Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovi...Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome.Recently, we generated a synthetic baculovirus Ac MNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). Here, we report the modification of Ac MNPV-WIV-Syn1 into a novel bacmid, Ac Bac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a Lac Z:att Tn7 and egfp cassette was constructed, and recombined with a linearized Ac MNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid Ac Bac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the att Tn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that Ac Bac-Syn can be readily used for foreign gene insertion and expression.Ac Bac-Syn has several advantages over the conventional Ac MNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration;its DNA copy numbers could be induced to a higher level in E. coli;and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.展开更多
基金This work was supported by the grants from the National Science Foundation of China(Grant Nos.31800143 and 31872640)the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences(Grant No.QYZDJ-SSW-SMC021)the Hubei Provincial Innovation Center of Agricultural Sciences and Technology(Grant No.2019-620-000-001-017)。
文摘Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome.Recently, we generated a synthetic baculovirus Ac MNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). Here, we report the modification of Ac MNPV-WIV-Syn1 into a novel bacmid, Ac Bac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a Lac Z:att Tn7 and egfp cassette was constructed, and recombined with a linearized Ac MNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid Ac Bac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the att Tn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that Ac Bac-Syn can be readily used for foreign gene insertion and expression.Ac Bac-Syn has several advantages over the conventional Ac MNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration;its DNA copy numbers could be induced to a higher level in E. coli;and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.
