• 한국해양바이오학회지
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• 제1권2호
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• pp.84-90
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• 2006

Vibrio fluvialis의 opp gene cluster내에 존재하는 oppABCDF 유전자를 allelic exchange 방법에 의해서 각각의 유전자가 deletion된 mutant를 제조하였다. 각각의 유전자가 deletion된 mutant의 확인은 PCR과 Southern hybridization으로 결정하였다. 각 mutant들을 BHI 배지에서 생육을 비교한 결과 배양후 4시간 이전까지는 wild strain이 모든 mutant들에 비해서 생육이 좋았으나 4시간 이후부터는 같은 수준의 생육을 보여주었다. Biofilm 생산을 비교한 결과 ${\Delta}oppA$ mutant에서 가장 높은 생산성을 보여주었다. ${\Delta}oppC,D,F$ mutant들의 biofilm 생산은 ${\Delta}oppA$ mutant 보다는 낮았으나 wild strain보다는 높은 biofilm 생산성을 보여주었다.

• 생명과학회지
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• 제14권2호
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• pp.362-367
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• 2004

장염비브리오균의 숙주 내 감염을 일으키는 기작을 이해하기 위하여 세포외 효소 중의 하나인 콜라겐분해효소의 유전자가 불활성화된 돌연변이체를 제작하였다. 콜라겐분해효소의 유전자인 vppC 유전자에 항생제 내성 유전자인 nptII를 삽입하여 제작된 재조합 DNA를 suicide vector인 pDMS197에 클로닝하여 pVCM03이라 명명하였다. 재조합 suicide 플라스미드 pVCM03을 E. coli 7213에 형질전환하여 접합을 통하여 원 균주인 V. varahaemolyticus 04에 전달하였다. 전달된 pVCM03 유래의 재조합 vvpC::npfII DNA는 homologous recombination에 의해 wild-type allele와 교환되어 돌연변이체를 형성하게 되고, 돌연변이체는 10% sucrose가 함유된 TCBS 배지에서 선별되었다. Allele exchange는 PCR에 의한 증폭된 DNA의 크기 비교로 확인하였다. 돌연변이체인 V. parahaemolyticus CM은 원 균주와 비교하였을 때 약 4배정도 낮은 콜라겐 분해 활성을 나타내었고, vero cell을 이용한 MTT assay에서도 원 균주에 비하여 낮은 세포독성을 보였다.

• 생명과학회지
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• 제16권7호
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• pp.1112-1118
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• 2006

돼지 위축성 비염과 개의 kennel cough의 원인균인 B. bronchiseptica는 각 숙주의 상부 호흡기관의 점막에 집락을 형성하는 병원균으로서 철이 부족한 환경에서 hydroxamate type의 alcaligin이 라는 siderophore를 생산한다. Alcaligin의 생합성에 관련하는 구조유전자 중 alcA 유전자의 기능을 밝히고자 alcA 결손돌연변이주 구축을 통하여 확인하였다. alcA 유전자 결손 돌연변이를 위해 0.6 kb alcA 5' flanking DNA와 0.7 kb alcA 3' flanking DNA fragment들을 pCP1.11을 주형으로 하여 PCR법으로 증폭한 후, 5' flanking과 3' flanking DNA가 연결된 재조합 suicide vector pDMl을 구축하여 세포 접합을 통해 B. bronchiseptica로 도입시켰다. 도입된 pDM1으로부터 allelic exchange법에 의해 alcA 유전자가 결손된 돌연변이주 B. bronchiseptica H1을 얻을 수 있었다. B. bronchiseptica H1은 야생형인 B. bronchiseptica에 비하여 alcaligin siderophore를 거의 생성하지 못하였다. alc 오페론 중 promoter와 alcA 유전자만을 가지는 재조합 플라스미드를 B. bronchiseptica Hl에 도입하였을 때 alcaligin siderophore의 생산이 회복됨을 확인할 수 있었다. 이상의 결과로부터 alcA 유전자가 alcaligin 생합성에서 매우 중요한 역할을 수행하는 것을 알 수 있었다.

• 생명과학회지
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• 제34권2호
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• pp.79-85
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• 2024

SlyA는 Salmonella와 같은 장내세균과(Enterobacteriaceae)에 속하는 E. coli에서 용혈소(HlyE)의 발현을 조절하는 전사 조절인자로 알려져 있다. 그러나 Salmonella에는 slyA 유전자가 있지만 hlyE 유전자는 없다. Salmonella에서 SlyA의 역할을 탐구하기 위해 slyA 유전자가 결실된 돌연변이주를 사용하였다. S. Typhimurium CK295 (ΔslyA)는 allelic exchange 방법으로 제작되었다. 야생형 균주와 CK295 균주의 비교시험에서 생육 특성, 운동성, 총 단백질 분석, 분비 단백질 분석 등에서 특별한 차이가 발견되지 않았다. CK295 균주는 야생형에 비해 생물막을 약간 적게 생성하는 패턴을 보였다. 흥미롭게도, 대식세포에서의 생존능력을 비교한 결과, CK295 균주는 야생형에 비해 생존능력이 60% 감소하는 것으로 나타났다. 마우스의 독성을 테스트하기 위해 6주령 BALB/c 마우스에 경구 투여한 후 사망률을 측정하였다. 그 결과, BALB/c에서 CK295 (ΔslyA)의 LD₅₀ 값이 야생형 S. Typhimurium 𝜒3339의 값보다 100배 이상 높게 나타났다. 종합적으로, SlyA는 살모넬라균의 in vivo 생존에 관여하는 독성 인자를 코딩하는 유전자의 발현을 조절하는 것으로 추정된다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 춘계학술대회 및 임시총회
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• pp.60-60
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• 2015

Hybrid histidine kinase is a part of two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. In the present study, Tco1, a homologue of human pathogen Cryptococcus neoformans Tco1 encoding a hybrid histidine kinase, was identified in corn smut pathogen Ustilago maydis by bioinformatic analysis. To explore the role of Tco1 in the virulence of U. maydis, mutants in which the tco1 gene was partially deleted were constructed by allelic exchange. The U. maydis tco1 mutants did show unaltered growth rate on axenic medium but were unable to produce conjugation tubes and develop fuzzy filaments, resulting in impaired mating of compatible strains. The expression levels of prf1, pra1, and mfa1 which are involved in the pheromone pathway significantly decreased in the tco1 mutants. In inoculation tests to host, the tco1 mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that Tco1 plays important roles in sexual development and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.

• 한국동물위생학회지
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• 제28권4호
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• pp.393-405
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• 2005

In prion pathogenesis, the structural conversion of the cellular prion protein $(PrP^c)$ to its abnormal isomer $(PrP^{Sc})$ is believed to be a major event. The susceptibility or resistance to natural sheep scrapie is associated with polymorphisms of host PrP gene (PRNP) at amino acid residues 136, to a lesser extent 154. The 112 residue in ovine PrP displays a natural polymorphism, Methionine to Threonine, which has not been thoroughly investigated. However the cell-free conversion assay showed that ARQ with Thr112 $(T_{112}ARQ)^{1)}$ presents lower convertibility to $PrP^{Sc}$than wild type ARQ $(M_{112}ARQ)$ [1] In this study we generated ovine recombinant PrPs of 112 allelic variants by metal chelate affinity chromatography and cation exchange chromatography. The final purity of the ovine PrP ARQ was more than $95\%$. These variants showed similar immunoreactivity against anti-PrP monoclonal antibodies in Western blot and ELISA. The refolded $M_{112}ARQ$ and $M_{112}ARQ$ presented the secondary structural content to similar extent via CD spectroscopy analysis. The inherited structural features of $M_{112}ARQ$ and $M_{112}ARQ$ under the different biophysical conditions are in the middle of investigation.

• 대한의생명과학회지
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• 제16권3호
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.1010-1022
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• 2017

Hybrid histidine kinase is part of a two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. The Tco1 gene in human pathogen Cryptococcus neoformans encodes a hybrid histidine kinase and is important for pathogenesis. In this study, we identified a Tco1 homolog, UmTco1, in the maize pathogen Ustilago maydis by bioinformatics analysis. To explore the role of UmTco1 in the survival of U. maydis under environmental stresses and its pathogenesis, ${\Delta}umtco1$ mutants were constructed by allelic exchange. The growth of ${\Delta}umtco1$ mutants was significantly impaired when they were cultured under hyperosmotic stress. The ${\Delta}umtco1$ mutants exhibited increased resistance to antifungal agent fludioxonil. In particular, the ${\Delta}umtco1$ mutants were unable to produce cytokinesis or conjugation tubes, and to develop fuzzy filaments, resulting in impaired mating between compatible strains. The expression levels of Prf1, Pra1, and Mfa1, which are involved in the pheromone pathway, were significantly decreased in the ${\Delta}umtco1$ mutants. In inoculation tests to the host plant, the ${\Delta}umtco1$ mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that UmTco1 plays important roles in the survival under hyperosmotic stress, and contributes to cytokinesis, sexual development, and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.59-62
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• 2002

The role of a TolC homologue in the virulence of Vibrio vulnificus, a marine bacterium causing serious wound infection and fulminant septicemia in persons with underlying conditions, has been studied. TolC, an outer membrane protein, has been implicated in a variety of bacterial functions including export of diverse molecules ranging from large proteins to antibiotics. A homologue of the tolC gene of V. cholerae, which has been shown to be required for bile resistance, cytotoxicity and colonization of this organism, was identified in the partially determined genome sequence of V. vulnificus. To determine the role of TolC in the virulence of V. vulnificus, a TolC-deficient (TD) mutant was isolated by in vivo allelic exchange. Compared with the parent strain, the TD mutant was more sensitive to bile, and much less virulent in mice challenged subcutaneously. This mutant was noncytotoxic to the HEp-2 cells, but its metalloprotease and cytolysin activities in the culture supernatant were comparable to the parent strain. In addition, the resistance of the TD mutant to human serum bactericidal activity as well as its growth in either human or murine blood was not affected. Collectively, our data suggest that TolC may be involved in colonization and/or spread of V. vulnificus to the blood stream, probably by secreting a cytotoxin other than the cytolysin.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.856-865
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• 2012

We describe the construction and immunobiological properties of a novel whooping cough vaccine candidate, in which the aroQ gene, encoding 3-dehydroquinase, was deleted by insertional inactivation using the kanamycin resistance gene cassette and allelic exchange using a Bordetella suicide vector. The aroQ B. pertussis mutant required supplementation of media to grow but failed to grow on an unsupplemented medium. The aroQ B. pertussis mutant was undetectable in the trachea and lungs of mice at days 6 and 12 post-infection, respectively. Antigen-specific antibody isotypes IgG1 and IgG2a, were produced, and cell-mediated immunity [CMI], using interleukin-2 and interferon-gamma as indirect indicators, was induced in mice vaccinated with the aroQ B. pertussis vaccine candidate, which were substantially enhanced upon second exposure to virulent B. pertussis. Interleukin-12 was also produced in the aroQ B. pertussis-vaccinated mice. On the other hand, neither IgG2a nor CMI-indicator cytokines were produced in DTaP-vaccinated mice, although the CMI-indicator cytokines became detectable post-challenge with virulent B. pertussis. Intranasal immunization with one dose of the aroQ B. pertussis mutant protected vaccinated mice against an intranasal challenge infection, with no pathogen being detected in the lungs of immunized mice by day 7 post-challenge. B. pertussis aroQ thus constitutes a safe, non-reverting, metabolite-deficient vaccine candidate that induces both humoral and cell-mediated immune responses with potential for use as a single-dose vaccine in adolescents and adults, in the first instance, with a view to disrupting the transmission cycle of whooping cough to infants and the community.