A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates
A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates
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DOI：
10.2144/000113119
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发表时间：
2009-05-01
期刊：
影响因子：
2.7
通讯作者：
Arts, Eric J.
中科院分区：
文献类型：
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作者：
Dudley, Dawn M.;Gao, Yong;Arts, Eric J.
对人类免疫缺陷病毒1（HIV-1）的复制研究依赖于与流行病中的主要HIV-1亚型不同的几种实验室毒株。不同HIV-1分离株和亚型之间的几个表型差异可能会影响疫苗的开发和治疗，但该研究领域缺乏强大的克隆/病毒生产系统来研究药物敏感性，复制动力学或开发个性化疫苗。极端的HIV-1异质性使得细菌克隆策略几乎没有限制性酶切位点。在这项研究中，我们描述了一种替代方法，涉及直接引入任何HIV-1编码区（例如，来自患者样品的任何基因）插入HIV-1DNA载体中。该技术使用酵母中的阳性和阴性选择标记，并且避免了对DNA底物和克隆产物的纯化和筛选的需要。然后从用该酵母衍生的HIV-1载体转染的修饰的哺乳动物293 T包装细胞系产生有复制能力的病毒。虽然HIV-1是原型，但这种克隆策略现在正在开发用于其他不同的病毒物种，如丙型肝炎病毒和流感病毒。
Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinetics, or to develop personalized vaccines. Extreme HIV-1 heterogeneity leaves few restriction enzyme sites for bacterial cloning strategies. In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus species such as hepatitis C virus and influenza virus.