摘要
本研究通过PCR扩增出猪圆环病毒2型(PCV-2)的全基因组(1768bp),克隆入pcDNA3载体的EcoR Ⅰ酶切应点,获得含有PCV-2全基因组的重组质粒,命名为pcDNApcv2。将重组质粒大量扩增后,用EcoR Ⅰ切出1768bp的PCV-2全基因组,在体外用T4 DNA连接酶使其连接环化。用脂质体法将体外连接产物转染无PCV污染的PK-15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV-2病毒。由此可见,本试验构建的环化的PCV-2全基因组DNA具有感染性。
The complete genome of type 2 Porcine circovirus (PCV-2) was amplified by polymerase chain reaction (PCR) and cloned directly into the EcoRI sites of plasmid pcDNA3, and recominant plasmid carrying the complete genome was constructed, designated pcDNApcv2. The entire genome of PCV-2 was purified and recycled from pcDNApcv2 with the digestion of EcoRI enzyme and then circular genomic DNA was generated by self-ligating with T4 DNA ligase in vitro. The non-infected PK-15 cells were transfected with the PCV-2 circular genome using Lipofectin Reagent. After four continuous passages, PCV-2 virus and specific antigens were visualized by electron microscopy and IFA, respectively. Thus, the cloned circular PCV-2 genomic DNA generated in this study was infectious in vitro.
出处
《中国病毒学》
CAS
CSCD
2003年第4期371-375,380,共6页
Virologica Sinica
基金
国家863高技术发展计划资助项目(2001AA249012)
上海市科技兴农攻关项目[农科攻字(2001)第3-5号]
关键词
猪圆环病毒2型
基因组
DNA
克隆
Porcine circovirus type 2
Complete genome
Transfection
