A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli
A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli
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一种通过用含有杆粒的二氨基庚二酸营养缺陷型大肠杆菌感染昆虫细胞来高通量生产重组杆状病毒的新经济方法
DOI：
10.1016/j.jbiotec.2009.10.003
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发表时间：
2010-01-01
影响因子：
4.1
通讯作者：
Yan, Hui Chao
中科院分区：
文献类型：
--
作者：
Yao, Lun Guang;Sun, Jing Chen;Yan, Hui Chao
由于杆状病毒DNA的制备和随后的转染过程是劳动密集型和耗时的，因此开发具有成本效益的方法在昆虫细胞中高通量生产重组杆状病毒是非常具有挑战性的。我们建立了一种将细菌重组Bacmid DNA导入昆虫细胞的新方法，只需利用侵袭性二氨基苯甲酸（DAP）缺陷营养大肠杆菌感染frugiperda 9细胞。带重组Bacmids的大肠杆菌细胞在假结核耶尔森菌侵袭因子的帮助下进入昆虫细胞。培养基中没有DAP， DAP营养不良大肠杆菌的细胞壁无法合成，使细菌细胞破坏并释放重组Bacmid。释放的Bacmids将在昆虫细胞中产生具有感染性的重组杆状病毒颗粒。我们将这种大肠杆菌侵染法与零背景转位法相结合，快速、简便地产生了重组杆状病毒。该方法不需要从大肠杆菌中制备和纯化重组杆状病毒，也不需要复杂的转染过程，不需要劳动密集型和复杂的转染过程，可以方便、经济地高通量生产重组杆状病毒。(c) 2009 Elsevier B.V.版权所有
Developing cost-effective methods for high throughput production of recombinant baculoviruses in insect cells is very challenging, because the baculovirus DNA preparation and the following transfection procedure are labour-intensive and time consuming. We developed a new method of introducing recombinant Bacmid DNA from bacteria into insect cells simply using invasive diaminopimelate (DAP) auxotrophic Escherichia coli to infect Spodoptera frugiperda 9 cells. The E coli cells with recombinant Bacmids enter insect cells with the help of the invasion factor from Yersinia pseudotubercolusis. Without DAP in medium, the cell wall of DAP auxotrophic E. coli cannot be synthesized so that the bacterial cell will disrupt and release recombinant Bacmid. The released Bacmids will generate infective recombinant baculovirus particles in insect cells. We combined this E coli invasion method with the zero background transposition system to generate recombinant baculovirus in a rapid and simple way. Without preparation and purification of recombinant Bacmids from E coli and the labour-intensive and complex transfection procedure, this transfection reagent free method enables a convenient and economic high throughput production of recombinant baculoviruses. (c) 2009 Elsevier B.V. All rights reserved.