通过含有转座子的 F 因子体外转座来克隆大型双链 DNA 病毒的完整基因组。

Cloning of complete genomes of large dsDNA viruses by in vitro transposition of an F factor containing transposon.

机构信息

Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Centre Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435 Neustadt an der Weinstrasse, Germany.

出版信息

J Virol Methods. 2010 Jul;167(1):95-9. doi: 10.1016/j.jviromet.2009.11.026. Epub 2009 Dec 3.

DOI:10.1016/j.jviromet.2009.11.026

PMID:19948191

Abstract

An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems.

摘要

一种改进的 bacmid 技术已经被开发并测试，用于克隆具有圆形基因组的大型 dsDNA 病毒的完整基因组。该系统称为 EZ::BAC，基于大肠杆菌 F 因子复制子，氯霉素抗性标记基因，其末端的嵌合序列可被 Tn5 转座酶特异性识别。体外转座作用针对杆状病毒穿梭载体 pMON14272（136kb）和美洲棉铃虫多角体病毒（AcMNPV）基因组（134kb）作为靶 DNA 进行。转座子 EZ::BAC 随机插入靶 DNA 中，导致插入部位侧翼末端的 9bp 重复。获得的 AcMNPV::BAC 之一在转染后在 Sf21 细胞中复制。使用 EZ::BAC 在体外随机生成病毒 bacmid，促进了完整和功能性病毒基因组在大肠杆菌细胞中的独立复制，并且不需要目标 DNA 的序列信息，而这是常规系统中生成 bacmid 所必需的。