Production of high-titer Epstein-Barr virus recombinants derived from akata cells by using a bacterial artificial chromosome system
Production of high-titer Epstein-Barr virus recombinants derived from akata cells by using a bacterial artificial chromosome system
复制标题
DOI：
10.1128/jvi.78.13.7004-7015.2004
复制
发表时间：
2004-07-01
影响因子：
5.4
通讯作者：
Takada, K
中科院分区：
文献类型：
--
作者：
Kanda, T;Yajima, M;Takada, K
将伯基特淋巴瘤衍生细胞系Akata中的EB病毒（EBV）基因组克隆到细菌人工染色体（BAC）载体中。BAC克隆，命名为AK-BAC，通过在大肠杆菌中的高效同源重组的方式被快速和精确地修饰。该系统用于生产具有转基因的重组EBV。将绿色荧光蛋白（GFP）的表达盒插入到AK-BAC中，并将所得BAC克隆AK-BAC-GFP转染到Akata细胞中。我们发现转染的BAC质粒在EBV阳性的Akata细胞中有效地形成附加体。然后产生野生型和AK-BAC-GFP病毒的混合物并用于感染EBV阴性Akata细胞。我们获得了仅含有AK-BAC-GFP但没有野生型附加体的细胞克隆。这些细胞克隆在刺激病毒产生后产生感染性病毒，并且AK-BAC-GFP重组病毒有效地使原代B淋巴细胞永生化。我们进一步修改了该方法，以便任何类型的cDNA都可以快速插入到人工引入AK-BAC的独特I-PpoI位点。AK-BAC系统将具有广泛的应用，例如各种病毒基因产物的遗传分析和用于人类基因治疗的病毒载体的开发。
An Epstein-Barr virus (EBV) genome in Burkitt's lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in Escherichia coli. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.