3种亚群PCV-2感染性克隆的构建及体内外感染性  

Construction of an infectious molecular clone of three kinds of subgroups of PCV-2 and its in vitro and in vivo experimental infection

作　　者：李薇[1] 罗维[1] 余兴龙[1] 李润成[1] 白霞[1] 葛猛[1] 王亚[1] 向卫军[1] 李晶[1] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《湖南农业大学学报（自然科学版）》2011年第1期68-72,共5页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家自然科学基金项目(30971951);湖南农业大学稳定人才基金项目(09WD18)

摘　　要：通过PCR扩增出猪圆环病毒2(PCV-2)3个亚群(1A、1B、1C)毒株的全基因组DNA序列,并分别克隆入pMD-18T载体,获得含有PCV-2全基因组的3个重组质粒,分别命名为RP8(1A)、P0(1B)和P4-1(1C)。将3个重组质粒分别用SacⅡ切出l 767 bp的PCV-2全基因组,并在体外分别用T4连接酶连接而环化。用脂质体将3种自身环化的基因组转染无PCV-1污染的PK-15细胞,并按常规病毒培养方法分别传代,第5代细胞培养物分别用间接免疫荧光试验(IFA)及PCR方法检测转染的细胞,检测结果均为阳性,说明已获得PCV-2共3个亚群1A、1B、1C的感染性克隆。用1A、1B、1C感染性克隆的第5代细胞培养物分别接种小鼠,可在接种小鼠的血液中分别检测到相应PCV-2亚型的基因组DNA,表明本试验构建的自身环化的1A、1B、1C亚型的PCV-2的全基因组DNA在体内、体外均具有感染性。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 SacⅡ 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 with the cell cultures which were passed 5 times,the blood samples were collected 5 d after inoculation,the PCV-2 genomes were confirmed by PCR.The results indicate that the cloned circular PCV-2 genomic DNA is infectious both in vitro and in vivo,which will provide good foundation for further study of the relationship between different genotype PCV-2 strains and their virulence.

关 键 词：猪圆环病毒2 感染性克隆 基因亚群 毒力 

分 类 号：S855.3[农业科学—临床兽医学]