猪增生性肠炎诊断方法的初步建立及胞内劳森氏菌LI0065基因的克隆与表达

发布时间：2018-06-01 02:49

本文选题：猪增生性肠炎 + 胞内劳森氏菌　；参考：《湖南农业大学》2016年硕士论文

【摘要】：猪增生性肠炎是由胞内劳森菌引起的一种接触性传染性疾病,以肠道增生为主要特征,其临床症状表现为腹泻、日增重量下降、生长缓慢等,病理变化主要为肠道出血、肠壁增粗、肠粘膜增厚,隐窝上皮细胞增生。该病传播范围广,发病率高,呈世界流行性趋势,严重影响猪场经济效益。本研究旨在建立一套能在临床上高效、准确、快速检测猪增生性肠炎的诊断方法；并实现对劳森氏菌基因L10065的克隆与表达。1、采集湖南省规模化猪场腹泻猪的粪便进行处理,对其进行DNA抽提、目的片段巢式PCR扩增及扩增条件的优化,然后通过敏感性、特异性、重复性实验及临床样本的检测验证此方法的可行性。对巢式PCR检测为阳性的猪进行剖检,取其小肠做成石蜡切片,进行HE染色与改良银染,分析其组织病理学变化。结果成功扩增出大小为263bp目的片段,与劳森菌全序列比对同源性高达99%。临床检测阳性率达25%,检测稳定可靠、重复性好、特异性高；HE染色发现小肠绒毛内杯状细胞明显减少甚至消失,改良银染发现小肠隐窝的非正常融合,且隐窝上皮细胞胞浆内可见疑似长杆状或弯曲杆状胞内劳森氏菌的扎堆生长。2、本实验扩增的目的片段L10065全长750bp,共编码249个氨基酸,编码蛋白分子量为29.2KD。生物信息学分析发现,L10065编码的蛋白具有不稳定性,为非信号肽,预测可能是参与细胞信号传导的膜受体蛋白。SDS-PAGE凝胶电泳鉴定L10065编码的蛋白为可溶性蛋白。
[Abstract]:Porcine proliferative enteritis is a kind of contact infectious disease caused by intracellular Lawson's bacteria. It is characterized by intestinal hyperplasia. Its clinical symptoms are diarrhea, daily weight loss, slow growth and so on. The pathological changes are mainly intestinal bleeding. Thickening of intestinal wall, thickening of intestinal mucosa and proliferation of crypt epithelial cells. The disease has a wide spread range and a high incidence, which has a worldwide epidemic trend and seriously affects the economic benefits of pig farms. The purpose of this study was to establish a highly effective, accurate and rapid diagnostic method for detecting porcine proliferative enteritis, and to clone and express L10065 gene of Lawson's bacillus, and to collect feces from diarrhea pigs in large-scale pig farms in Hunan Province. The DNA was extracted and the nested PCR amplification conditions were optimized. The feasibility of the method was verified by sensitivity, specificity, reproducibility and clinical sample detection. The piglets with positive nested PCR were dissected, and their small intestine was made into paraffin sections. The histopathological changes were analyzed by HE staining and improved silver staining. Results the target fragment of 263bp was amplified successfully, and the homology of the fragment was as high as 99g with the whole sequence of Lawson's bacillus. The positive rate of clinical examination was 25%, the detection was stable and reliable, reproducible, specific and HE staining showed that goblet cells in small intestinal villi decreased or even disappeared, improved silver staining found abnormal fusion of intestinal crypt. In addition, in the cytoplasm of crypt epithelial cells, the growth of L10065 is 750bp, encoding 249 amino acids, and the molecular weight of encoded protein is 29.2kD. the growth rate of L10065 is 750bp. the length of L10065 is 750bp, and the molecular weight of protein is 29.2kD. Bioinformatics analysis showed that the protein encoded by L10065 was unstable and non-signal peptide. The membrane receptor protein, SDS-PAGE, which might be involved in cell signal transduction, was identified as soluble protein by SDS-PAGE gel electrophoresis.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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