Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome
Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome
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基于合成杆状病毒基因组的新型杆粒 AcBac-Syn 的构建和表征
DOI：
10.1007/s12250-021-00449-w
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发表时间：
2021-09-27
影响因子：
5.5
通讯作者：
Hu, Zhihong
中科院分区：
文献类型：
--
作者：
Shang, Yu;Hu, Hengrui;Hu, Zhihong
杆状病毒是一种大型DNA病毒，广泛用作表达载体和生物杀虫剂。同源重组和Bac-to-Bac系统已成为杆状病毒基因组操作的主要方法。最近，我们合成了一种人工合成的杆状病毒AcMNPV-wiv-SYN1，它与其亲本病毒加利福尼亚州Autograph a calfornica多核多角体病毒(AcMNPV)完全相似。在这里，我们报道了将AcMNPV-wiv-SYN1修饰成一种新型的杆状病毒，AcBac-Syn，它可以作为Bac-to-Bac系统的骨架。为了实现这一点，构建了含有LacZ：attTn7和EGFP的载体，并通过转化相关重组与线性化的AcMNPV-wiv-SYN1基因组在酵母中重组，获得了bacmidAcBac-Syn。将该杆状病毒转入昆虫细胞，在芽生病毒的产生动力学、包涵体的形态和对昆虫幼虫的口腔感染性等方面表现出与野生型病毒相似的生物学特性。通过常规的Bac-to-Bac方法，将红色荧光蛋白基因Dsred转座到attTn7位点，并进行了转染和感染实验，结果表明，AcBac-Syn可以方便地用于外源基因的插入和表达。与传统的AcMNPV杆状病毒相比，AcBac-Syn具有许多优点，如它含有一个EGFP报告基因，便于病毒繁殖和滴定的可视化；它的DNA拷贝数可以在大肠杆菌中诱导到更高的水平；它的基因组中保留了天然的多角体基因，使其成为研究与咬合体组装和口腔感染相关基因功能的一个有吸引力的系统。
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 AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-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 attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV 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.