Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy
Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy
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DOI：
10.1016/s0166-0934(98)00130-x
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发表时间：
1998-12-01
影响因子：
3.1
通讯作者：
Gould, EA
中科院分区：
文献类型：
--
作者：
Gritsun, TS;Gould, EA
在不到一个月的时间里，我们利用长高保真PCR和改良的细菌克隆系统，从100 μ l未纯化的病毒悬液中构建了减毒蜱传脑炎病毒（Vasilchenko株）的感染性克隆。3'反义引物浓度的优化对于实现来自感染性病毒的RNA的11kb cDNA拷贝的PCR合成是必要的。一种新的系统，利用两个反义引物，14聚体的逆转录和35聚体的长PCR，产生高产量的基因组长度的cDNA。使用低拷贝数的Able（TM）K细胞和28 ℃的孵育温度增加了克隆cDNA的遗传稳定性。含有11 kb cDNA插入片段的克隆产生的菌落大小减少？从而为全长克隆提供阳性选择系统。感染性克隆的测序强调了与常规PCR和克隆方法相比该方法的保真度提高。一个简单而快速的策略进行遗传操作的感染性克隆。这些发展代表了重组技术的重大进步，应该适用于不能容易地纯化或遗传操作的正链RNA病毒。(C)1998 Elsevier Science B.V.保留所有权利。
In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 mu l of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able(TM) K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size? thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated. (C) 1998 Elsevier Science B.V. All rights reserved.