Successful Rescue of Synthetic AcMNPV with a ~17 kb Deletion in the C1 Region of the Genome.
Successful Rescue of Synthetic AcMNPV with a ~17 kb Deletion in the C1 Region of the Genome.
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成功拯救基因组 C1 区域约 17 kb 缺失的合成 AcMNPV
DOI：
10.3390/v14122780
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发表时间：
2022-12-13
期刊：
影响因子：
--
通讯作者：
Hu Z
中科院分区：
文献类型：
--
作者：
Guo Y;Hu H;Xiao H;Deng F;Li J;Wang M;Hu Z
杆状病毒已被广泛用作表达载体。然而，杆状病毒基因组中的许多基因对于细胞感染和蛋白质表达不是必需的，这使得杆状病毒表达载体的优化成为可能。采用合成生物学方法，对应用最广泛的杆状病毒表达载体——加州多核多角体病毒（AcMNPV）的部分区域基因进行了减少。AcMNPV的C1区为46.4 kb，分为B1、B2和B3片段。我们首先通过删除非必需基因来设计修饰的B1、B2和B3片段，然后在酵母中通过转化相关重组合成含有单个修饰B片段或联合修饰B片段的完整病毒基因组。然后将合成的基因组转染到Sf9细胞中以拯救子代病毒并测试其传染性。设计-构建-测试的循环不断重复，直到最终获救的病毒能够有效地产生子代病毒。最后，通过删除C1区约17.2 kb，包括20个orf，得到AcMNPV-Syn-mC1-1.1。这对于合成能够产生感染性子代病毒的最小AcMNPV基因组至关重要，并可进一步用于优化杆状病毒表达载体的基础。
Baculoviruses have been widely used as expression vectors. However, numerous genes in the baculoviral genome are non-essential for cellular infection and protein expression, making the optimisation of baculovirus expression vectors possible. We used a synthetic biological method to reduce the number of genes in a partial region of the autograph californica multiple nucleopolyhedrovirus (AcMNPV), the most widely used baculovirus expression vector. The C1 region of the AcMNPV is 46.4 kb and is subdivided into B1, B2, and B3 fragments. We first designed modified B1, B2, and B3 fragments by deleting the non-essential genes, and then synthesised complete viral genomes containing either individual modified B fragments or joint modified B fragments through transformation-related recombination in yeast. The synthetic genomes were then transfected into Sf9 cells to rescue the progeny viruses and test their infectivity. The design-build-test cycle was repeated until the ultimately rescued virus could produce progeny viruses efficiently. Finally, AcMNPV-Syn-mC1-1.1 by deleting approximately 17.2 kb, including 20 ORFs, in the C1 region, was obtained. This is essential to the synthesis of a minimal AcMNPV genome that can generate infectious progeny viruses and can be further used to optimise the foundation of baculovirus expression vectors.
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