【关键词】 禽I型腺病毒; 传染性法氏囊病毒; VP2基因; 重组腺病毒;

【英文关键词】 Fowl Adenovirus type I; Infectious bursal disease virus （IBDV）; VP2 gene; recombinant Adenovirus;

【中文摘要】 禽腺病毒宿主细胞广泛,繁殖后病毒滴度高,大多数呈隐性感染,是构建基因工程疫苗最为理想的载体之一。本实验室何秀苗等早期从健康鸡群中分离到一株无致病性的I型禽腺病毒,并构建了禽腺病毒通用转移载体pFAVI-CMV。本研究在此基础上,用EcoRⅠ、NheⅠ双酶切通用转移载体pFAVI-CMV,通过电泳、回收载体DNA;同时,用同样的酶切pCDNA3.1-VP2,电泳、回收VP2片段。通过连接酶连接构建成插有VP2基因的转移载体,经酶切分析正确,命名为:pFAVI-VP2。 以Lipofectin转染试剂将pFAVI-VP2 DNA与野生型禽I型腺病毒在鸡胚肾细胞(CEK)上进行同源重组,通过间接免疫荧光试验(IFA)进行检测,结果获得一株表达传染性法氏囊病病毒(IBDV)VP2基因的重组禽I型腺病毒,命名为:rFAVI-VP2。以梯度稀释和双层琼脂覆盖两种方法对重组病毒进行纯化,通过IFA方法进行检测,结果获得了纯化的rFAVI-VP2。用抗VP2特异性单克隆抗体建立的IFA方法对感染rFAVI-VP2病毒的细胞进行检测,证明该重组病毒能表达VP2蛋白抗原。用ELISA和PCR方法对在体外连续传递...

【英文摘要】 Fowl Adenoviruses have a wide host-cell range, most of which are nonpathetic and could be propagated in large amount easily. HE Xiu-Miao previously isolated a strain of Fowl Adenovirus group I and constructed universal transfer vector pFAVI-CMV. Infectious bursal disease virus (IBDV) VP2 gene from pcDNA3.1-VP2 was subcloned into pFAVI-CMV by EcoRⅠand NheⅠdouble digestion. The recombinant transfer vector was designated as pFAVI-VP2. Restriction endonuclease digestion identified it right. In the presence...