【作者】 郗鑫；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 猪圆环病毒(porcine circovirus，PCV)属于圆环病毒科，是一种无囊膜的二十面体的单链环状DNA病毒。该病毒分为两种基因型，即PCV-1和PCV-2。前者由Tischer等1974年在PK-15(ATCC-CCL)细胞中发现，没有致病性；后者是仔猪断奶后多系统衰竭综合征的重要病原。 设计合成一对扩增猪Ⅱ型圆环病毒(PCV-2)全基因组的特异性引物，从3份患断奶后仔猪多系统衰弱综合征(postweaning multisystemic wasting syndrome，PMWS)的死亡仔猪病料中，PCR扩增和克隆了3株PCV-2全基因组序列。将所测序列与已公布的PCV毒株序列进行同源性比较，并绘制系统发育进化树。结果显示，所测毒株间的核苷酸同源性为95.7％-99.4％，与其它毒株的同源性较高，达95.1％以上；ORF1的核苷酸同源性高达97.8％以上，氨基酸同源性大于98％；ORF2的核苷酸同源性较低，为90％-98％不等，推导的氨基酸序列同源性介于87％-99.1％。进化树分析表明各分离毒株在进化上存在地域上的相关性。将克隆到的PCV-2基因组环化后，脂质体介导转染PK-15细胞。盲传3代。间接免疫荧光检测表明，克隆到的青岛(QD)毒株基因组具有感染性。 将克隆到的PCV-2基因组SacⅡ酶切释放，体外连接生成二聚体，然后插入pBluescript SK+载体，构建PCV-2分子克隆pSK2PCVG，脂质体介导转染PK-15细胞，间接免疫荧光检测表明，pSK2PCVG在体外具有感染性。 根据已测定的序列设计引物，PCR扩增和克隆到了ORF1基因的完整编码区段，并测定了核苷酸序列。克隆的ORF1基因全长945bp，编码314个氨基酸。进一步将此片段亚克隆到原核表达载体pET-28a和pGEX-KG中，转化大肠杆菌BL21(DE3)菌株，Western印迹检测获得了分子量分别为38kD和63kD的非融合蛋白和与谷胱甘肽-S转移酶(GST)相融合的表达蛋白。 本研究克隆了3株PCV-2的全基因组，并测定了序列，通过系统进化分析表明各分离毒株在进化上存在地域上的相关性，在此基础上进一步构建了PCV-2感染性分子克隆。对ORF1基因成功的进行了原核表达。这些工作为研究PCV-2的流行和变异、探索PCV-2的致病机理、开发新的疫苗和诊断方法奠定了基础。更多还原

【Abstract】 Porcine Circovirus(PCV) is a small, nonenveloped, icosahedral mammalian virus containing a circular single-stranded DNA genome, which now has been assigned to the Circoviridae family. PCV-lwas first described in 1974 by Tisher as a picornavirus-like contaminant of the permanent pig kidney cell culture PK-15(ATCC-CCL33) and accepted to be a nonpathogemic virus. However, PCV-2 has been associated with postweaning multisystemic wasting syndrome(PMWS).In this research ,a pair of specific primers was designed according to the published genome sequences of PCV-2 to amplify the full length of viral genome by PCR from three samples with clinical signs of PMWS. The PCR product was cloned into pMD-18T vector and sequenced. After comparison the sequence with other PCV strains in GenBank. phylogenetic analysis was performed on the PCV strains. The results showed that there was 95.7%-99.2% nucleotide identity among the three strains and more than 95.1% identity between our cloned genomes and other PCV-2 strains in GenBank. The ORF1 of the cloned genomes shared more than 97.8% nucleotide identities and more than 98.0% deduced amino acid identities with other PCV-2 isolates, while the ORF2 has 90%-98% nucleotide sequence identity and 87%-99.1% amino acid identity. Phylogenetic tree analysis showed there is correlation between PCV-2 isolates and geographic regions. The circulated genomes are transfected into PK-15 cell line with Ipofectamine?2000. IFA was conducted after 3 passages of the transfected cells and the results showed that the genome of QD strain was infectious.The PCV-2 genomic DNA was digested with Sac II and then ligated to form concatemers ,. the concatemers was cloned into pBluescript SK + vector to construct the molecular PCV-2 clone: pSK2PCVG. The molecular PCV-2 clone was transfected into PK-15 cells by Ipofectamine mediated transformation., IFA showed that pSK2PCVG was infectious in vitro.In another research, a pair of specific primers were designed and synthesized according to the sequence of ORF1 gene of PCV-2 QD strain.The complete DNA fragment of ORF1 gene was obtained by PCR. Its nuclectide sequence was determined by the dideoxy-mediated chain termination method. Sequence analusis showed that the complete open reading frame (ORF) of ORF1 gene encoding 314 amino acid was 945bp in length. Then, the DNA fragment of ORF 1 gene was subcloned into prokaryotic expression vectorpET-28a and pGEX-KG, the specific non-fusion and fusion protein with GST of molecular weight 38kD and 63kD was expressed in E.coli BL21(DE3).Western blotting assay indicated that the polyclonal antibody against PCV-2 could recognize these two proteins.In this study, three complete genomes of PCV-2 were amplified and sequenced. Phylogenetic tree analysis showed there is correlation between PCV-2 isolates and geographic regions. Consequently, We construct the infectious molecular clone of porcine circovirus type 2. Moreover, ORF1 gene was expressed in E.coli. This study will give some help on studying epidemiology, vaccine , diagnosis and control of PCV-2 in China.更多还原