【作者】 崔晓华；

【导师】 胡桂学；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 本研究根据GenBank中已发表的猪圆环病毒(Porcine Circovirus，PCV)基因序列，设计一对PCV2特异性引物。利用PCR方法，对吉林省部分猪场的疑似断奶仔猪多系统衰竭综合征(PMWS)的9个自然病例的淋巴结、脾脏组织进行PCR检测。检测到阳性病料2份，检测阳性率为22.2％，说明该病在吉林省普遍存在。将检测阳性的病料组织研磨、离心、过滤除菌，接种到无PCV污染的PK-15细胞进行病毒分离。病毒在PK-15细胞上盲传4代后，提取病毒DNA，利用PCV2特异性引物对细胞培养物进行PCR检测，获得阳性结果，表明PCV2在无PCV污染的PK-15细胞中增殖培养成功。将PK-15细胞增殖所得的病毒样品处理，在电镜下可观察到直径约为17nm的圆形病毒样粒子。间接免疫荧光试验可观察到PCV2特异性免疫荧光。由此说明，分离出的病毒为猪圆环病毒2型，并命名为PCV2JL01和JL02株。 根据已发表的猪圆环病毒2型(PCV2)ORF2的基因序列，设计一对PCV2的ORF2基因特异性引物，以病料组织和病毒的细胞DNA为模板，对PCV2的ORF2基因进行PCR扩增，产物经琼脂糖凝胶电泳，出现一条大小约0.7kb的特异性目的条带，与预期目的片段大小相符。利用DNA凝胶快速回收试剂盒对PCR产物进行纯化回收，送大连宝生物公司测序。测序结果表明：PCV2 JL01和JL02株的ORF2基因序列均由702bp组成，通过比较发现，分离的JL01和JL02株ORF2基因同源性为99.9％，与其他PCV2毒株ORF2基因的同源性为91.9％～99.4％。 猪圆环病毒2型吉林株的分离鉴定、ORF2序列分析，为进一步开展猪圆环病毒病的病原学、诊断和防制研究奠定了基础，也为下一步开展的ORF2基因编码蛋白的生物活性及建立PCV2诊断试剂盒奠定基础。更多还原

【Abstract】 According to the published gene sequence of porcine circovirus (PCV) from Genbank, a pair of primer specific to porcine circovirus type 2(PCV2) were designed and synthesized. Lymph nodes and spleen of a swine suspected PMWS broke out in some porcine farm in Jilin province were detected by polymerase chain reaction (PCR). The result showed that 2 PCV2 positive samples were detected in 9 samples. The average positive rate is 22.2%. It approved that PMWS existed Jilin province. The PCV2 positive samples were abraded, sedimented by centrifugation, filtered and inoculated to the PK-15 cell line, which were not contaminated by PCV1. The extract viral DNA in PK-15 cells after 6 passages was detected with a pair of primers of PCV2 and obtained positive result. The result showed that the PK-15 cells free of PCV were infected by PCV2. Then the viral samples were pured and observed with electronic microscope. They were 17nm in diameter. In the cytoplasm, the specific fluorescence particles were observed in the frozen section strained by indirect immunofluorescence test specific to PCV2. According to above results, the isolated strain belongs to PVC2 and named PCV2 JL 01 and 02 strain.According to the published ORF2 gene sequence of PCV2, a pair of primer were designed synthesized and ORF2 gene was amplified by PCR. The product was about 0.7 kb in size detected by agarose gel electrophoresis. It was accord with the anticipated fragment. The products were purified and sequenced. The result showed that the ORF2 genes of JL01 and JL02 isolated strains are 702bp in size and they shared 99.9% homology with the referential strains and 91.9-99.4% homology with other isolated strains.That the viruses were isolated, identified and sequence analysis of ORF2 gene will supply good foundation for aetiology, diagnosis and control of PCV2. And the result also gives some help on researching the bioactivity of ORF2 protein-code and establishing the diagnostic kit.更多还原