Mohammadzadeh Sara, Roohvand Farzin, Memarnejadian Arash, Jafari Anis, Ajdary Soheila, Salmanian Ali-Hatef, Ehsani Parastoo
a Department of Molecular Biology .
b Department of Virology .
Pharm Biol. 2016;54(3):465-73. doi: 10.3109/13880209.2015.1048371. Epub 2015 May 20.
Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition.
We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine.
A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector.
Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants.
The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.
基于病毒载体转化的植物已成为在瞬时条件下快速表达大量且廉价抗原的有前景的工具。
我们研究了在烟草（Nicotiana tabacum）叶片中瞬时表达一种与HBsAg融合的多聚体构建体（HCVpc）[包含C区（核心区；氨基酸132 - 142）、E6区（包膜蛋白2；氨基酸614 - 622）、N区（NS3；氨基酸1406 - 1415）和E4区（包膜蛋白2；氨基酸405 - 414）的H - 2d和HLA - A2限制性CD8 + CTL表位肽，呈CE6NE4串联形式]用于开发基于植物的丙型肝炎病毒（HCV）疫苗的可能性。
设计了一个编码Kozak序列、六组氨酸（6×His）标签肽并与HCVpc串联的密码子优化基因，进行化学合成，与HBsAg基因融合，并在重复的马铃薯X病毒（PVX）外壳蛋白启动子（CPP）控制下插入马铃薯X病毒（PVX - GW）载体。将得到的重组质粒（经酶切和测序分析确认后）转入根癌农杆菌菌株GV3101，并通过真空渗透法导入烟草叶片。还通过共渗透p19表达载体评估了基因沉默抑制因子番茄丛生矮化病毒的p19蛋白对HCVpc - HBsAg表达产量的影响。
密码子优化基因提高了烟草中的适配指数（CAI）值（从0.61提高到0.92）。通过蛋白质免疫印迹法和基于HBsAg的检测ELISA在烟草叶片的总可提取蛋白上证实了HCVpc - HBsAg的表达。在p19共农杆菌渗透的植物中，融合蛋白的表达水平显著更高。
结果表明在烟草中表达具有适当蛋白质构象的HCVpc - HBsAg构建体用于最终作为植物源HCV疫苗应用的可能性。
