鸡马立克氏病病毒细菌人工染色体的构建-预防兽医学专业论文.docxVIP

AbstractMarek’S Abstract Marek’S disease virus(MDVl is member of the Alphaharpesvirinae subfamily of the Herpesviridae，which is representative oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry．there three sero哆pes of MDV,MDVol include all virulent and very virulent oncogenic viruses，MDV-2 contain all unoncogenic viruses,and MDV-3 represents the herpesvirus of turkeys (HVn which has been widely used for vaccination against MD． 1t is especially true that construction or MDV viral mutants iS difficult and tediOUS procedure．Fonunatelg completely novel approach for the construction of herpesvirus mutants which is based cloning of the virus genome鹕a bacterial artificial chromosome(BAC)in Eseherichia coil has been developed．Once the viral genome is cloned as BAC，mutagenesis Can be done by homologous recombination in E．coli which is mainly based RED,ET mutagenesis system． Thc novel approach have clear advantages,and the powerful methods of bacterial gefietics allow the rapid and efficient introduction of any kind of mutations(deletion，insertion，and point mutation)into the cloned viral genome．even mutagenesis ofessential viral genes nOW becomes feasible． Thc aim ofthis study Was tO clone full—length genome ofMarek’S disease virus serotype l(MDV-1) 914 strain(nature attenuated strain)as an infectious BAC．In this study,Using the 8．8kb fragment containing self-designed selection marker(Eco-gpt，1．3kb)and the BAC vector pBeloBACl 1(7．5kb)。a recombination plasmid pUAB·印t-BACl 1 was constructed．For cloning MDV814-BAC the recombination plasmid and MDV Total-DNA were cotransfected into secondary CEF bY Lipofectamini 2000．virus-containing cells wld[-e passaged eight rounds in selection medium containing xanthine and mycophenolic acid tO obtain purified recombinant viruses．Recombinant viral Genomes Was extracted and Eleetroperated into E．cofi and thirty-eight BACs we犯obtained and identified，Finally,DNA from various MDV-1 BACs WSS transfected into secondary chic