Initial Establishment Of Porcine Proliferative Enteritis Diagnostic Methods & Cloning And Expression Of The Gene LI0065

Porcine proliferative enteritis, a contacted contagious disease resulting in diarrhea, daily weight gain decreasing, slow growth and other clinical symptoms and some pathological changes, such as intestinal bleeding, intestinal wall thickening, intestinal mucosal thickening, crypt epithelial cell hyperplasia etc., is caused by Lawsonia intracellularis which is characterized by intestinal hyperplasia. The wide-spread disease, with a worldwide trend and high morbidity, seriously affect the economic benefits of hogpens. The purpose of this study is to establish a set of efficient, fast and accurate detection in the clinical diagnosis of porcine proliferative enteritis and realize the clone and expression of LI0065.1.We collected pig diarrhea sample for processing in Hunan Province scale pig farm and optimizing the conditions of DNA extraction and nested PCR amplification of target fragment. The feasibility of this method was verified by sensitivity, specificity, reproducibility and detection of clinical samples. The pigs, result of nested PCR assay was positive, were necropsied. The small intestine was made into paraffin sections which were performed with HE staining and modified silver staining. And the histopathological changes of the tissues were analyzed. The target fragment,263bp, was successfully amplified, whose homology with the whole sequence of Lawson was as high as 99%. The positive rate of clinical test was 25%, which was stable, reliable, good repeatability and high specificity. HE staining showed that the goblet cells in the small intestine were significantly reduced or even disappeared. The result of modified silver staining revealed small intestinal crypt were fused irregularly. And the suspected long rod or curved rod Lawsonia intracellularis got together and grew in the cytoplasm of the crypt epithelial cells.2.The total length of amplified target fragment LI0065 is 750bp, which encodes 249 amino acids. And the molecular weight of the encoded protein is 29.2KD. The result of bioinformatics analysis implies that the protein, a non signal peptide, encoded by LI0065 is not stable, which may be a membrane receptor protein involved in cell signal conduction. The identification of SDS-PAGE gel electrophoresis showed that the protein encoded by LI0065 is a soluble protein.