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DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus

文献类型： 外文期刊

作者： Zou, Xiao-Hui 1 ; Bi, Zhi-Xiang 2 ; Guo, Xiao-Juan 1 ; Zhang, Zun 1 ; Zhao, Yang 2 ; Wang, Min 1 ; Zhu, Ya-Lu 2 ; Jie, Ho 1 ;

作者机构： 1.Chinese Ctr Dis Control & Prevent, Natl Inst Viral Dis Control & Prevent, State Key Lab Infect Dis Prevent & Control, 100 Ying Xin Jie, Beijing 100052, Peoples R China

2.Jiangsu Acad Agr Sci, Inst Vet Immunol & Engn, Natl Vet Prod Engn Res Ctr, Nanjing 210014, Jiangsu, Peoples R China

3.Jiangsu Coinnovat Ctr Prevent & Control Important, Yangzhou 225009, Jiangsu, Peoples R China

关键词： DNA assembly; Infectious clone; Fowl adenovirus 4; Purification; One-Step growth curve

期刊名称：JOURNAL OF VIROLOGICAL METHODS （ 影响因子：2.014； 五年影响因子：2.001 ）

ISSN： 0166-0934

年卷期： 2018 年 257 卷

页码：

收录情况： SCI

摘要： Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coil BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.

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