禽腺病毒江苏株（JS）复制非必需片段的确定

利用PCR方法从一株Ⅰ群禽腺病毒的分离物(FAVI-JS)中扩增出其基因组的两个末端L片段和r片段、ITR片段,并分别克隆进pGEM-T easy载体,然后将L片段、r片段和ITR片段同时克隆进pHC粘粒载体中,获得质粒pHC-FAVI-r-ITR-L,再在该克隆片段中插入增强型绿色荧光蛋白(eGFP)基因,获得转移质粒载体pFAVI-eGFP.将pFAVI-eGFP转染已被该野生型Ⅰ群禽腺病毒分离物感染了的鸡胚肾细胞进行同源重组,通过无限稀释法筛选重组病毒,结果获得了表达增强型绿色荧光蛋白的重组Ⅰ群禽腺病毒rFAVI-eGFP,证明位于基因组右末端r片段和ITR片段之间的位点为病毒复制非必需区,为禽腺病毒的重组基因工程疫苗的研究奠定了基础.

Identification of Non-essential Region for Fowl Adenovirus Replication of JS Strain

The non-essential region of a strain of fowl adenovirus group I virus (FAVI-JS) isolated from chicken was identified. The left terminal (L fragment) and right terminal (r fragment and ITR fragment) of virus genome were respectively amplified by PCR. PCR products were cloned into pGEM-T easy vector respectively. L fragment, ITR and r fragment were then cloned into pHC cosmid to construct pHC-FAVI-r-ITR-L. The enhanced green fluorescence protein (eGFP) coding sequence was cloned between r fragment and ITR fragment to construct transfer vector pFAVI-eGFP. Then the pFAVI-eGFP was transfected CEK infected with wt-FAVI-JS.The recombinant FAVI (rFAVI-eGFP) were recovered by homologous recombination in CEK between wt-FAVI-JS genome and pFAVI-eGFP DNA. rFAVI-eGFP was purified by serial dilution of the supernatant of transfected cells. These results showed that the region between r fragment and ITR fragment was dispensable for virus replication in vitro.