title: [Construction of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome].
abstract: The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.
authors.affiliation: National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Cui
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Wang
authors.forename: Xingming
authors.initials: X
authors.lastname: Shi
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Tong
authors.forename: Desong
authors.initials: D
authors.lastname: Lan
authors.forename: Lai
authors.initials: L
authors.lastname: He
authors.forename: Huaji
authors.initials: H
authors.lastname: Qiu
authors.forename: Changjun
authors.initials: C
authors.lastname: Liu
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
mesh_terms: D000818:Animals; D002645:Chickens / Q000276:immunology / Q000821:virology*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D025922:Mardivirus / Q000145:classification / Q000235:genetics* / Q000502:physiology; D012703:Serotyping; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
chemical_list: D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins