• 한국미생물·생명공학회지
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• 제33권2호
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• pp.106-111
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• 2005

비금속성 이성화효소로 추정되는 Staphylococcus aureus의 tagatose-6-phosphate isomerase(E.C. 5.3.1.26)의 기질다 양성을 조사하기 위해서 그 구조유전자(lacB;510bp와 lacA;430bp)를 대장균에서 동시발현하였다. 알려진 기질 이외에 D-ribose와 D-allose에 대해 이성화활성이 새롭게 관찰되었다. EDTA 1 mM 존재하에서도 D-ribose와 D-allose에 대하여 각각 EDTA 비존재 조건에 대비하여 $95\%,\;75\%$의 이성화활성을 나타내는 것으로 미루어 tagatose-6-phosphate isomerase가 비금속성 이성화효소임을 밝혔다. 이때 lacA 또는 lacB의 단독발현시에는 이성화활성이 전혀 밝견되지 않았다. D-Ribose와 D-allose에 대한 기질친화상수 ($K_m$)은 각각 26 mM와 142 mM였다.

• 한국미생물·생명공학회지
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• 제36권4호
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• pp.353-359
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• 2008

Hepatitis C virus (HCV) is known as the causative agent of blood transmitted hepatitis. Two viral proteins, E2 and NS5A, are known to exert interferon resistance of HCV via PKR pathway. Here, we report a third protein, the RNA-dependent RNA polymerase (NS5B) of HCV, induced interferon resistance inhibiting p56 pathway. p56 was shown to interact with p48 subunit of eukaryotic initiation factor 3 (eIF3). This interaction inhibited formation of ternary complex in translation initiation. Using dual reporter assay system, we observed that the translation decreased when interferon alpha was added to the culture. But, in the presence of HCV NS5B, the translation partly recovered. NS5B and p48 subunit of eIF3 were shown to interact. This interaction seems to inhibit the interaction between p48 and p56. This is the first report that a virus exerts interferon resistance via p56 pathway.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.310-314
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• 1998

DW-116 is a new fluoroquinolone antimicrobial agent with a broad spectrum. In order to elucidate the resistance mechanism to DW-116 in Acinetobacter spp. bacteria, total chromosomal DNA was isolated from 10 strains of Acinetobacter spp. resistant to DW-116. Quinolone resistance determinant region (QRDR) of DNA gyrase gene was amplified by PCR. The 345 bp nucleotide fragment yielded was inserted into pKF 3 which was used as the vector. Comparisons of the DNA sequences of 8 strains with that of the wild type strain revealed a Ser-83 to Leu mutation in mutants and all ten strains contained one silent mutation$(T{\rightarrow}G)$in QRDR. From Acinetobacter MB4-8 strain, DNA gyrase was isolated and purified, through novobiocin-sepharose, heparin-sepharose affinity column chromatography. The enzyme was composed of two subunits and the molecular mass of subunits A and B were 75.6 and 51.9 kDa, respectively. The supercoiling activity of the reconstituted DNA gyrase composed of subunit A from Acinetobacter MB4-8 and subunit B from E. coli was not inhibited by $128{\mu}\textrm{g}$ml of ciprofloxacin. It might be said that one of the resistance mechanisms to DW-116 in Acinetohacter MB4-8 was subunit A alteration of DNA gyrase.

• Journal of Microbiology and Biotechnology
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• 제6권1호
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• pp.26-29
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• 1996

The genes coding for the urease of alkalophilic Bacillus pasteurii have been previously cloned and recently sequenced. (You, J. H., B. H. Song, J. H. Kim, M. H. Lee, and S. D. Kim (1995) Molecules and Cells 5, 359-369.) The recombinant Bacillus pasteurii urease expressed in an E. coli HB101 strain was purified 31.2 fold by using combinations of anion-exchange and hydrophobic chromatography followed by Mono-Q chromatography on a FPLC. In spite of the presence of three discrete structural peptide genes in the Bacillus pasteurii urease gene cluster, only one or two enzyme subunits have been observed to date. Here we report for the first time that the recombinant Bacillus pasteurii urease expressed in a E. coli strain consists of three distinct subunits. One large subunit was estimated to be of $M_r$=65, 200 and the two small-subunit peptides are of $M_r$=14, 500 and $M_r$=13, 700, respectively.

• Journal of Microbiology
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• 제43권4호
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• pp.354-360
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• 2005

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately $1.9\%$ of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.489-499
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• 2019

Subunit vaccines are safer and more stable than live vaccines although they have the disadvantage of eliciting poor immune response. To develop a subunit vaccine, an effective delivery system targeting the key elements of the protective immune response is a prerequisite. In this study, oxidized carbon nanospheres (OCNs) were used as a subunit vaccine delivery system and tuberculosis (TB) was chosen as a model disease. TB is among the deadliest infectious diseases worldwide and an effective vaccine is urgently needed. The ability of OCNs to deliver recombinant Mycobacterium tuberculosis (Mtb) proteins, Ag85B and HspX, into bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) was investigated. For immunization, OCNs were mixed with the two TB antigens as well as the adjuvant monophosphoryl lipid A (MPL). The protective efficacy was analyzed in vaccinated mice by aerosol Mtb challenge with a virulent strain of Mtb and the bacterial burdens were measured. The results showed that OCNs are highly effective in delivering Mtb proteins into the cytosol of BMDMs and BMDCs. Upon immunization, this vaccine formula induced robust Th1 immune response characterized by cytokine profiles from restimulated splenocytes and specific antibody titer. More importantly, enhanced cytotoxic $CD8^+$ T cell activation was observed. However, it did not reduce the bacteria burden in the lung and spleen from the aerosol Mtb challenge. Taken together, OCNs are highly effective in delivering subunit protein vaccine and induce robust Th1 and $CD8^+$ T cell response. This vaccine delivery system is suitable for application in settings where cell-mediated immune response is needed.

• Archives of Pharmacal Research
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• 제18권5호
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• pp.308-313
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• 1995

This study has been undertaken to examine the acetylcholinesterase (AChE) of electric organ from korean electtric ray(Narke japonica). Korean electric ray was caughted at Chungmu sea and transported to the laboratory, where electric organs were removed and stored at $-70^{\circ}C$ until used. Acelycholinesterase(AChE) of electric organ was purified by affinity column that was prepared with dicaproyl-methylpyridinium linked to Sepharose 4B. Upon purification, the specific activities in Ellman unit were increased by 52 and 39 times for high salt soluble AChE (HSSE, 870.86 $\DeltaOD/min/geam$ of tissue) and detergent soluble AChE(DSE, 105.42 .$\DeltaOD/min/geam$ of tissue), respectively. Each subunit of AChE separated by SDS polyacrylamide gel electrophoresis(SDS-PAGE)was transferred to immonilon P by western boltting and detected by mAbs raised against each subunit of AChE from electric organ og Torpedo califomica. Collagenic tails of AChE from Torpedo califomica, likewise 103Kd protein of AChE from Narke japonica was detected by monoclonal antibody specific to 103Kd of AChE from Torpedo califomica. However, molar ratio of three subunits of AChE from Narke japonica is different from that of Torpedo calicormica. Furthermore, catalytic subunit of AChE from Narke japonica was not identified by monoclnal antibody specific to catalytic subunit of AChE from Torpedo californica. These results showed differences in molecular structure of AChE from Narke japonica and AChE from Torpedo califormica eventhough they showed same enzymatic activities.

• 한국작물학회지
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• 제49권3호
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• pp.256-260
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• 2004

보리 국가목록등재 48품종의 hordein 밴드패턴의 다양성을 보고 이러한 hordein 밴드패턴을 D/B화하여 기존(참고)품종으로 관리, 활용하면서 품종구별 및 출원품종과 비교될 대조품종의 선별 가능성에 대하여 검토한 결과를 요약하면 다음과 같다. 보리 48품종의 hordein SDS-PAGE 결과, 총 22개의 hordein polypeptide밴드를 읽을 수 있었으며 다양한 15종류 의 hordein 밴드패턴을 볼 수 있었다. Hordein 밴드패턴에 따라 48품종을 집괴분석한 결과, 전체 유사도지수 0.54∼1.00 범위에서 3개군으로 분류되었고 주로 보리의 조성에 따라 분류되었다. Hordein polypeptide 밴드패턴 자체가 보리품종의 형태적 특성을 나타내는 유전적거리와 반드시 비례하는 것은 아니므로 현재의 UPOV관점과 마찬가지로 품종보호권 설정에 직접적인 근거를 제공할 순 없다고 판단되었지만 hordein polypeptide 밴드패턴 그 자체를 신품종의 구별성에 대한 보완적 자료로 이용할 수 있으리라 사료되었다.

• International Journal of Oral Biology
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• 제42권2호
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

• 미생물학회지
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• 제48권4호
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• pp.319-324
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• 2012

본 연구는 남조세균 흔들말목(Cyanobacteria, Oscillatoriales)의 16S ribosomal RNA (rRNA) 및 RNA polymerase beta subunit(rpoB) 유전자를 대상으로 염기서열 변이 및 분자계통학적 특성을 분석한 것이다. 흔들말목 rpoB 유전자는 16S rRNA보다 유전자 변이(유전거리: rpoB=0.270, 16S=0.109)가 큰 것으로 조사되었으며, 통계적으로 유의한 차이를 보였다(Student t-test, p<0.001). 흔들말목 16S rRNA와 rpoB의 계통분석에서 유사한 계통 분지형태를 보였으며, rpoB 유전자가 높은 해상도를 갖고 있어 흔들말목 분류군을 더 명확하게 구분하였다. 또한, parsimony 분석을 통해 rpoB 유전자가 16S rRNA 보다 2.40배 빠르게 진화하는 것으로 파악되었다. 본 연구결과는 rpoB 유전자가 흔들말목의 분자계통 및 종 분류 연구에 매우 유용하다는 것을 제시해 준다.