• 대한수의학회지
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• 제48권4호
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• pp.431-440
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• 2008

Brucellosis is one of the most important zoonosis in worldwide. As one of the control measures, attempts have been made to develop new diagnostic methods using filed isolates as a national policy in many countries. Currently, bovine brucellosis in Korea have been received attention in both public health and economical aspects due to sudden increase of outbreak. Based on the situation, we compared standard strain (B. abortus 1119-3) with field isolates to reveal the differences among them. Biological and biochemical charateristics, antibiotic resistance profiles, outer membrane proteins (OMPs) and lipopolysaccharide analysis of the strains were included in this study. For the diagnostic purpose, an attempt was made to find out a novel antigen from the Korean isolates by serological analysis. There were differences about 55 kDa, 36-38 kDa and 20 kDa in analysis of OMPs by SDS-PAGE and Western blot with positive sera ($\geq$ 1:400 in SAT titer). Also, a serological diagnostic method, ELISA was conducted using OMPs of the strains as novel antigen. Relationships between O.D. and SAT titer were analyzed using field sera showing different SAT titer. High correlation coefficient was observed between SAT titer and ELISA. Results from this study suggested that a new diagnsotic method should be developed using their own field isolates in each country.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1519-1528
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• 2017

Foodborne contamination and salmonellosis caused by Salmonella Enteritidis (S. Enteritidis) are a significant threat to human health and poultry enterprises. Outer membrane vesicles (OMVs), which are naturally secreted by gram-negative bacteria, could be a good vaccine option because they have many biologically active substances, including lipopolysaccharides (LPS), outer membrane proteins (OMPs), and phospholipids, as well as periplasmic components. In the present study, we purified OMVs derived from S. Enteritidis and analyzed their characteristics through silver staining and sodium dodecyl sulfate polyacrylamide gel electrophoresis. In total, 108 proteins were identified in S. Enteritidis OMVs through liquid chromatography tandem mass spectrometry analysis, and OMPs, periplasmic proteins, and extracellular proteins (49.9% of total proteins) were found to be enriched in the OMVs compared with bacterial cells. Furthermore, native OMVs used in immunizations by either the intranasal route or the intraperitoneal route could elicit significant humoral and mucosal immune responses and provide strong protective efficiency against a lethal dose (~100-fold $LD_{50}$) of the wild-type S. Enteritidis infection. These results indicated that S. Enteritidis OMVs might be an ideal vaccine strategy for preventing S. Enteritidis diseases.

• Biomolecules & Therapeutics
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• 제6권2호
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• pp.191-198
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• 1998

Vsrio vulnificus is an estuarine gram-negative human pathogen that affects people with chronic hepatitis, alcoholic cirrhosis, diabetes mellitus or other underlying diseases. V. vulnificus infection is mediated primarily by consumption of raw fish or by exposure of pre-existing wounds to seawater, causing permanent tissue damages or fatal septic shock. We have been developing a vaccine against V. vulnificus composed of whole cell Iysate of a V. vulnificus O-antigen serotype 4 strain. Oral administration of the V. vulnificus;oral vaccine;immunogenicity;protective efficacy vaccine elicited a high serum antibody response in rabbits. The induced antibodies were reactive not only to the homologous strain but also to heterologous O-antigen serotype strains, indicating cross-reactivities among serotypes. Western blot analysis revealed that the antibodies are mainly specific for outer membrane proteins (OMPs) and reacted equally well with OMPs purified from 9 O-antigen serotypes. The rabbit antisera showed opsonophagocytic killing activity against heterologous strains as well as the homologous strain. Passively transferred rabbit antisera into mice were protective against a lethal V. vulnificus infection. These data demonstrate that oral administration of the V. vulnificus vaccine induced a systemic antibody response which had a protective efficacy against V. vulnificus infections, suggesting that this vaccine preparation could be used to develop an oral vaccine against V. vulnificus.

• 한국동물위생학회지
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• 제20권1호
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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.

• 수산해양교육연구
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• 제27권5호
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• pp.1508-1521
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• 2015

Vibrio harveyi is a pathogenic marine bacterium causing systemic symptoms resulting in mass mortalities in fishes and shrimps in aquaculture. Outer membrane proteins(OMPs) are related to the pathogenicity and thus good targets for diagnosis and vaccination for Gram negative bacteria. Recently vaccination strategies using the OMPs have been suggested to control vibriosis in several fish species. In this study, we have isolated V. harveyi from diseased marine fishes from different regions of Korea and investigated genetic variations of four OMP genes including OmpK, OmpU, OmpV and OmpW. Consequently, OmpK and U genes could be divided into 3 subgroups of type I, II, III and type A, B, C, respectively, without any correlation with geographical regions and species while OmpV and W were highly homologous. OmpW gene of V. harveyi FP4138 was fully sequenced and predicted the deduced amino acid sequence to form ${\beta}-barrel$ with hydrophobic channel. Indeed, the immunogenicity of recombinant OmpW produced in Escherichia coli was assessed by vaccinating flounder. As a result, the high antibody response with antibody titer of $4.2{\pm}0.7$ and protection with relative percent survival of 60% against artificial infection of V. harveyi were demonstrated. This result indicates that OmpW is a virulence related factor and it can be a vaccine candidate to prevent a high mortality caused by V. harveyi infection in olive flounder, Paralichthys olivaceus.

• 대한수의학회지
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• 제38권2호
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• pp.371-378
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• 1998

국내의 무지개송어, 금붕어, 은어양식장에서 아가미와 지느러미의 부식과 괴사를 나타내는 물고기로부터 균주를 분리하여 형태학적, 생화학적, 항원적시험 그리고 전기영동을 통하여 분리주와 미국과 일본에서 분리된 표준주(NCMB $2248^T$, EK 28)의 성상을 비교하였다. 즉, 형태학적으로 cytophaga agar에서 강한 점착성, rhizoid 그리고 노랗고 편평한 집락을 나타내었으며 flexirubin type pigment를 산생하고 동종과 이종에 대한 항원적 시험에서도 응집역가는 1024, 512를 나타냈다. 비록 분리주는 각기 다른 어종에서 분리되었음에도 불구하고 형태학적, 생화학적 그리고 혈청학적인 특성에 있어서 미국과 일본에서 분리된 표준주와 유사하였다. SDS-PAGE에 의한 세포외막단백질(outer membrane proteins, OMPs)의 분석에서도 표준주와 gel pattern이 일치하는 형태를 나타내었다. 이상의 결과를 미루어볼 때 국내 무지개송어, 금붕어 및 은어양식장에서 분리된 균주들은 Cytophaga columnaris로 분리 동정할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.421-424
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• 2005

Effect of antibiotics belonging to three different groups, including third generation cephalosporins, aminoglycosides, and quinolones, on the outer membrane protein (OMP) profile of Klebsiella pneumoniae was examined. It was found that a new OMP (porins) of 40 kDa molecular mass was expressed in Klebsiella pneumoniae, when grown in the presence of ceftazidime, whereas new proteins with 30 kDa and 22 kDa masses were detected in the presence of ofloxacin. The immunoblot analysis showed that the new proteins of 40 kDa and 30 kDa molecular masses were expressed on the outer envelope, when being exposed to antibiotics ceftazidime and ofloxacin, respectively. This finding is important, as the outer surface comes in contact with the immune system, and therefore may have a bearing on the outcome of the disease.

• 한국미생물·생명공학회지
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• 제33권4호
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• pp.274-280
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• 2005

Pasteurella. multocida 균은 광범위한 질병을 야기시키는 악성 감염원으로 알려져 있다. 그람 음성균의 동종 및 이종간에 의한 감염에 대한 강력한 백신 후보 물질로써 OmpH라고 불리는 porin 단백질이 고려되어 왔다. 이 OmpH가 이번 연구에서 분리 및 정제되었다. 선도 단백질을 제거한 재조합 OmpH 단백질은 pRSET A발현벡터를 이용하여 40kDa로 발현되었으며, 친화성 크로마토그래피 정제되었다. OmpH에 대한 면역혈청을 얻기 위해, 한마리의 실험용 쥐에 한번에 50ug의 단백질을 복강 주사를 통해 2회에 걸쳐 주사했다. 항 OmpH 면역혈청의 증가는 ELISA로 측정되었다. 이번 실험에서 확인된 면역혈청의 증가는 OmpH단백질이 병원성 파스튜렐라 균이 일으키는 가금 콜레라를 예방하기 위한 백신의 강력한 후보임을 보여주고 있다.

• 한국어병학회지
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• 제19권1호
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• pp.45-54
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• 2006

E. tarda의 독성 비교를 위하여 일반 TSB, iron-chelate인 2,2‘-dipyridyl 첨가 TSB 및 Fe 첨가 TSB의 세 가지 조건에서 E. tarda를 배양하였다. 독성시험의 결과는 iron-chelate 첨가 시험구의 E. tarda에서 가장 낮은 독성을 나타내었다. 그리고 각각의 OMPs와 IROMPs을 분리하여 SDS-PAGE로 항원성을 비교한 결과, iron-chelate 첨가 시험구의 E. tarda IROMPs에서 68, 73 kDa 크기의 분자들이 더 많이 발현된다는 것을 확인할 수 있었다. 두 가지 vaccine 투여구 중에서 2,2‘-dipyridyl 첨가 배지로 배양한 균체로 제작한 DP-FKC 항원 투여구가 응집 항체가가 더 높게 나타났다. 면역반응의 변화를 알기 위한 응집 항체가 조사에서는 FKC 항원과 DP-FKC 항원 투여구에서 모두 대조구보다 높은 값을 나타내었고, 3주 째에 최고치를 나타내었다. ELISPOT을 이용한 항체 생성 세포 수의 검출에서도 응집 항체가와 유사한 pattern을 나타내었는데, 두 가지 vaccine 투여구에서 2주 째에 가장 많은 항체 생성 세포 수를 나타내었다. 공격실험에서는 3주 째에 FKC 항원 투여구가 50%, DP-FKC 항원 투여구에서는 80%의 생존율을 나타내었다. 전체적으로 DP-FKC 항원 투여구의 생존율이 높게 나타났다. 이상의 결과로부터 넙치에 대한 E. tarda의 vaccine으로는 현재 연구되고 있는 FKC vaccine보다 철 결핍 조건에서 배양된 균체의 항원인 DP-FKC vaccine이 동일한 조건으로 사용하였을 때 더 나은 방어력을 가진다는 것을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제30권10호
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• pp.1552-1558
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• 2020

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4℃-55℃). The optimum temperature and pH ranges of LysECP26 were identified at 37℃-42℃ and pH 7-8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serve as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.