【作者】 张伟；

【导师】 冉多良；

【作者基本信息】 新疆农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 本研究通过从昌吉周边规模化猪场采集疑似PMWS病料,将样品经研磨、冻融、过滤等处理后,接种于PK15细胞,盲传6代。根据GenBank中猪圆环病毒基因组序列,设计两对PCV特异性引物,从病毒液中提取病毒核酸,应用PCR方法扩增含有EcoRⅠ酶切位点的PCV2特异性片段,酶切鉴定后证实采集的病料中有PCV2存在。将盲传6代细胞病毒经免疫荧光试验检测,可见细胞的胞质中含PCV特有的免疫荧光,表明该猪场中已存在PCV2感染。用扩增PCV2全基因组的特异性引物P2-1/P2-2,扩增病毒全基因组序列,回收PCR扩增产物,克隆到pMD18-T载体中,经PCR、酶切鉴定以及序列测定后,最终获得2个阳性克隆株,分别命名为XJPCV2-a株和XJPCV2-b株。通过多序列比对和遗传进化树分析,表明2个分离株全基因组序列与国内外其它16个PCV2毒株之间,核苷酸同源性分别为95.8%-99.4%；而与2个PCV1参考毒株之间核苷酸的同源性则仅为75.9%-76.1%,2个分离株之间核苷酸同源性高达99.3%。遗传进化树分析证实了本试验分离株与GenBank中登陆的16个PCV2毒株之间不存在地理位置上的相关性。2个毒株的成功分离为本地区规模化养猪场有效地诊断防治猪圆环病毒提供了科学依据,丰富了新疆地区规模化猪场PCV2感染的流行病学资料。根据分离株XJPCV2基因组序列,设计一对引物,以重组质粒pMD18-PCV2-a为模板,扩增出大小为654bp的ORF2基因片段。通过对ORF2基因的核苷酸和氨基酸序列进行分析可知,ORF2编码的蛋白在其氨基端含有许多稀有密码子,稀有密码子的存在影响到ORF2基因的表达,并且该蛋白的抗原位点主要位于羧基端。因此,通过限制性酶切对ORF2基因进行修饰,最终获得393 bp的ORF2基因。将截短的ORF2基因亚克隆到融合表达载体pGEX-6p-1中。经PCR、酶切鉴定以及序列分析后,将阳性重组子转入大肠杆菌BL21(DE3)中,进行IPTG诱导表达。SDS-PAGE电泳和Western blotting分析结果表明,重组质粒在大肠杆菌中表达的融合蛋白相对分子量为40.7kDa,融合蛋白能被PCV2单克隆抗体所识别,说明该融合蛋白可作为一种诊断PCV2感染用的诊断抗原。融合蛋白成功表达为猪圆环病毒病的临床诊断、建立血清学诊断技术以及疫苗的研制奠定了基础。更多还原

【Abstract】 In this study, some samples were collected from (contain lymph nodes, spleens, lungs and livers and so on) pigs with PMWS in Changji.The samples were frozen in-20℃and homogenized.The inocula composed of homogenized tissues were centrifuged and filtered to eliminate bacterial contaminant.The monolayer of PK15 cells free of PCV1 and PCV2 were inoculated with the inocula, and then incubated at 37℃for 48h in 5%CO2 atmosphere.The specific primers were designed, then the presence of PCV2 was tested by PCR from PK15 cell.The PCR products were digested with appropriate restriction enzymes and PCR-positive cell were identified by immunofluorescence.Results showed that the tissues infected the PCV2 viruses.The specific primers were designed and synthesized on the basis of the published sequence of PCV2 and the complete genomes were amplified by PCR from viral DNA of the PK15 cell.The purified PCR products were cloned into pMD18-T simple vector for sequencing.The identification of PCR and digested enzymes and sequencing results showed that the two isolates were obtained, and were named XJPCV2-a and XJPCV2-b.The phylogenetic analysis showed that the two PCV2 isolates were closely related to each other, shared 99.3% nucleotide sequence identities and displayed 95.8%-99.4% nucleotide homology and 75.9%-76.1% nucleotide homology with other PCV1 isolates in GenBank.Phylogenetic analysis confirmed the two isolates with other PCV2 strains have no pertinence in geograpfy.The specific primers were designed to amplify the ORF2 gene about 654 bp from the extracted DNA of isolate XJPCV2-a.According to the ORF2 gene nucleotide and amino acid analysis:The N end have a lot of rare codes, which influnce the expression.Main antigen eptiod of capsid protine located in the C end, therefore, the ORF2 gene was modified by enzymed digestion.The fragment of 393 bp of ORF2 gene and were cloned into pGEX-6P-1 vector.The recombinant plasmid (pGEX-6P-dORF2) was identified by PCR and digested enzymes and sequencing, then the postive recombinant plasmid (pGEX-6P-dORF2) was transformed into E.coli BL21.The fusion protein was expressed under the induction of IPTG and identification by SDS-PAGE and western blotting.Results showed that the fusion protein identical with the predicted molecular weight of 40.7 kDa, and that the protein could be recognized by monoantibodies.These research results would provide a basis for further study on the diagnosis technology of PCV2, clinic diagnosis and the devolopment of vaccine.更多还原