3种亚群PCV-2感染性克隆的构建及体内外感染性精选.pdfVIP

37 1 () Vol.37 No.1 2011 年 2 月 Journal of Hunan Agricultural University (Natural Sciences) Feb ．2011 DIO:10.324SP.J.1238.2011.00068 3 种亚群 PCV−2 感染性克隆的构建及体内外感染性 ( 410128) 摘 要：PCR 2(PCV-2) 3(1A1B1C)DNA pMD-18TPCV-23RP8(1A)P0(1B)P4-1(1C)3 Sac Ⅱl 767 bpPCV-2T43 PCV-1PK-155 (IFA)PCRPCV-231A1B1C 1A1B1C5PCV-2 DNA1A1B1CPCV-2DNA 关 键 词：2 中图分类号：S855.3 文献标志码：A 文章编号：1007− 1032(2011)01−0068−05 Construction of an infectious molecular clone of three kinds of subgroups of PCV-2 and its in vitro and in vivo experimental infection * LI Wei, LUO Wei, YU Xing-long , LI Run-cheng , BAI Xia, GE Meng, WANG Ya, XIANG Wei-jun, LI Jing (College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China) Abstract: The complete genome of three subgroups(1A, 1B, 1C) of porcine circovirus 2 (PCV-2)was amplified by polymerase chain reaction(PCR) and cloned directly into plasmid pMD-18T, and recombinant plasmid carrying the complete genome was constructed, designated RP8(1A), P0(1B), P4-1(1C) respectively. The three entire genomes of PCV-2 were purified and recycled from recombinant pMD-18T with the digestion of Sac Ⅱ enzyme and then circular genomic DNA were generated by self- ligating with T4 DNA ligase in vitro, resulting in three circular virus genome. The PCV1-free PK-15 cells were transfected with the PCV-2 circular genome using lipofectin reagent. At the fifth passages, PCV-2 virus and specific antigens could be detected by PCR and indirect immunofluorescent assay(IFA).Mice were inoculated