猪Ⅱ型圆环病毒豫A株的全基因组克隆与序列分析

Cloning and Sequencing of Complete Genome of Porcine Circovirus Type 2(PCV-2)YU A Strain

参照国外发表的猪Ⅱ型圆环病毒 (porcinecircovirustype 2 ,PCV - 2 )全基因组序列 ,设计一对PCV - 2特异性引物 ,用该室分离的PCV - 2豫A株感染PK - 15细胞 ,从中提取PCV - 2复制型基因组DNA ,并以之为模板进行PCR扩增。回收PCR产物 ,构建重组测序质粒T -PCV - 2。测序结果表明 ,猪Ⅱ型圆环病毒豫A株的全基因组为 176 7bp ,与GenBank收录的PCV - 2国外分离株核苷酸的同源性可高达 97%。序列分析表明 ,复制型豫A株的基因组包含 10个读码框架 ,其中ORF1、ORF2是其两个最主要的读码框架 ,分别编码 314、2 34个氨基酸。豫A株和PCV - 1间的ORF1、ORF2的氨基酸序列同源性分别为 85 %、6 6 % ,与其它PCV - 2毒株间的ORF1氨基酸同源性均在 98%以上 ,而ORF2的氨基酸同源性为 92 %～ 97%。

The replicative genomic DNA of porcine circovirus type 2(PCV-2)of YU A strain was extracted from the infected PK-15 cells The complete genome of YU A strain was amplified by utilizing an inverse PCR approach with one pair of special primers. A 1 76kb fragment was obtained and cloned into pMD-18T vector resulting the recombinant plasmid T-PCV-2 that contained the complete genomic DNA of YU A strain The sequence analysis showed that YU A strain genome consisted of 1767nt,which shares 97% sequence identity with other PCV-2 strains in GenBank We also found replicative genome of YU A strain contained ten open reading frames(ORFs) The major ORFs were ORF1 and ORF2,that encoded 314 and 234 amino acids respectively Amino acid sequence homology of ORF1 and ORF2 between YU A strain and PCV-1 were 85% and 66% respectively The ORF1 of YU A strain displayed 98%-99% identity based on amino acids with other PCV-2 strains,but 92%-97% in ORF2