Tn7-mediated Introduction of DNA into Bacmid-cloned Pseudorabies Virus Genome for Rapid Construction of Recombinant Viruses  

作　　者：Fang-fang ZHUAN Zhen-feng ZHANG Di-ping XU Yan-hong SI Han-Zhong WANG Ghopur MIJIT 

机构地区：[1]College of Life Science and Technology, Xinjiang University, Urumchi, 830046, China [2]State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China [3]Institute of Animal and Veterinary Science, Academy of Agricultural Sciences of Hubei, Wuhan, 430064, China

出　　处：《中国病毒学》2007年第4期316-325,共10页Virologica Sinica

基　　金：Key technologies R&D program (2006BAD06A01) from the Ministry of Science and Technology of China.

摘　　要：lacZα-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome (BAC) by homologous recombination in E. coli. The resulting recombinant BAC (pBeckerZF1) was confirmed by PCR and sequencing. Green fluorescent protein (GFP) gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect (CPE) observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3. vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.lacZa-mini-attTn7 was inserted into the intergenic region between the gG and gD genes in a PRV bacterial artificial chromosome （BAC） by homologous recombination in E. coli. The resulting recombinant BAC （pBeckerZF1） was confirmed by PCR and sequencing. Green fluorescent protein （GFP） gene was then transposed into pBeckerZF1 by transposon Tn7 to generate pBeckerZF2. Recombinant viruses vBeckerZF1 and vBeckerZF2 were generated by transfection with the corresponding BAC pBeckerZF1 or pBeckerZF2. The titers and cytopathic effect （CPE） observed for by vBeckerZF1 and vBeckerZF2 was comparable to that of the parental virus vBecker3, vBeckerZF2 was serial passaged for five rounds in cell culture, and the mini-Tn7 insertion was stably maintained in viral genome. These results show that recombinant viruses can be rapidly and reliably created by Tn7-mediated transposition. This technology should accelerate greatly the pace at which recombinant PRV can be generated and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies.

关 键 词：狂犬病病毒 无性系 DNA转导 Tn7转座子 

分 类 号：R373[医药卫生—病原生物学]