Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus.
Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus.
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DOI：
10.1007/978-1-4939-6964-7_6
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Britton P
中科院分区：
文献类型：
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作者：
Bickerton E;Keep SM;Britton P
我们已经为禽冠状病毒传染性支气管炎病毒（IBV）开发了一种反向遗传学系统，在该系统中，与IBV基因组相对应的全长cDNA在T7启动子序列的控制下被插入痘苗病毒基因组中。痘苗病毒作为全长IBV cDNA的载体具有这样的优势：可以利用同源重组将修饰引入IBV cDNA，同源重组是一种常用于在痘苗病毒基因组中插入和缺失序列的方法。在此，我们描述了使用瞬时显性选择作为一种将修饰引入IBV cDNA的方法，该方法已成功用于特定核苷酸的替换、基因组区域的缺失以及完整基因的交换。在将含有修饰的IBV cDNA的痘苗病毒DNA转染到感染了表达T7依赖DNA的RNA聚合酶的重组禽痘病毒的细胞中之后，具有感染性的重组IBV在原位产生。
We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependant RNA polymerase.