High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system
High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system
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DOI：
10.1111/j.1467-7652.2007.00316.x
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发表时间：
2008-02-01
影响因子：
13.8
通讯作者：
Mason, Hugh S.
中科院分区：
文献类型：
--
作者：
Huang, Zhong;LePore, Kate;Mason, Hugh S.
重组乙型肝炎表面抗原（HBsAg）是目前常用的乙型肝炎疫苗，并已成功地作为外源表位的载体。为了开发一种廉价、易于施用的全球免疫疫苗来源，一些研究小组已经在植物系统中表达了HBsAg。转基因植物源性HBsAg可组装成病毒样颗粒（vlp），在小鼠和人体内均具有免疫原性。然而，HBsAg在转基因植物系统中的表达相对较低。产生转基因植物的耗时和劳动密集型过程也极大地限制了各种HBsAg融合抗原的高通量分析。本文介绍了利用一种新型病毒瞬时表达系统在植物叶片中高效快速生产HBsAg的方法。通过多克隆酶联免疫吸附法测定，经MagnICON病毒载体侵染的本烟叶片产生高水平的HBsAg，感染后10天平均叶片鲜重295 μ g/g。瞬时表达的HBsAg作为全长产物积累，形成二硫化物连接二聚体，显示构象‘a’抗原行列式并组装成VLPs。用部分纯化的HBsAg免疫小鼠可引起HBsAg特异性抗体。此外，研究还发现，利用全株真空浸润而不是叶片注射器浸润的方法瞬时生产HBsAg，易于扩展，并且大大提高了正确折叠的HBsAg的积累，这些HBsAg显示了保护性的“a”决定因素。
Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 mu g/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.