Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid
Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid
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家蚕体腔内注射含有 BmNPV-Bacmid 的侵入性二氨基庚二酸营养缺陷型大肠杆菌生产重组家蚕核多角体病毒
DOI：
10.1042/ba20100148
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发表时间：
2010-11-01
影响因子：
2.8
通讯作者：
Kan, Yunchao
中科院分区：
文献类型：
--
作者：
Sun, Jingchen;Yao, Lunguang;Kan, Yunchao
本研究阐述了一种经济高效、无转染剂的方法，通过将携带 BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] 的侵入性大肠杆菌注射到幼虫血腔中，在蚕幼虫和蛹中产生重组家蚕核多角体病毒。通过腔内注射含有 10(6) DAP（二氨基庚二酸）营养缺陷型和含有表达入侵素和李斯特菌溶血素的 BmBacmid 的大肠杆菌细胞，在家蚕中产生高达 10(9) PFU（空斑形成单位）/ml 的感染性重组杆状病毒。因此，1 毫升过夜培养的大肠杆菌足以注射超过 2000 只幼虫，而成本高达 1 美元的 DAP 足以注射约 4000 只幼虫。通过调整注射紧密度，也可以控制重组蛋白主要在蛹中表达。在这种新方法中，许多原始操作被取消，包括BmBacmid制备和随后的复杂转染程序。因此，与传统转染方法相比，这是一种节省时间和成本的大规模注射家蚕用于重组杆状病毒生产的手段，这可能在BmNPV-家蚕生物反应器的工业化发展中发挥重要作用。
The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 10(9) PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 10(6) DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1 ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 Larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection close, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.