Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.

Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in . Recombinant human-acidic fibroblast growth factor was recovered following infiltration of . The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. -derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of-derived recombinant human acidic fibroblast growth factor. Additionally, derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that-derived recombinant human acidic fibroblast growth factor effectively protects skin cell from UVB, suggesting its potential use as a cosmetic or therapeutic agent against skin photoaging.

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Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of protoplast extraction, which was 6.36 ± 0.51 × 106 protoplasts/g fresh weight (FW), was approximately 3.7 times higher than that previously reported for potato protoplasts. To obtain data, wounded leaves were used by partially cutting both sides of the midrib, and isolated protoplasts were purified by the sucrose cushion method, with a sucrose concentration of 20%. We confirmed a significant effect on the extraction efficiency by measuring enzymolysis during a 6 h period, with three times more washing buffer than the amount normally used. Protoplasts fixed in alginate lenses with appropriate space were successfully recovered and developed into microcalli 2 weeks after culture. In addition, to induce high efficiency regeneration from protoplasts, calli in which greening occurred for 6 weeks were induced to develop shoots in regeneration medium solidified by Gelrite, and they presented a high regeneration efficiency of 86.24 ± 11.76%.

The ability of potato-derived major surface antigen of hepatitis B virus (P-HBsAg) to elicit antibody responses to different dosages of P-HBsAg ranging from 0.02 to 30 g administered orally in mice was examined. All immunized groups produced specific serum IgG and fecal IgA antibodies against P-HBsAg, even at low levels (<5 g), after administration of a 0.5-g yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) booster.The major surface antigen of hepatitis B virus (HBV) (HBsAg) is one of the best-developed examples of a plant-derived antigen (6,9,11,13,18); however, low expression levels prevent plant-derived antigens from being economically competitive (5,15,16). Average antigen expression levels are in the range of 0.01% to 0.4% of total soluble protein (TSP). Aspects related to eliciting protective antibody responses, e.g., oral administration doses (1, 10), efficient delivery of oral vaccines (3, 4), and functional effects of adjuvants (12), were identified to stimulate immune responses after vaccination with low levels of plant-derived antigen.The objective here was to examine the effects of plant-based oral immunization on HBV-specific immune responses over a broad range of doses, from the lowest dose of 0.02 g potatoderived HBsAg (P-HBsAg) to the maximum dose of 30 g P-HBsAg. IgG humoral and IgA mucosal responses were observed at various P-HBsAg doses, and these results are discussed in relation to the optimization of plant-derived vaccines. Furthermore, the analysis of IgG subclass distribution following oral administration with varied doses of P-HBsAg was carried out to understand the mechanism of the immune response.To evaluate the immunogenicity upon oral administration of various doses of P-HBsAg from a plant line showing the highest production of HBsAg (7, 18), mice were immunized orally with tuber extract on days 1, 7, and 14. The detailed procedure was described previously (18). Mice were immunized with 150 g of yeast-derived HBsAg (Y-HBsAg; LG Life Sciences, Republic of Korea) mixed with 10 g of Cholera toxin (CT; Sigma) as a positive control. Each concentration of P-HBsAg administered to mice was divided into one of the the following three groups: lower level, consisting of 0.02, 0.1, and 0.5 g; middle level, consisting of 1.0, 2.5, and 5.0 g; and higher level, consisting of 10 g, 15 g, and 30 g. Only five concentrations (0.1, 1.0, 5.0, 10, and 30 g) were shown among the nine concentrations depicted in the figures to help with understanding by using a concise arrangement. Antigen-specific IgG responses to representative doses in mouse sera against P-HBsAg were graphically monitored up to week 12 (Fig. 1a). The groups administered 10 g and 30 g of antigen stimulated slight primary responses of 41 mIU and 51 mIU, respectively, at 7 weeks before booster administration compared to responses of the mice immunized with potato extract from the nontransformed (NT) plant (NT group) (4 mIU). Mice administered higher levels of P-HBsAg (10, 15, and 30 g) exhibited significantly increased immu...