• 대한의생명과학회지
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• 제8권3호
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• 대한수의학회지
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• 제40권3호
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• pp.525-531
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• 2000

Verotoxin을 산생하지 않은 Escherichia coli O157과 verotoxin을 산생하는 E coli(VTEC)를 건강한 소의 분변에서 분리하여 생화학적 및 유전적인 특성에 대해서 비교하였다. E coli O157 : nonH7(운동성은 있으나 H혈청형이 7이 아님)의 sorbitol 분해능과 ${\beta}-glucurondase$ 활성은 E coli O157 : H7이 나타내는 것과는 차이가 있었다. 그리고 uidA 유전자는 verotoxin 산생능과 상관없이 sorbitol과 ${\beta}-glucuronidase$ 음성인 E coli O157 : H7에서 특이적으로 검출되었다. 한편 6개 목장에서 수거한 소 분변 45예에서 VTEC를 분리한 결과, 7주(15.6%)가 분리되었으며 이들은 모두 sorbitol을 분해하였으며 ${\beta}-glucurondase$ 활성이 있었으나 장벽 부착인자를 지배하는 eaeA 유전자가 없었다. 비록 소가 VTEC의 보균원으로 추정되나, 정상 소에서 분리한 VTEC는 eaeA 유전자가 결여된 균주가 많으므로 공중위생학상 eaeA 유전자를 보유한 E coli 보다 위해성이 낮으며, 이러한 결과는 왜 사람에서 유행하는 VTEC 혈청형과 소에서 유행하는 것과 차이가 있는지를 일부 설명해준다.

• Journal of Microbiology
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• 제38권2호
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• pp.93-98
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• 2000

Nine hundred patients diagnosed with diarrhea or hemorrhagic uremic syndrome in the Kyungpook Province, Korea, were examined from November 1998 to February 2000. One patient in Kumi appeared to possess the Escherichia coli O157:H7 strain, which is very important in clinical decision making and public health action. The isolated strain, an E. coli O157:H7 KM, contained a 60 MDa plasmid and typical virulence genes including the verotoxin 2 gene, ehxA gene (encoding enterohemorrhagic hemolysin), and eae (encoding attaching and effacing protein-intimin) gene. This strain produced only verotoxin 2. Pulsed field gel electrophoretic analysis showed that the genomic organization of the E. coli O157:H7 KM strain may differ greatly from those of representative strains previously reported in the United States and Japan.

• 대한의생명과학회지
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• 제4권1호
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• pp.43-56
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• 1998

최근 전세계적으로 문제가 되고 있는 장출형성 대장균 O157:H7을 분리배양 및 동정 없이 바로 시료를 분석하여 신속하게 검출하기 위한 다중 중합효소 연쇄반응 (multiplex PCR) 기법을 확립하고, 이 기법을 이용하여 국내 분리 균주 중에서 SLT-I.II, eaeA, 60-MDa plasmid gene을 가지고 있는 대장균을 유전자 수준에서 검출하고자 하였다. 장출혈성 대장균 O157:H7이 가진 SLT-I.II, 60-MDa plasmid 유전자들에 대한 특이 oligonucleotide primers (MK1'-MK2', NAE19-NAE20, MFSIF-MFSIR)를 함께 동시에 반응 완충액에 넣어 다중 중합효소 연쇄반응을 시행한 결과 317bp (eaeA), 228bp (SLT-I.II), 167bp (60-MDa plasmid)의 PCR 증폭 DNA생성물을 표준균주 (E. coli ATCC 35150)에서는 확인할 수 있었지만, 기타 다른 병원성 장내세균 13세균 13균주에서는 band를 확인할 수 없었다. 한편 다중 중합효소 연쇄반응의 template DNA 추출 방법에 따른 PCR 결과를 비교하였다. 각각의 DNA 추출 방법 중 boiling lysis 방법이 신속하고 간편하여 장출혈성 대장균 O157:H7에 의한 식중독의 임상진단에 다중 중합효소 연쇄반응 (multiplex PCR) 적용하는 데에는 boiling lysis법을 이용하는 것이 가장 적합한 방법으로 확인되었다.

• IMMUNE NETWORK
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• 제20권5호
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• pp.40.1-40.15
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• 2020

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM-19 OVN. GRIM19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

• 한국융합학회논문지
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• 제6권5호
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• pp.77-84
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• 2015

본 연구는 항균제에 내성을 보이는 대장균의 특성을 알아보기 위해 설사환자에서 분리된 대장균에 대한 항균제 감수성 및 병원성 인자의 상관성을 분자융합적 기술을 통해 조사하였다. 분리한 대장균의 항균제 내성은 60주에서 ESBL(extendede spectrum ${\beta}$-lactamase) positive균주가 8주이고, negative균주는 52주였다. ESBL 양성 8주 중 2주는 병원성 유전자가 검출되지 않았으며, stb(3주), flich7(1주), flich7-eae(2주)로 나타났다. ESBL 음성 52주 중 26주는 병원성 유전자가 검출되지 않았고, stx1(3주), stb(10주), flich7 및 eae(각 2주), stx1-flich7(2주), stx1-stb(4주), flich7-stb(2주), flich7-stb-eae(1주)이었다. 결론적으로 항균제 내성이 증가하는 시대에 분자 융합적 관점에서 독력 유전자의 분포와 항균제 내성과의 관계는 적게 나타났으나, 향후 다양한 독력 유전자의 분석을 통한 융합기술연구가 이루어진다면 보다 정확한 병원성 인자를 추정할 수 있을 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.525-529
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• 2009

Cytolethal distending toxins (CDTs) represent an emerging family of newly described bacterial products that are produced by a number of pathogens. The genes encoding these toxins have been identified as a cluster of three adjacent genes, cdtA, cdtB, and cdtC, plus 5 cdt genetic variants, designated as cdt-I, cdt-II, cdt-III, cdt-IV, and cdt-V, have been identified to date. In this study, a general multiplex PCR system designed to detect Escherichia coli cdts was applied to investigate the presence of cdt genes among isolates. As a result, among 366 E. coli strains, 2.7% were found to carry the cdtB gene. In addition, the use of type-specific primers revealed the presence of cdt-I, cdtIV, and cdt-V types of the cdt gene, yet no cdt-II or cdt-III strains. The presence of other virulence genes (stxl, stx2, eae, bfp, espA, espB, and espD) was also investigated using a PCR assay. Among the 10 cdtB gene-positive strains, 8 were identified as COT-producing typical enteropathogenic E. coli (EPEC) strains ($eae^+$, $bfp^+$), whereas 2 were identified as CDT-producing atypical EPEC strains ($eae^+$, $bfp^-$). When comparing the cytotoxic activity of the CDT-producing typical and atypical EPEC strains, the CDT-producing atypical EPEC strains appeared to be less toxic than the CDT-producing typical EPEC strains.

• 한국동물위생학회지
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• 제28권4호
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• pp.407-412
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• 2005

Ten isolates of Verotoxin-producing Escherichia coli (VTEC) were detected in slaughtered cattle and investigated their phenotypic characteristics and antimicrobial susceptibility. None of the isolates was positive for eae gene. Only one isolate was positive for uidA gene. Eight out of ten isolates of VTEC were originated from broker's cattle. Thus microbiological monitoring for broker farms should be performed to minimize VTEC contamination. In the antimicrobial susceptibility test, all the isolates were highly resistant to bacitracin and lincomycin whilst they are susceptible to apramycin and neomycin.

• 대한수의학회지
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• 제39권2호
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• pp.338-342
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• 1999

Twenty three strains of Escherichia (E) coli O157 : H7 isolated from Korea, Japan, USA were analyzed by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA and multiplex polymerase chain reaction. Various PFGE patterns of E. coli O157 : H7 were found on the same farm. Most of the E, coli O157 : H7 strains had shiga-like toxin (slt) II gene only (43.5%) or both slt I and slt II genes(30.4%). eaeA gene was highly conserved in the E. coli O157 : H7. There was no correlation between PFGE and slt gene patterns. The results indicate that various genotypes of E. coli O157 : H7 have spread throughout the country and genomic DNA patterns generated by PFGE are highly specific for different strains and have significant value in epidemiologic investigations of infectious disease outbreaks.

• Journal of Microbiology and Biotechnology
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• 제25권9호
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• pp.1460-1466
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• 2015

In this study, the prevalence of Shiga toxin-producing Escherichia coli (STEC) was investigated among raw meat or meat products from slaughterhouses and retail markets in South Korea, and their potential for antibiotic resistance and virulence was further analyzed. A total of 912 raw meats, including beef, pork, and chicken, were collected from 2008 to 2009. E. coli strains were frequently isolated in chicken meats (176/233, 75.9%), beef (102/217, 42.3%), and pork (109/235, 39.2%). Putative STEC isolates were further categorized, based on the presence or absence of the Shiga toxin (stx) genes, followed by standard O-serotyping. Polymerase chain reaction assays were used to detect the previously defined virulence genes in STEC, including Shiga toxins 1 and Shiga toxin 2 (stx1 and 2), enterohemolysin (ehxA), intimin (eaeA), STEC autoagglutination adhesion (saa), and subtilase cytotoxin (subAB). All carried both stx1 and eae genes, but none of them had the stx2, saa, or subAB genes. Six (50.0%) STEC isolates possessed the ehxA gene, which is known to be encoded by the 60-megadalton virulence plasmid. Our antibiogram profiling demonstrated that some STEC strains, particularly pork and chicken isolates, displayed a multiple drug-resistance phenotype. RPLA analysis revealed that all the stx1-positive STEC isolates produced Stx1 only at the undetectable level. Altogether, these results imply that the locus of enterocyte and effacement (LEE)-positive strains STEC are predominant among raw meats or meat products from slaughterhouses or retail markets in Korea.