Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems
Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems
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DOI：
10.1007/s11103-016-0484-5
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发表时间：
2016-07-01
影响因子：
5.1
通讯作者：
Clarke, Jihong Liu
中科院分区：
文献类型：
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作者：
Gottschamel, Johanna;Loessl, Andreas;Clarke, Jihong Liu
据世界卫生组织称，登革热是热带和亚热带许多地区的一种疾病，全球约一半人口面临感染风险。登革热由四种相关的登革热病毒血清型DEN - 1、 - 2、 - 3和 - 4中的任何一种引起，这些病毒通过埃及伊蚊传播给人类。经过半个世纪的不懈努力，自2015年以来，市场上仅有（且仅限于少数几个国家）一种疫苗（登革热疫苗（Dengvaxia®））可供使用。因此，仍然迫切需要价格合理且易于获取的登革热疫苗。登革热包膜蛋白结构域III（EDIII）能够引发血清型特异性中和抗体，已成为亚单位疫苗研发的重点。为了促进一种易于获取且价格合理的登革热疫苗的研发，在当前的研究中，我们利用植物疫苗生产系统在烟草中生成了一种登革热亚单位疫苗候选物。我们应用叶绿体基因组工程技术，通过组成型和乙醇诱导型表达系统在烟草叶绿体中表达血清型特异性重组EDIII蛋白。我们还尝试表达一种包含所有四种血清型的EDIII多肽的四价抗原融合构建体。我们获得了表达EDIII的转基因烟草植株，并通过Southern杂交分析验证了其同质性。Northern杂交分析表明编码EDIII抗原的基因得到了表达。不同重组EDIII蛋白和不同表达系统的EDIII蛋白积累水平有所差异，达到细胞总蛋白的0.8% - 1.6%。我们的研究证明叶绿体区室适合作为生产基于EDIII的登革热疫苗候选物的场所，并展示了一种用于诱导转基因表达的Gateway®质体转化载体。
Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(A (R))) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(A (R)) plastid transformation vector for inducible transgene expression.