Huy Nguyen-Xuan, Tien Nguyen-Quang-Duc, Kim Mi-Young, Kim Tae-Geum, Jang Yong-Suk, Yang Moon-Sik
1Department of Molecular Biology, Chonbuk National University, Jeonju, Republic of Korea.
5Biology Department, Hue University of Education, 34 Le Loi, Hue, Vietnam.
Plant Cell Tissue Organ Cult. 2016;127(2):369-380. doi: 10.1007/s11240-016-1059-5. Epub 2016 Aug 23.
Porcine epidemic diarrhea virus (PEDV) belongs to the family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into and subsequently infiltrated into leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB-S1D fusion protein were confirmed by Western blot and G-ELISA. The highest expression level of CTB-S1D fusion protein was 0.07 % of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB-S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB-S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB-S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.
猪流行性腹泻病毒（PEDV）属于该家族，可引起猪的急性肠炎。大刺突糖蛋白的一个片段，称为S1D表位（氨基酸636 - 789），单独以及与霍乱毒素B亚基融合后，分别独立克隆到植物表达载体中，得到质粒pMYV717和pMYV719。将植物表达载体转化到[未提及具体受体]中，随后浸润到[未提及具体植物]叶片中。发现S1D在浸润后2天（dpi）表达水平最高，达到总可溶性蛋白的0.04%，此后迅速下降。通过蛋白质免疫印迹法（Western blot）和酶联免疫吸附测定（G - ELISA）证实了CTB - S1D融合蛋白的表达和组装。CTB - S1D融合蛋白在4 dpi时最高表达水平为TSP的0.07%，此后迅速下降。在存在番茄丛生矮化病毒的p19蛋白的情况下，S1D和CTB - S1D蛋白水平在6 dpi时达到峰值，分别比不存在p19时高4至7倍。口服瞬时表达的CTB - S1D融合蛋白，或与细菌霍乱毒素或表达突变霍乱毒素61F的水稻愈伤组织一起口服后，小鼠针对细菌CTB和S1D抗原的血清IgG和sIgA水平显著更高，在第6周达到峰值。将瞬时表达的CTB - S1D融合蛋白口服给猪，以评估对PEDV的免疫反应。
