为鉴定含J亚群禽白血病病毒(ALV-J)全长cDNA分子克隆(pBlALV)的感染性,首先将pBlALV转染CEF细胞,拯救出病毒(rALV),然后连续传代3次,对拯救病毒进行增殖培养。分别利用RT-PCR、westernblot、IFA等方法,对拯救病毒进行鉴定,并应用实时荧光定量PCR方法对拯救病毒的复制动力学进行了测定。研究结果证明,本研究成功拯救出了具有感染性的rALV-J,为研究禽白血病的致病机理和探讨新的防制措施等提供了良好的ALV的反向遗传操作技术平台。
To confirm the infectivity of the full-length cDNA clone(pBlALV) of subgroup J avian leucosis virus(ALV-J) HPRS103 strain,pBlALV was transfected into chicken embryo fibroblast(CEF) cells and the ALV was rescued.The rescued ALV(rALV-J) was identified by RT-PCR,western blot and indirect immunofluorescence assay,respectively.Besides,the real-time PCR was applied to evaluate the replication dynamic of the rescued virus.The results showed that the rALV-J was able to well replicate in CEF.This study provides an useful platform for investigation of the pathogenesis and molecular biology of ALV-J.