Improved expression of porcine epidemic diarrhea antigen by fusion with cholera toxin B subunit and chloroplast transformation in Nicotiana tabacum
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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 Nicotiana tabacum cv. Maryland could be assembled to pentameric form which increases the possibility to develop a mucosal vaccine against PEDV.
This study describes a protocol for improved plant expression of a porcine epidemic diarrhea immunogen by utilisation of a CTB fusion protein and chloroplast transformation. The results show that the production of CTB fusion antigen was twenty times higher than antigen alone in transplastomic plant.
KeywordsChloroplast expression CTB fusion protein Porcine epidemic diarrhea virus S1D
This research was supported by the Ministry of Agriculture, Food and Rural Affairs (714001-07), and Nguyen-Quang-Duc Tien was supported by the BK21 plus program, Republic of Korea.
M-YK and N-Q-DT conceived, designed and performed the overall study. N-XH performed Southern blot analysis. N-Q-DT and M-YK wrote and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
All authors declare no conflict of interest.
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