摘 要：
根据GenBank中发表的猪圆环病毒(porcine circovirus,PCV)的核苷酸序列,设计3对特异性引物。通过PCR方法,以引物P1和P2从接毒细胞中扩增到猪型圆环病毒(PCV2)全基因组,克隆入pBluescriptⅡ SK(+)载体构建质粒SK-PCV2,以之为模板,用引物P5和P6扩增不包含PCV2-ORF2的部分基因(SK-PCV2△ORF2);另一对引物P3、P4用于特异性扩增猪型圆环病毒(PCV1)的ORF2基因,与前面得到的SK-PCV2△ORF2基因连接,构建嵌合型质粒SK-PCV2-1,以此为基础构建双联体质粒SK-PCV2-1(DB)。体外转染PK-15细胞,盲传4代后,用RT-PCR和间接免疫荧光方法检测,结果表明本试验构建的嵌合病毒PCV2-1克隆具有感染性。
译 名：
Construction and identification of the chimeric infectious DNA clone of porcine circovirus Ⅱ(PCV2-1)
作 者：
LIU Xin-wen,YAO Qing-xia,CAO Sheng-bo,GUO Dong-chun,CHEN Huan-chun* (College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China)
关键词：
PCV2-1;the chimeric infectious DNA clone;construction;identification
摘 要：
Three pairs of specific primers were designed according to the published PCV genome sequence in GenBank.The primer pair,P1 and P2,was used to amplify the full-length genome DNA by PCR from the cells which were infected by PCV2(GenBank:AY291318).The PCR product was cloned into pBluescriptⅡSK(+)(pSK) vector to construct the recombinant plasmid designated as SK-PCV2.The PCR primer pair,P5 and P6,amplified the pSK vector and the PCV2 genome without the ORF2 gene(SK-PCV△ORF2),a fragment of 4 023 bp,using the plasmid of SK-PCV2 as the template and introduced NheⅠ and SphⅠ restriction enzyme sites;the primer pair of P3 and P4 amplifed the ORF2 gene of PCV1,a fragment of 702 bp,and introduced NheⅠ and SphⅠ restriction enzyme sites.The SK-PCV2△ORF2 and PCV1 ORF2 which were digested by NheⅠ and SphⅠ,were ligated to construct the chimeric plasmid of SK-PCV2-1.By SacⅡ digestion,the PCV2-1 genome was excised and dimerized to produce the reciprocal chimeric plasmid of SK-PCV2-1(DB).Transfect PK-15 cells with SK-PCV2-1(DB) in vitro.After four passages,the infectivity of the cloned chimeric PCV2-1 genome was confirmed by RT-PCR and IFA.
