猪圆环病毒2型全基因组的克隆及病毒拯救

Cloning and virus rescue of full length genome of porcine circovirus type 2

为构建猪圆环病毒(Porcine circovirus,PCV)2型(PCV2)感染性DNA克隆,以淋巴结病料组织中分离的PCV2细胞培养适应毒株为材料,通过基因序列分析比对,在PCV2基因组序列XbaⅠ酶切位点区设计重叠引物,进行全长扩增并克隆至pMD-18T和pBluescript SK,获得载体pMD-PCV2和pBluescript SK-PCV2。将pMDPCV2重组质粒大量扩增后,用XbaⅠ切出PCV2全基因组,在体外用T4DNA连接酶使其自环化。通过脂质体法将PCV2自环化产物转染至PK15细胞,并且经15次连续传代,分别用PCR、RT-PCR和间接免疫荧光测定(Indirect immunofluorescence assay,IFA)检测到PCV2的复制、转录和蛋白表达。该研究成功构建具有感染性的PCV2全基因组DNA,为PCV2致病机理和基因功能研究奠定基础。

In order to construct the infectious DNA clone of Porcine circovirus 2（PCV2）,the adapted strain cultured with PCV2 cell separated from lymphadenopathy tissues was used as the material,the overlap primer was designed in the Xba Ⅰ enzyme cutting site area of PCV2 genome sequence through gene sequence analysis and comparison to conduct full length amplification and clone it to pMD-18 Tand pBluescript SK so as to gain the carriers pMD-PCV2 and pBluescript SK-PCV2.After amplification of pMD-PCV2 recombinant plasmid in quantity,Xba Ⅰ was used to cut PCV2 whole genome,and T4 DNA ligase was used in vitro to make it auto-cyclized.Auto-cyclized product of PCV2 was transfected to PK15 cell by lipidosome method.After 15 continuous passages,PCR,RT-PCR and indirect immunofluorescence assay（IFA）were used to detect replication,transcription and protein expression of PCV2.In this research,DNA of whole genome of infective PCV2 was successfully constructed.Thus,this research lays a foundation for the research on PCV2 pathogenesis and gene functions.