Abstract：Abstract Zera tag allowed its recombinant fusion antigens to form fully active protein microparticles in planta and markedly enhanced the immune response. Thus Zera tag-induced microparticles may be regarded as promising production and delivery vehicles for subunit vaccines. To test this hypothesis, Zera-GFP sequences were synthesized in vitro, and pJ Zera-GFP plant expression vector was constructed by subclone technology. The vectors were co-inoculated on Nicotiana benthamiana plants with pCBNoX HC-Pro vector, a plant binary vector expressing the RNA silencing suppressor of HC-Pro protein, through agroinfiltration. The epidermal cells expressing Zera-GFP in inoculated Nicotiana leaves were then surveyed by confocal laser scanning microscopy, and the relative expression level of Zera-GFP were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot respectively. All data were collected for the assessment of the potential of Zera tag-induced microparticles as delivery vehicles for subunit vaccines. And a simple purification technique for Zera tag-induced microparticles was established. The effect of Agrobacterium tumefaciens strains on the expression level of pJ Zera-GFP vector was tested firstly and the better results were obtained when using the EHA105 strains instead of GV3101strains. It was also found that Zera-GFP was expressed in the form of protein particles and they were not found to be toxic to the N. benthamiana tissues. The morphological characteristics of Zera-GFP particles in tobacco leaf cells were further observed. The particles were composed of small round spheres with different size and clear edge. The diameter of the sphere particles was between 0.5~2.5 μm, the spheres were close to each other and were closely clustered together. Based on the analysis of the factors such as particle size, morphology, uniformity and antigen content of Zera tag-induced particles, it is suggested that the particles had the potential to be a delivery vehicles of subunit vaccine. Then Zera-GFP particles were purified by sucrose density gradient and sucrose cushion centrifugation. The results showed that Zera-GFP particles could be purified from the inoculated tobacco leaves by both centrifugal technology and 60% sucrose cushion was more suitable for the purification of Zera-GFP particles. Otherwise, we speculated that the formation of Zera tag-induced particles in the plant cells was a dynamic process from soluble to aggregation. The results of this study provide a theoretical and practical basis for the study of the novel particulate vaccine delivery systems based on Zera tag.
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