureus by traditional methods, such as trisaccharide and Baird Parker plate, 37 suspected strains were obtained, and 35 strains of Staphylococcus aureus were identified by molecular biological method. The DNA extracted from MD20 strain was selected as template to amplify the ClfA gene amplification product to connect Trans T1 competent cells. The results showed that the length of amplification was 1168 BP, which was 98% homology with gene Bank: HQs 424292 of Clf A factor. PGEMT-ClfA and pcDNA3.1-HisB were digested with Hind 鈪,
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