Tien Nguyen-Quang-Duc, Huy Nguyen-Xuan, Kim Mi-Young
1Bioactive Material Science, Chonbuk National University, Jeonju, South Korea.
3College of Sciences, Hue University, Hue City, Vietnam.
Plant Cell Tissue Organ Cult. 2019;137(2):213-223. doi: 10.1007/s11240-019-01562-1. Epub 2019 Feb 2.
The porcine epidemic diarrhea virus (PEDV) belongs to the coronavirus family, which causes acute diarrhea in pigs with higher mortality in piglets less than 2 weeks old. The PEDV is one of the major concerns of the pig industry around the world, including Asian countries and Noth America since first identified in Europe. Currently, there is no PEDV licensed vaccine to effectively prevent this disease. This study was performed for the development of a mucosal PEDV vaccine and B subunit of cholera toxin (CTB) as a carrier was employed to surpass the tolerogenic nature of GALT and induce potent immune responses against the target antigen fused to CTB. An epitope (S1D) alone or conjugated with CTB was constructed into the tobacco chloroplasts expression vector which is controlled under the chloroplast rRNA operon promoter with T7g10 5' UTR and the psbA 3'UTR as a terminator. The homoplastomic lines were obtained by third round screening via organogenesis from the leaf tissues which were verified by PCR with antigen and chloroplast specific primers and then confirmed by Southern blot analysis. While the expression level of the S1D alone as detected by Western blotting was approximately 0.07% of total soluble protein, the CTB-S1D fusion protein was expressed up to 1.4%. The fusion protein showed binding to the intestinal membrane GM1-ganglioside receptor, demonstrating its functionality. The result shows that the highest expression of S1D could be achieved by fusion with a stable CTB protein and chloroplast transformation. Furthermore, the CTB-S1D expressed in chloroplasts of cv. Maryland could be assembled to pentameric form which increases the possibility to develop a mucosal vaccine against PEDV.
猪流行性腹泻病毒（PEDV）属于冠状病毒科，可引起猪的急性腹泻，对2周龄以下仔猪的致死率较高。自首次在欧洲发现以来，PEDV一直是包括亚洲国家和北美在内的全球养猪业主要关注的问题之一。目前，尚无获得许可的PEDV疫苗能有效预防这种疾病。本研究旨在开发一种黏膜PEDV疫苗，并采用霍乱毒素B亚基（CTB）作为载体，以克服肠道相关淋巴组织的免疫耐受特性，并诱导针对与CTB融合的靶抗原产生有效的免疫反应。将单独的一个表位（S1D）或与CTB偶联的表位构建到烟草叶绿体表达载体中，该载体由叶绿体rRNA操纵子启动子控制，带有T7g10 5'非翻译区，并以psbA 3'非翻译区作为终止子。通过器官发生从叶片组织进行三轮筛选获得同质性系，并用抗原特异性引物和叶绿体特异性引物进行PCR验证，然后通过Southern印迹分析进行确认。通过蛋白质印迹法检测发现，单独的S1D表达水平约占总可溶性蛋白的0.07%，而CTB-S1D融合蛋白的表达量高达1.4%。该融合蛋白显示出与肠膜GM1神经节苷脂受体的结合能力，证明了其功能。结果表明，通过与稳定的CTB蛋白融合及叶绿体转化可实现S1D的最高表达。此外，在马里兰品种烟草叶绿体中表达的CTB-S1D可组装成五聚体形式，这增加了开发抗PEDV黏膜疫苗的可能性。
