He Junyun, Peng Li, Lai Huafang, Hurtado Jonathan, Stahnke Jake, Chen Qiang
The Biodesign Institute, Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287, USA.
The Biodesign Institute, Arizona State University, 1001 S. McAllister Avenue, Tempe, AZ 85287, USA ; School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.
Biomed Res Int. 2014;2014:952865. doi: 10.1155/2014/952865. Epub 2014 Apr 3.
We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μ g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μ g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.
我们描述了在植物中快速生产包膜（E）蛋白的结构域III（DIII）作为西尼罗河病毒（WNV）的候选疫苗。利用解构病毒载体表达系统的各种载体模块组合，通过瞬时表达在本氏烟草叶片的三个亚细胞区室中生产了DIII。当靶向内质网（ER）时，DIII的表达水平远高于靶向叶绿体或细胞质时，在浸润后4天内积累水平高达每克叶片鲜重73μg DIII。植物内质网来源的DIII是可溶的，易于纯化至>95%的纯度，无需耗时的变性和复性过程。进一步分析表明，植物产生的DIII加工正确，并证明与识别构象表位的抗DIII单克隆抗体具有特异性结合。此外，用5μg和25μg纯化的DIII对小鼠进行皮下免疫引发了强烈的全身反应。本研究为在植物中低成本、可扩展地快速生产针对WNV的免疫原性候选疫苗提供了原理证明。
