【作者】 李进；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪圆环病毒(PCV)的开放阅读框2(ORF2)是其主要结构蛋白(Cap)的编码基因，也是区分猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)的重要基因，本研究参照GenBank已发表的15株PCV2基因序列，设计合成一对引物，对PCV2的ORF2基因进行PCR扩增，产物经琼脂糖凝胶电泳，呈现一条大小约0.67kb的特异条带，经条件筛选，建立了猪病料中PCV2感染的PCR检测方法。采用建立的PCR方法对四川9个规模化猪场104头猪的病料进行PCV2检测，结果表明，受检猪的组织及血清样品中PCV2的总检出率为77.9％，证实四川规模化猪场存在PCV2感染。 对PCV2感染猪的症状及病理变化进行观察，结果表明，患猪主要表现为仔猪生长不良、腹泻及怀孕母猪流产等症状，患猪精神不振、消瘦，发生黄疸。剖检可见全身淋巴结显著肿大，肺脏出血、水肿以及发生肉样变等病理损伤，病理组织学观察发现组织器官内淋巴细胞减少，生发中心内淋巴细胞消失，在巨嗜细胞内发现嗜碱性包涵体。 将PCR检测为阳性的病料接种PK15细胞，盲传6代后进行电镜观察，结果在PK15细胞的细胞核及细胞质中发现包涵体。细胞质中包涵体电子密度深，位于核膜周围，内有呈晶格状排列的病毒粒子，直径12±2nm；细胞核内染色质消散，在细胞核内发现多个电子密度深的、圆形或环状、由大量异染色质组成的包涵体，内有细微的粒状物质。分离鉴定获得PCV2四川毒株1株。将病毒在PK15细胞上传6代后，提取病毒DNA，设计引物扩增全序列，并进行全基因组序列的测定与分析。结果表明病毒的全基因组为1767bp，与国内外分离毒株(15株)的全基因组序列比较发现，该分离株与我国湖南分离株(AY556477)的同源率为97.5％，与欧洲、美洲、南非毒株的同源率在94.2-96％之间。 本研究建立了针对PCV2 ORF2基因的PCR检测方法，可对病料、血清提取DNA进行检测，在7小时内得出结果。该方法可用于PCV2感染猪的快速诊断，可用于PCV2感染的流行病学调查、引种检疫等。本研究丰富了我国PCV2感染的流行病学资料，为规模化猪场有效诊断防治猪圆环病毒病提供了科学依据。更多还原

【Abstract】 The ORF2 gene is the most important protein-code(Cap) gene for porcine circovirus(PCV), and is the main gene differing porcine circovirus typel(PCVl) from porcine circovirus type2 (PCV2). To construct a specific PCR detecting method, we designed a pair of primers to amplify a partial fragment of ORF2 gene from PCV2, referring to fifteen strains’ complete genome published in Genbank. The PCR product was about 0.67kb in size detected by gelose gel electrophoresis. PCV2 in tissues or serum from 104 diseased or dead pigs collected from 9 large-scale pig farms in Sichuan was detected by PCR. It showed that the positive rate was 77.9%. So it proved that there was PCV2 infection in Sichuan.The symptom of pigs infected PCV2 were mainly unthrifty, diarrhoea and abortion. The gross lesions in pigs with PCV2 infection showed wasting, depression, gastric ulcerations and icterus. The abnormalities in the lungs and enlargement of all of the inguinal, mesenteric, bronchial and mediastinal lymph nodes were consistent findings. The lung was hemorrhagic, and was dropsy with granulomatous interstitial pneumonia. Histologically the lymphocytes in many organs were decreased, the lymphocytes of germinal center were disappeared, and basophilic inclusion bodies were found in macrophage.The ultrastructure of porcine circovirus was examined in persistently infected (six times) porcine kidney PK15 cells, which was positive detected by PCR. It turned out that many inclusions were found in the nucleolus and the cytoplasm of PK15 cell. The paracrystalline arrays was found in the inclusion in cytoplasm whose electron dense is deep; The nuclear of PK15 cell dissolved and there were margination of multiple chromatin aggregates and suspected intranuclear viral inclusion in PK15 cell. It was identical with the reports concerned.Extract viral DNA through PK15 cell infected PCV2 as template, and design a pair of primers to amplify the complete PCV2 genome. After sequenced and analyzed, we found that the complete genome of PCV2 isolated is 1767bp in size. For the complete genome sequence, the PCV2 isolate shared 97.5% homology with the isolate from Hunan(AY556477) and shared higher homology with the isolates from Europe, America and S. Africa isolates, which is from 94.2% to 96%.We developed specific PCR method based on ORF2 gene to detect PCV2 DNA through tissue samples or serum from PCV2-infected pigs, and we can get the perfect result in 7 hours. This method can be used in epidemiology investigation, and in quarantine of PCV2 infection. This study enriched molecular epidermiologic study of PCV2 infection, and provided scientific gist to prevent and treat PCV2 infection effectually on large-scale pig farms.更多还原