猪圆环病毒1型感染性克隆的构建与病毒拯救

Construction and virus rescue of an infectious clone of porcine circovirus type 1

用PCR法扩增猪圆环病毒1型(PCV1)全长基因组,将2个基因组顺式连接插入到pUC19质粒载体中构建感染性分子克隆。通过引物设计替换碱基,在病毒基因组内插入SalⅠ酶切位点作为分子靶标,拯救出带有分子标记的克隆病毒,命名为recPCV1/G株。通过免疫过氧化物酶单层细胞试验(IPMA)在病毒感染细胞中检出病毒抗原,其抗原性仅与PCV1/Cap蛋白单价特异性抗体发生反应,而与抗PCV2/Cap蛋白抗体无交叉。克隆毒株基因组内插入一个SalⅠ酶切位点,可用PCR结合限制性片段长度多态性分析法(PCR-RFLP)与亲本病毒相鉴别。该毒株经细胞连续传10代,体外培养增殖性能稳定,病毒滴度达104.8 TCID50/mL。构建的PCV1感染性克隆,为今后开展该病毒的起源与演化、遗传变异规律、分子鉴别诊断等研究奠定了基础。

The full-length genome of porcine circovirus type 1 （PCV1） was amplified by polymemse chain reaction （PCR）, Two copies of the genome were ligated in tandem to construct an infectious clone ofPCV1. A Sal I restriction enzyme site was inserted into the clone as a genetic marker. A recombinant virus designated as recPCV1/G was rescued from the infectious clone. The antigenicity of the virus was confirmed by immunoperoxidase monolayer assay （IPMA） using anti-PCV1/Cap antibody. No crossreaction was detected with anti-PCV2/Cap antibody. The viral genome could be differentiated from the parental virus by PCR combined with restriction fragment length polymorphism （PCR-RFLP）. The recombinant virus was stable in cell culture during ten passages and had a titre up to 10^4.8 TCID50/mL. This study suggests that the cloned recPCV1/G stain should be a useful tool in studies on genesis and evolvement, genetic variation, and molecular differentiating diagnosis ofPCV1.