• Toxicological Research
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• v.34 no.3
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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.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.719-727
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• 2011

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) antigenically diverse neurotoxins (BoNTs). BoNTs are the most poisonous substances known to humans, with a median lethal dose ($LD_{50}$) of approximately 1 ng/kg of body weight. Owing to their extreme potency and lethality, they have the potential to be used as a bioterrorism agent. The mouse bioassay is the gold standard for the detection of botulinum neurotoxins; however, it requires at least 3-4 days for completion. Attempts have been made to develop an ELISA-based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. The present study was designed using a synthetic gene approach. The synthetic gene encoding the catalytic domain of BoNT serotype B from amino acids 1-450 was constructed with PCR overlapping primers (BoNT/B LC), cloned in a pQE30 UA vector, and expressed in an E. coli M15 host system. Recombinant protein production was optimized at 0.5 mM IPTG final concentration, 4 h post induction, resulting in a maximum yield of recombinant proteins. The immunogenic nature of the recombinant BoNT/B LC protein was evaluated by ELISA. Antibodies were raised in BALB/c mice using various adjuvants. A significant rise in antibody titer (p<0.05) was observed in the Alum group, followed by the Titermax Classic group, Freund's adjuvant, and the Titermax Gold group. These developed high-titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.5
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• pp.1009-1015
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• 2002

The purpose of this research was to investigate the effect of Yonggak-san (YGS) on the anti-allergic reaction in vivo and in vitro. Administration of YGS(500 mg/kg) enhanced hemaggutination(HA)titer against SRBC. On the while, YGS inhibited hyaluronidase activity in vitro and passive cutaneous anaphylaxis reaction, lethal anaphylaxis and mortality induced by compound 48/80 in mice, YGS decreased Arthus reaction, acute hind paw edema induced by histamine. But YGS did not affect delayed type hypersensitivity induced by SRBC. These results suggest that YGS have anti-allergic action

• Journal of Sasang Constitutional Medicine
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• v.10 no.2
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• pp.589-613
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• 1998

The purpose of this research was to investigate effects of Bangpoongtongsungsan water extract(BTSE) on the immune reaction, anti-allergy action and anti-inflammatory action in BALB/c mice. The administration of BTSE (500mg/kg) enhanced the cell viability of thymocytes and the population of helper T cells in splenic T-lymphocytes. BTSE suppressed the production of nitric oxide, but enhanced the phagocytic activity in peritoneal macrophages. BTSE enhanced hemagglutination titer in mice. BTSE inhibited passive cutaneous anaphylaxis induced by egg albumin in rat, the lethal anaphylaxis induced by platelet activating factor and compound 48/80 in mice, and then inhibited the degranulation of peritoneal mast cells induced by compound 48/80. BTSE did not inhibit Arthus reaction, but inhibited the delayed type hypersensitivity induced by SRBC and contact dermatitis induced by DNFB. BTSE inhibited the acute hind paw edema induced by histamine after 30 minutes, the permeability of evans blue into peritoneal cavity induced by acetic acid and the writhing syndrome induced by acetic acid. These results suggest that BTSE has an immunopotentiative action, anti-allergy action and anti-inflammatory action via the inhibition of histamine release.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1960-1965
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• 2015

Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.12 no.1
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• pp.113-142
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• 1999

The purpose of this research was to investigate the effect of Kamidangkwieumja(KDEJ) water extract on the allergy reaction in mice. The results were obtained as follows: 1. The passive cutaneous anaphylaxis induced by egg albumin in fat was not affected. 2. The lethal anaphylaxis induced by platelet activating factor in mice. was inhibited. 3. The degranulation of peritoneal mast cells induced by compound 48/80 was not affected. 4. The acute hind paw edema was inhibited after 2hours later when it was induced by histamine. 5. The permeability of evans blue into peritoneal cavity induced by acetic acid was not affected. 6. Arthus reaction in mice was not affected. 7. The delayed type hypersensitivity induced by SRBC was inhibited. 8. The contact dermatitis induced by DNFB was not affected. 9. The hemagglutination titer induced by SRBC was inhibited. 10. The writhing syndrome induced by acetic acid was inhibited. 11. The population of heper T cells in mice thymus was enhanced. 12. The phagocytic activity of peritoneal macrophages was enhanced. 13. The production of nitric oxide from peritoneal macrophages was not affected. These results suggest that the anti-allergy effect of KDEJ is caused by steroidlike and enhanced immune action. The steroidlike action of KDEJ correspond with steroid-applying-method that frequently used in clinic, so it is used io treatment of psoriasis. The research on anti-allergy of KDEJ might has to be continued.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1529-1536
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• 2006

We cloned a gene of ompH(D:4) from pigs infected with P. multocida D:4 in Korea [16]. The gene is composed of 1,026 nucleotides coding 342 amino acids (aa) with a signal peptide of 20 aa (GenBank accession number AY603962). In this study, we analyzed the ability of the ompH(D:4) to induce protective immunity against a wild-type challenge in mice. To determine appropriate epitope(s) of the gene, one full and three different types of truncated genes of the ompH(D:4) were constructed by PCR using pET32a or pRSET B as vectors. They were named ompH(D:4)-F (1,026 bp [1-1026] encoding 342 aa), ompH(D:4)-t1 (693 bp [55-747] encoding 231 aa), ompH(D:4)-t2 (561 bp [187-747] encoding 187 aa), and ompH(D:4)-t3 (540 bp [487-1026] encoding 180 aa), respectively. The genes were successfully expressed in Escherichia coli BL21(DE3). Their gene products, polypeptides, OmpH(D:4)-F, -t1, -t2, and -t3, were purified individually using nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. Their $M_rs$ were determined to be 54.6, 29, 24, and 23.2 kDa, respectively, using SDS-PAGE. Antisera against the four kinds of polypeptides were generated in mice for protective immunity analyses. Some $50{\mu}g$ of the four kinds of polypeptides were individually provided intraperitoneally with mice (n=20) as immunogens. The titer of post-immunized antiserum revealed that it grew remarkably compared with pre-antiserum. The lethal dose of the wild-type pathogen was determined at $10{\mu}l$ of live P. multocida D:4 through direct intraperitoneal (IP) injection, into post-immune mice (n=5, three times). Some thirty days later, the lethal dose ($10{\mu}l$) of live pathogen was challenged into the immunized mouse groups [OmpH(D:4)-F, -t1, -t2, and -t3; n=20 each, two times] as well as positive and negative control groups. As compared within samples, the OmpH(D:4)-F-immunized groups showed lower immune ability than the OmpH(D:4)-t1, -t2, and -t3. The results show that the truncated-OmpH(D:4)-t1, -t2, and -t3 can be used for an effective vaccine candidate against swine atrophic rhinitis caused by pathogenic P. multocida (D:4) isolated in Korea.

• Journal of fish pathology
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• v.12 no.2
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• pp.89-99
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• 1999

New sixteen heat shock proteins (Hsps) and ten ethanol shock proteins were appeared on the analysis with SDS-PAGE when cultivation temperature for the Vibrio vulnifrcus ATCC 27562 strain was shifted-up to $42^{\circ}C$ from $30^{\circ}C$ for 20 mins and treated with of 6% ethanol for 10 mins, respectively. Even the induction of thermotolerance in V. vulnificus was coincided with the induction of Hsps if the pre-shock was adjusted to thermal temperature. Outer membrane proteins (OMPs) that were purified from the membrane of cells after heat shock showed more immunodominant pattern to the immunized rabbit anti-V. vulnificus O serum in enzyme-linked immunosorbent assay (ELISA). On the western immunoblot analysis it was confirmed that both 62 kDa IMP and 69 kDa OMP in the Hsps and 48 kDa IMP a major OMP in the ethanol shock proteins were reacted with rabbit anti-V. vulnificus O sera. Agglutination titer of the heat shocked V. vulnificus with rabbit anti-V. vulnificus O serum was higher than that of the untreated bacteria.

• Korean Journal of Veterinary Service
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• v.20 no.1
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• pp.103-125
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• 1997

An epidemiologic study on pleuropneumonia in the slaughter pigs(Chonan and Asan area, Chungnam province, Korea) during the period of January 1994 through December 1995 was conducted. Isolation of A pleuropneumoniae was attempted in 425 pigs with pneumonic lesions. Biochemical properties, antimicrobial susceptibility, serotypes and pathogenicity of isolated A pleuropneumoniae were investigated. In addition, outer membrane protein(OMP) of the Isolates were extracted to determine its properties and immunogenicity in both mice and piglets The results obtained through this study were summarized as followed ; 1. Of 3, 395 slaughter pigs, pleuropneumonia was observed in 425 pigs(10.6%). A pleuropneumoniae was isolated from 22 pigs(5.2%) out of 425 pigs with pneumonic lesions. The biochemical properties of all isolates were same as those of reference A pleuropneumoniae strain. Among 22 isolates, 9, 1 and 12 isolates were serovar 2, 3 and 5, respectively. 2. The results of antimicrobial susceptibility test revealed that the isolates showed high susceptibility to ciprofloxacin and cephalothin, moderate susceptibility to amikacin, gentamicin, kanamycin and streptomycin, and low susceptibility to erythromycin, tylosin and sulfadimethoxin. 3. The isolates were varied in pathogenicity to mice. Median lethal dose of LE9402(serovar 2) and LE9511(serovar 5) were $9.2{\times}10^7$ CFU and $2.8{\times}10^7$%CFU, respectively. Specific pneumonic lesions were observed from the infected mice with clinical signs. Bacteria recovery rate was high in the lung, but low In heart blood and tracheas. 4. Serovar 2 was found to be more pathogenic than serovar 5 in guinea pig. Mortality on guinea pigs inoculated with serovar 2($5.4{\times}10^8-5.4{\times}10^6$CFU) and serovar 5($2.8{\times}10^8-2.8{\times}10^6$ CFU) was 20~40% and 40~80%, respectively. A severe hemorrhagic lesions and focal pneumonic lesions were observed from dead guinea pigs. Bacteria recovery rate was relatively higher in the lung than that of other organs. 5. In the SDS-PAGE analysis, OMP-enriched fractions of both isolates and reference strains contain common peptide bands equivalent to molecular weight of 17, 27, 42, 52 and 95Kd. In addition to common peptide bands, the bands which are specific to each isolate were also observed. The profiles of Sephadex G25 fractions showed 3 major peaks. The common peptide bands which were observed by SDS-PAGE of the crude OMPs were found in the peaks 1 and 2. 6. The OMPs extracted from serovar 2(LE9402) and serovar 5(LE9511) provided high level of protection in mice(70~80%) and pigs(100%). All animals inoculated with OMPs were seroconverted, showing micro-agglutination titer of 640 to 1280.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2095-2105
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• 2018

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.