Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae
Generation of recombinant virus-like particles of human and non-human polyomaviruses in yeast Saccharomyces cerevisiae
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DOI：
10.1159/000067922
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发表时间：
2002-07-01
期刊：
影响因子：
4.6
通讯作者：
Ulrich, R
中科院分区：
文献类型：
--
作者：
Sasnauskas, K;Bulavaite, A;Ulrich, R
目的：非病毒基因转移方法在基因治疗方法中受到青睐，原因有几个，特别是其安全性、简单性和将异源 DNA 引入细胞的便利性。多瘤病毒类病毒颗粒（VLP）是一种有前景的外源核酸衣壳化载体，用于基因治疗。为了开发此类基因传递系统以及提供改进病毒诊断的试剂，建立了用于生成不同多瘤病毒 VLP 的有效酵母表达系统。方法：使用半乳糖诱导型酿酒酵母表达系统。通过氯化铯超速离心、琼脂糖凝胶电泳和电子显微镜证实空VLP的形成。使用针对 VP1 蛋白的兔和小鼠血清，通过蛋白质印迹分析不同多瘤病毒的主要衣壳蛋白 (VP1) 的交叉反应性。结果：人类多瘤病毒（JC多瘤病毒和BK多瘤病毒血清型AS和SB）、恒河猴（猿猴病毒40）、仓鼠（仓鼠多瘤病毒）、小鼠（鼠多瘤病毒）和鸟类（虎皮鹦鹉雏鸟病病毒）的VP1在酵母中高水平表达。由所有酵母表达的 VP1 蛋白形成的空 VLP 被解离成五聚体，并在规定的离子和 pH 条件下重新结合成 VLP。观察到 VP1 蛋白与异源小鼠和兔血清的不同交叉反应模式。结论：所开发的异源酵母表达系统适合多瘤病毒VLP的高水平生产。酵母来源的 VLP 通常不含毒素、宿主细胞 DNA 和蛋白质。这些 VLP 可能有助于生成新的诊断工具、基因传递系统和抗病毒疫苗。版权所有 (C) 2003 S. Karger AG，巴塞尔。
Objectives: Non-viral methods of gene transfer have been preferred in gene therapy approaches for several reasons, particularly for their safety, simplicity and convenience in introducing heterologous DNA into cells. Polyomavirus virus-like particles (VLPs) represent a promising carrier for encapsidation of foreign nucleic acids for gene therapy. For the development of such gene delivery systems as well as for providing reagents for improving virus diagnostics, an efficient yeast expression system for the generation of different polyomavirus VLPs was established. Methods: A galactose-inducible Saccharomyces cerevisiae yeast expression system was used. Formation of empty VLPs was confirmed by cesium chloride ultracentrifugation, agarose gel electrophoresis and electron microscopy. Cross-reactivity of the major capsid proteins (VP1) of different polyomaviruses was analyzed by Western blot using rabbit and mice sera raised against the VP1 proteins. Results: VP1 of polyomaviruses from humans (JC polyomavirus and serotypes AS and SB of BK polyomavirus), rhesus monkeys (simian virus 40), hamsters (hamster polyomavirus), mice (murine polyomavirus) and birds (budgerigar fledgling disease virus) were expressed at high levels in yeast. Empty VLPs formed by all yeast expressed VP1 proteins were dissociated into pentamers and reassociated into VLPs by defined ion and pH conditions. Different patterns of cross-reactivity of the VP1 proteins with heterologous mice and rabbit sera were observed. Conclusion: The developed heterologous yeast expression system is suitable for high-level production of polyomavirus VLPs. Yeast-derived VLPs are generally free of toxins, host cell DNA and proteins. These VLPs might be useful for the generation of new diagnostical tools, gene delivery systems and antiviral vaccines. Copyright (C) 2003 S. Karger AG, Basel.