猪圆环病毒Ⅱ型感染性DNA克隆的构建  

Construction of an Infectious DNA Clone of Porcine Circovirus Type Ⅱ

作　　者：谢俊岭[1] 邵小雪[1] 孟凡伟[1] 周双海[1] 

机构地区：[1]北京农学院动物科学技术学院,北京102206

出　　处：《中国农学通报》2012年第32期84-88,共5页Chinese Agricultural Science Bulletin

基　　金：国家自然科学基金项目"微血管内皮细胞在猪圆环病毒II型感染中的作用"(30600442);北京市自然科学基金项目"猪圆环病毒II型感染对伪狂犬病疫苗免疫反应的影响"(6082008);北京市教委科技计划项目"嵌合猪圆环病毒疫苗株的构建"(KM200810020013)

摘　　要：为了构建猪圆环病毒Ⅱ型(PCV2)感染性DNA克隆,并为PCV2的突变体构建及其致病机理研究建立基础。用PCR方法从PCV2 DNA质粒中扩增出PCV2全基因组片段,将其插入到pBluescript-SK载体中,构建出单拷贝PCV2 DNA克隆,并进一步构建了双拷贝PCV2 DNA克隆,将PCV2 DNA克隆转染PK-15细胞,以获得PCV2拯救病毒,并初步测定了拯救病毒的体外增殖能力。结果显示:成功获得了PCV2拯救病毒,并能够在PK-15细胞中进行稳定传代,其TCID50在连传5代后达到10-6.05/mL,与其亲本病毒相近,显示出较高的感染滴度。研究结果表明,成功构建了具有较高感染性的PCV2感染性克隆,为PCV2的生物学特性及致病机理研究奠定了基础。The aim of this study was to construct an infectious DNA clone of Porcine Circovirus Type II （PCV2） and lay the foundations for the mutant construction and pathogenesis researches of the PCV2. The complete genome of PCV2 was amplified from the PCV2 DNA plasmid by PCR method, and inserted into the pBluescript-SK vector to construct the single copy of PCV2 DNA clone. Furthermore, the double copy of PCV2 DNA clone was developed. Then, the transfection of porcine kidney （PK）-15 cells with the PCV2 DNA clones was conducted to obtain the rescue viruses of PCV2, and the reproducibility of the rescue virus in vitro was determined. The results showed that the rescue viruses of PCV2 were acquired successfully and could propagate stably in PK-15 cells, its 50% tissue culture infectious doses （TCIDso） was 10-6~5/mL after 5 serial passages, which was close to its parent virus and demonstrated its relatively high infectious titer. The research results revealed that an infectious clone of PCV2 with relatively high infectivity was developed successfully, which established a foundation for further research on the virus biological characteristics and pathogenesis.

关 键 词：猪圆环病毒Ⅱ型 感染性DNA克隆 拯救病毒 增殖能力 

分 类 号：S855.3[农业科学—临床兽医学]