A High Efficient Method of Constructing Recombinant Bombyx mori (Silkworm) Multiple Nucleopolyhedrovirus Based on Zero-Background TO-Mediated Transposition in Escherichia coli
A High Efficient Method of Constructing Recombinant Bombyx mori (Silkworm) Multiple Nucleopolyhedrovirus Based on Zero-Background TO-Mediated Transposition in Escherichia coli
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DOI：
10.1002/btpr.125
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发表时间：
2009-03-01
影响因子：
2.9
通讯作者：
Jin, Peng Fei
中科院分区：
文献类型：
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作者：
Sun, Jing Chen;Zhang, Er Hui;Jin, Peng Fei
研究了用tn7介导的大肠杆菌转位法高效制备重组家蚕多核多角体病毒的方法。新系统由条件复制供体载体pRCDM和attTn7位点阻断的含有BmNPV-Bacmid的大肠杆菌组成。供体载体含有来自R6K γ的复制源，该复制源仅在转位背景下pir基因表达显著降低的宿主细胞中繁殖。与pUC衍生的原载体相比，用条件复制载体pRCDM转座原BmDH10Bac时，转座效率从5.7提高到66%（约10倍）。通过阻断宿主大肠杆菌基因组中的attTn7位点，进一步努力降低转位背景。由此产生的attTn7占据BmDH10Bac Delta Tn7导致通过转位产生重组BmNPV Bacmid的功效从5.7%显著增加到23%（约4倍）。此外，BmDH10Bac Delta Tn7与pRCDM的转座通常导致100%的白色菌落，这表明实现了零转座背景。该高效、零背景转位系统为构建重组BmNPV提供了一种简便、快速的新方法，可用于家蚕靶基因的表达或基因传递病毒颗粒的制备。(C) 2009美国化学工程师学会。掠夺。中文信息学报，25:524-529,2009
A high efficient way for generation of recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli was performed. The new system consists of a conditional replication donor vector pRCDM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor vector contains a replication origin derived from R6K gamma, which propagated only in host cells with pir gene expression decreased in the transposition background greatly. Compared with original vector derived from pUC, the transposition efficiency increased from 5.7 to 66% (approximate to 10 fold) when using conditional replication vector pRCDM transposition into original BmDH10Bac. A further effort to decrease the transposition background was made by blocking the attTn7 site in host E. coli genome. The resulting attTn7 occupied BmDH10Bac Delta Tn7 resulted in a significant increase from 5.7 to 23% (approximate to 4 fold) in the efficacy of generate recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10Bac Delta Tn7 with pRCDM resulted typically in 100% white colonies, and it indicated that a zero transposition background was accomplished. This high efficient and zero background transposition system provides a new simple and rapid method for construction of recombinant BmNPV used to express tat-get genes or produce gene-delivery virus particles in silkworm. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 524-529, 2009