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。
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 pBluescript Ⅱ SK （＋） （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/kORF2）oa fragment of 4 023 bp, using the plasmid of SK-PCV2 as the template and introduced Nhe Ⅰ and Sph Ⅰ restriction enzyme sites ;the primer pair of P3 and P4 amplifed the ORF2 gene of PCVI,a fragment of 702 bp,and introduced Nhe Ⅰ and Sph Ⅰ restriction enzyme sites. The SK-PCV2 △ORF2 and PCV1 ORF2 which were digested by Nhe Ⅰ and Sph Ⅰ ,were ligated to construct the chimeric plasmid of SK-PCV2-1. By Sac Ⅱ digestion,the PCV2-1 genome was excised and dimerized to produce the reciprocal chimeric plasmid of SK-PCV2-1 （DB）. Transfeet 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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2007年第1期1-5,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(36170701)
关键词
嵌合猪圆环病毒
感染性克隆
构建
鉴定
PCV2-1
the chimeric infectious DNA clone
construction
identification
