广西陆川猪肺炎支原体病原分离及其P97R1区基因的克隆及序列分析

发布时间：2018-07-18 13:29

【摘要】：猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)是引发陆川猪支原体肺炎(Mycoplasmal pneumonia of swine, MPS)的主要原因。本研究旨在对广西陆川猪Mhp进行病原分离鉴定及其P97 R1区基因序列分析,为进一步了解和研究广西陆川猪Mhp的病原特性、流行病学以及该病的综合防制提供理论依据。方法：(1)采集广西陆川猪疑似患有MPS的肺组织,常规Mhp病原分离培养后进行涂片染色和PCR方法鉴定,筛选阳性病料；(2)常规病理组织切片,HE染色观察组织病理学变化特征。采用免疫组织化学方法对组织中病原分布进行定位；用原位杂交技术对组织中Mhp的核酸进行检测、定位；(3)参考Mhp基因组序列设计扩增P97基因R1区片段的一对引物,提取基因组DNA,PCR扩增黏附因子P97基因R1区,对PCR产物进行测序,并将广西陆川猪Mhp不同地方流行毒株的P97基因R1区序列的碱基组成和推导的氨基酸序列进行比较。结果：(1)采用液体培养方法成功从疑似患有MPS的广西陆川猪肺组织中分离到M11p野毒株,液体培养物通过瑞氏一吉姆萨染色镜检,观察到环状、两极状等多形态的菌体；PCR检测液体培养物能扩增出目的片段；(2)Mhp阳性肺的病理组织HE染色结果显示,病变肺组织呈现典型的间质性肺炎以及融合性支气管肺炎变化,肺泡隔增厚,间质有大量炎性细胞增生和浸润,肺泡腔狭窄、缩小。细支气管管壁增厚,上皮细胞脱落,大量炎性细胞渗出,细支气管周围淋巴小结样增生。免疫组化结果显示,Mhp阳性信号主要分布在细支气管管壁柱状上皮细胞胞质和黏膜,管腔中的分泌物以及周围的炎性细胞胞浆中：原位杂交结果显示,Mhp DNA主要分布在气管和支气管黏膜表面。(3)广西陆川猪Mhp P97 R1区基因的碱基组成和推导的氨基酸序列比较结果显示,所扩增的15株广西陆川猪Mhp毒株与3株广西二元杂商品猪Mhp毒株的P97基因R1区多处碱基发生变异,通过推导氨基酸序列统计P97基因R1区氨基酸重复基序(AAKPV/E)数为9-18,平均值为12,TN重复数在1-4,发现广西陆川猪Mhp流行毒株变异使其的毒力和黏附能力增强。结论：本研究成功从疑似患有MPS的广西陆川猪肺组织中分离到Mhp野毒株,并定位了肺组织中Mhp病原菌和核酸的分布情况：此外,15株广西陆川猪Mhp毒株与3株广西二元杂商品猪的Mhp毒株的P97基因R1区序列分析、比较结果表明,广西陆川猪MhpP97基因R1区发生变异。
[Abstract]:Mycoplasma hyopneumoniae (MHP) is the main cause of Mycoplasma pneumonia of swine, pneumonia. The purpose of this study was to identify the pathogen of Mhp in Luchuan pig in Guangxi and to analyze the gene sequence of P97 R1 region in order to provide a theoretical basis for further understanding and studying the pathogenic characteristics, epidemiology and comprehensive prevention and control of MHP in Luchuan pig in Guangxi. Methods: (1) lung tissues of Guangxi Luchuan pig suspected to have MPs were collected, smear staining and PCR method were performed after isolation and culture of Mhp, and positive venereal disease materials were screened. (2) histopathological changes were observed by HE staining in routine histopathological sections. Immunohistochemical method was used to locate the pathogen in tissues; in situ hybridization was used to detect and locate Mhp nucleic acid; (3) a pair of primers were designed to amplify the R1 region of P97 gene with reference to the MHP genome sequence. The R1 region of P97 gene was amplified by PCR from genomic DNA. The PCR products were sequenced. The base composition and deduced amino acid sequence of R1 region of P97 gene of different endemic strains of Mhp in Luchuan pig in Guangxi were compared. Results: (1) the M11p strain was isolated successfully from the lung tissue of Guangxi Luchuan pig with MPs by liquid culture method. (2) HE staining showed that the pathological tissue of MHP positive lung showed typical interstitial pneumonia and fusion bronchopneumonia, and the alveolar septum thickened. A large number of inflammatory cells proliferate and infiltrate in the interstitium, and the alveolar lumen is narrow and narrowed. The wall of the bronchiole thickened, the epithelial cells fell off, a large number of inflammatory cells exudated, and lymphoid nodule hyperplasia around the bronchioles. Immunohistochemical results showed that MHP positive signals were mainly distributed in the cytoplasm and mucous membrane of columnar epithelial cells in the wall of bronchioles. Secretion in the lumen and the cytoplasm of the surrounding inflammatory cells: in situ hybridization results showed that the Mhp DNA was mainly distributed on the surface of trachea and bronchi mucosa. (3) the base composition and deduced amino acid sequence of Mhp P97 R1 region gene of Guangxi Luchuan pig Column comparison shows that, The P97 gene R1 region of 15 strains of Guangxi Luchuan pig Mhp virus and 3 Guangxi dualistic commercial pig MHP strains were mutated. According to the deduced amino acid sequence, the number of amino acid repeat motifs (AAKPVP / E) in R1 region of P97 gene was 9-18, with an average of 12 TN repeats in 1-4. It was found that the variation of Mhp epidemic strain of Guangxi Luchuan pig enhanced its virulence and adhesion ability. Conclusion: Mhp wild strain was isolated from lung tissue of Guangxi Luchuan pig with suspected MPs in this study. The distribution of Mhp pathogenic bacteria and nucleic acid in lung tissue was also determined. In addition, the R1 region of P97 gene of 15 strains of Guangxi Luchuan pig Mhp virus and 3 Guangxi dualistic commercial pigs were analyzed. The R1 region of MhpP97 gene in Luchuan pig was mutated.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.62

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