• Journal of Plant Biotechnology
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• 제7권2호
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• pp.97-103
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• 2005

This study was carried out to obtain basic information for gene manipulation in potent edible tobacco (Nicotiana tabacum cv. TI 516). N. tabacum cv. TI 516 is a plant for a possible candidate to use as an edible vaccine, since it contains a low level of nicotine. The effective plant regeneration system through leaf disc culture was achieved using a MS basal medium supplemented with 0.1 mg $1^{-1}$ NAA and 0.5 mg $1^{-1}$ BA. In order to transform the N. tabacum cv. TI 516 with the green fluorescent protein (GFP) gene, Agrobacterium tumefaciens LBA 4404 containing the GFP gene was used. Genomic PCR confirmed the integration of the GFP gene into nuclear genome of transgenic plants. Expression of the GFP gene was identified in callus, apical meristem and root tissue of transgenic N. tabacum cv. TI 516 plants using fluorescence microscopy. Western blot analysis revealed the expression of GFP protein in the transgenic edible tobacco plants. The amount of GFP protein detected in the transgenic tobacco plants was approximately 0.16% of the total soluble plant protein (TSP), which was determined by ELISA.

• Parasites, Hosts and Diseases
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• 제51권2호
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• pp.155-163
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• 2013

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) ($50{\mu}g/mouse$). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and antipathology vaccine candidate against S. mansoni infection.

• 한국식품영양학회지
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• 제26권3호
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• pp.375-382
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• 2013

The present study is designed to explore an anti-tumor activity on crude extracts of Oplopanax elatus. Water extractions of Oplopanax elatus were performed at $100^{\circ}C$(OeE-100). OeE-100 doses up to $62.5{\mu}g/m{\ell}$ had no cytotoxicity on the tumor cell lines in vitro. In experimental lung metastasis of colon26-M3.1 carcinoma or B16-BL6 melanoma, the prophylactic intravenous ($4{\sim}100{\mu}g/mouse$) or oral (2 mg/mouse) administration of OeE-100 significantly inhibited tumor metastasis as compared with tumor controls. Peritoneal macrophages stimulated with OeE-100 produced various cytokines such as TNF-${\alpha}$, IL-6 and IL-12. In an analysis of NK-cell activities, i.v. administration of OeE-100 ($10{\sim}100{\mu}g/mouse$) significantly augmented the cytotoxicity to YAC-1 tumor cells. Vaccination of mice with boiling-treated tumor cells (BT-vaccine) in combination with OeE-100 ($100{\mu}g/mouse$) showed higher inhibitions in tumor metastasis when compared with the mice of BT-vaccine treatment. In addition, the splenocytes from OeE-100 admixed BT-vaccine immunized mice secreted a higher concentration of Th1 type cytokine such as IFN-${\gamma}$. These results suggested that the OeE-100 stimulated immune system and was a good candidate adjuvant of anti-tumor immune responses.

• 대한수의학회지
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• 제46권2호
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• pp.127-133
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• 2006

Pasteurella multocida is a terrible veterinary pathogen that causes widespread infections in husbandry. To induce homologous and/or heterologous immunity against the infections, outer membrane protein Hs (OmpH) in the envelope of different strains of P. multocida are thought to be attractive vaccine candidates. Previously we cloned and characterized a gene for OmpH from pathogenic P. multocida (A : 3) (In Press, Korean J. Microbiol. Biotechnol. 2005, 33, December). The gene is composed of 1,047 nucleotides (nt) coding 348 amino acids (aa) with signal peptide of 20 aa. The truncated ompH, a gene without nt coding for the signal peptide, was generated using pRSET A to name "pRSET A/OmpH-F2". This truncated ompH was well expressed in Escherichia coli BL21 (DE3). Truncated OmpH was purified for induction of immunity against live pathogen of fowl cholera (P. multocida A : 3) in mice. Some $50{\mu}g$ of the purified polypeptide was intraperitoneally injected into mice two times with 10 day interval. Lethal dose ($25{\mu}l$) of live P. multocida A : 3 was determined by directly injecting the pathogen into wild mice (n = 25). To demonstrate the vaccine candidate of the truncated OmpH, the live pathogen ($25{\mu}l$) was challenged with the OmpH-immunized mouse group as well as positive & negative controls (n = 80). The results show that the truncated OmpH can be used for an effective vaccine production to prevent fowl cholera caused by pathogenic P. multocida (A : 3).

• Journal of Veterinary Science
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• 제24권1호
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• pp.15.1-15.13
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• 2023

Background: Inactivated vaccines are limited in preventing foot-and-mouth disease (FMD) due to safety problems. Recombinant virus-like particles (VLPs) are an excellent candidate for a novel vaccine for preventing FMD, given that VLPs have similar immunogenicity as natural viruses and are replication- and infection-incompetent. Objectives: The 3C protease and P1 polyprotein of type O FMD virus (FDMV) was expressed in yeast Hansenula polymorpha to generate self-resembling VLPs, and the potential of recombinant VLPs as an FMD vaccine was evaluated. Methods: BALB/c mice were immunized with recombinant purified VLPs using CpG oligodeoxynucleotide and aluminum hydroxide gel as an adjuvant. Cytokines and lymphocytes from serum and spleen were analyzed by enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and flow cytometry. Results: The VLPs of FMD were purified successfully from yeast protein with a diameter of approximately 25 nm. The immunization of mice showed that animals produced high levels of FMDV antibodies and a higher level of antibodies for a longer time. In addition, higher levels of interferon-γ and CD4⁺ T cells were observed in mice immunized with VLPs. Conclusions: The expression of VLPs of FMD in H. polymorpha provides a novel strategy for the generation of the FMDV vaccine.

• Clinical and Experimental Vaccine Research
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• 제12권4호
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• pp.304-312
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• 2023

Purpose: Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Materials and Methods: Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe₃O₄ NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. Results: The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. Conclusion: The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

• 대한바이러스학회지
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• 제27권1호
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• pp.39-47
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• 1997

A large number of viruses belonging to Genus Hantavirus in Family Bunyaviridae are etiologic agents for hemorrhagic fever with renal syndrome (HFRS), or hantavirus pulmonary syndrome (HPS). Hantaan (HTN), Seoul (SED), Belgrade (BEL), Puumala (PUU) serotype viruses are well known causative agents for HFRS in Eurasian continent. Among those viruses Hantaan and Seoul serotypes are well known to cause HFRS in Korea, but there are some sporadic incidence by other than Hantaan or Seoul viruses. Recently we have developed the combined Hantaan-Puumala virus vaccine to prevent world-wide occuring HFRS. This combined vaccine is formalin inactivated, suckling mouse and suckling hamster brain extracts for Hantaan and Puumala viruses, respectively. Protein contents of this purified candidate vaccine is $27\;{\mu}g/ml$, which contains 1,024 ELISA antigen units to each virus, but content of myelin basic protein which is causing experimental allergic encephalomyelitis is less than 0.1 ng/ml. Thirty hamsters were given twice at one month interval intra-muscularly and bled on 30 days after each vaccination from retro-orbital sinus vein. Antibody titers were tested against 5 major serotype viruses, Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses by IFA and PRNT. The mean IF antibody titers on 30 days after primary shot were 78.4, 68.8, 68.8, 37.9, and 15.6; mean neutralizing antibody titers were 65.4, 12, 6.1, 65.6 and 0.5 against Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses, respectively. The mean IF antibody titers on 30 days after booster shot were 686.9, 567.5, 550.4, 516.3, and 430.9; and neutralizing antibody titers were 710.8, 41.9, 24.3, 409.9, and 1.6 against Hantaan, Seoul, Belgrade, Puumala and Sin Nombre viruses, respectively.

• Clinical and Experimental Vaccine Research
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• 제13권3호
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• pp.232-241
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• 2024

Purpose: Brucellosis, a zoonotic infectious disease, is a worldwide health issue affecting animals and humans. No effective human vaccine and the complications caused by the use of animal vaccines are among the factors that have prevented the eradication of the disease worldwide. However, bio-engineering technologies have paved the way for designing new targeted and highly efficacious vaccines. In this regard, the study aimed to evaluate immunity induced by mannosylated niosome containing Brucella recombinant trigger factor/Bp26/Omp31 (rTBO) chimeric protein in a mouse model. Materials and Methods: rTBO as chimeric antigen (Ag) was expressed in Escherichia coli BL21 (DE3) and, after purification, loaded on niosome and mannosylated niosome. The characteristics of the nanoparticles were assessed. The mice were immunized using rTBO, niosome, and mannosylated niosome-rTBO in intranasal and intraperitoneal routes. Serum antibodies (immunoglobulin [Ig]A, IgG, IgG1, and IgG2a) and splenocyte cytokines (interferon-gamma, interleukin [IL]-4, and IL-12) were evaluated in immunized mice. Finally, immunized mice were challenged by B. melitensis and B. abortus. A high antibody level was produced by niosomal antigen (Nio-Ag) and mannosylated noisomal antigen (Nio-Man-Ag) compared to the control after 10, 24, and 38 days of immunization. The IgG2a/IgG1 titer ratio for Nio-Man-Ag was 1.2 and 1.1 in intraperitoneal and intranasal methods and lower than one in free Ag and Nio-Ag. Cytokine production was significantly higher in the immunized animal with Ag-loaded nanoparticles than in the negative control group (p<0.05). Moreover, cytokine and antibody levels were significantly higher in the injection than in the inhalation method (p<0.05). Results: The combination of mannosylated noisome and rTBO chimeric proteins stimulate the cellular and humoral immune response and produce cytokines, playing a role in developing the protective acquired immune response in the Brucella infectious model. Also, the intraperitoneal route resulted in a successful enhancement of cytokines production more than intranasal administration. Conclusion: Designing an effective vaccine candidate against Brucella that selectively induces cellular and humoral immune response can be done by selecting a suitable nanoniosome formulation as an immunoadjuvant and recombinant protein as an immune response-stimulating Ag.

• Toxicological Research
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• 제34권3호
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• pp.191-197
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• 2018

In 2015, a candidate for the second national reference standard (NRS) of Gloydius snake venom was produced to replace the first NRS of Gloydius snake venom. In the present study, the potencies of the candidate were determined by a collaborative study, and the qualification of the candidate was estimated. The potencies of the candidate were determined by measuring the murine lethal titers and lapine hemorrhagic titers of venom against the regional working reference standard (RWRS) for antivenom using the methods described in the previous report for the first NRS of Gloydius snake venom. Three Korean facilities contributed data from a total of 30 independent assays. Subsequently, two foreign national control research laboratories contributed to this collaborative study. The results were calculated using the Reed-Muench method for lethality and determined using a mixed-effects model for hemorrhage. The general common potencies of the lethal and hemorrhagic titers were obtained from the results of the 30 tests performed at three Korean facilities. The results are expressed in micrograms for 1 test dose (TD) with a 95% confidence interval as follows: a lethal titer of $90.13{\mu}g/TD$ (95% confidence interval = $87.39{\sim}92.86{\mu}g$) and a hemorrhagic titer of $10.80{\mu}g/TD$ (95% confidence interval = $10.46{\sim}11.14{\mu}g$). In addition, the candidate preparation showed good quality evaluation according to the results of the quality estimation of the candidate and is judged to be suitable to serve as the Korean NRS for snake venom. In conclusion, the second NRS of Gloydius snake venom was established in this study and will be used for national quality control, including a national lot release test of Korean antivenom products.

• 약학회지
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• 제48권1호
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• pp.47-54
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• 2004

ClpP is a stress-inducible protein and proteolytic subunit of the ATP-dependent Clp protease in prokaryotes and eukaryotes. Although its physiological roles in bacterial virulence were widely studied in various organsims, including Streptococcus pneumoniae, until now the immunological effect has not been investigated. Here, we have examined the cross reactivity of S. pneumoniae ClpP antibody with other organisms's cell lysate proteins. Although the protein sequence of S. pneumoniae ClpP was highly conserved among various organisms including human, the antibody rasised by S. pneumoniae ClpP was not cross-reacted with other organism's cell lysates, which were Saccharomyces cerevisiae , human lung A549 cell, Bacillus subtilis, Pseuomonas aeruginosa, E. coli, and Salmonella typhi. It was only reacted with S. pneumoniae and Lato-bacillus thermophilus. Thus we examined the immunoprotective effect of ClpP by immunizing mice with the purified ClpP. The mean survival time of mouse was significantly increased with the ClpP immunization. These results suggest that S. pneumoniae ClpP could be used as a vaccine candidate for prevention of S. pneumoniae infection.