基于T7 RNA聚合酶的传染性法氏囊病病毒反向遗传系统的建立

    为了建立传染性法氏囊病病毒反向遗传系统,应用PCR法在鸡传染性法氏囊病病毒基因组A、B两节段的5'末端和3'末端分别引入T7启动子和核酶HDV序列,然后分别将含有T7启动子和核酶HDV序列的A、B节段构建到载体PT-Pluc当中,构建感染性载体PT-mA和PT-mB.在脂质体介导下将PT-mA和PT-mB共转染经痘病毒vTF7-3(含T7 RNA聚合酶基因,能稳定表达T7 RNA聚合酶)预先感染1 h的vero细胞.细胞病变观测、间接免疫荧光试验、电镜观察和分子标记检测证实成功实现了鸡传染性法氏囊病病毒的遗传拯救.

Establishment of infectious bursal disease virus(IBDV) reverse genetics system using T7 RNA polymerase

In order to establish the reverse genetics of IBDV,the sequences of T7 promoter and ribozyme HDV were introduced into the 5' and 3' terminals of segments A and B of IBDV genome using PCR.Then the segments,which containing promoter and ribozyme HDV sequences,were cloned into PT-Pluc vector and named as PT-mA and PT-mB, respectively.Both PT-mA and PT-mB were co-transfected into vero cells which were infected by recombinant vaccinia virus vTF7-3(vTF7-3 can express the T7 RNA polymerase in vero cell) for 1 hour.The surveying of cytopathic effect(CPE),indirect immunofluorescence test and molecular marker detection confirmed the succeeding of the rescuing of IBDV.