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Immunological Characterization of Bacillus thuringiensis Antigens (Bacillus thuringiensis 항원들의 면역학적 분석)

  • Jung, Jae-Deuk;Park, Jung-Sun;Jo, Young-Soo;Hong, Soon-Bok;Lee, Hyung-Hoan;Cho, Myung-Hwan
    • Microbiology and Biotechnology Letters
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    • v.23 no.1
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    • pp.110-117
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    • 1995
  • This study was carried out to immunologically characterize Bacillus thuringiensis (B.t) antigens. Protein patterns of ultrasonicated- antigens of B. thuringiensis subspecies using SDS- PAGE revealed marked similarities among all the strains analyzed except for the difference between quantative variations of bands and some protein antigens. The comparison of the protein patterns showed that the protein antigen of 45 kilodalton (kd) was common in 11 strains and that the difference between B. thuringiensis subsp. canadensis and galleriae was noticed in quantative variations of bands despite of ambiguous serogrouping, suggesting a useful method for identification. All strains examined showed similar antigenic patterns in SDS-PAGE, while immunodominant bands differed in antigenic reactivity in western blot using polyclonal antibodies. Polyclonal antibody to B. thuringiensis subsp. thuringiensis and israelensis in indirect immunofluorescence assay reacted with flagella and cell surface antigens. The present study indicates that SDS-PAGE and western blot analysis may be used as tools for differentiation and identification of B. thuringiensis subspecies.

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Detection of cockroach (Blattella germanica L.) specific allergens by monoclonal antibodies (단크론 항체법을 이용한 바퀴의 특이항원 분리)

  • 옥미선;김광혁;유태현
    • Journal of Life Science
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    • v.5 no.2
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    • pp.63-69
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    • 1995
  • Cockroach antigen have been known as a cause of allergic disease. German ockroach(Blattella germanica L.) was chosen because it has the highest distribution range and poulation density. To identify the common and specific antigens of adult and larval stage of german cockroach, we made monoclonal antibodies which were confirmen by SDS-PAGE and EITB. Anti-B. germanica antibody producing hybridomas were 24 among the total 960wells. Only 4 hybridomas did not have cross reaction to other species of cockroach and hluse dust mites(Dermatophagodies farinae and D. pteronyssius). SDS-PAGE revealed about 20 bands from 90Kd to 15Kd to 15Kd. ETB showed specific antigens a6 60, 72 and 82Kd which were experimented by the culture supernatant of 4 selected hybridomas. Especially 60Kd coincided with a band of immunized mouse sera.

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Detection of cockroach (Blattella germanica L.) specific allergens by monoclonal antibodies (단크론 항체법을 이용한 바퀴의 특이항원 분리)

  • Ock, Mee-Sun;Kim, Kwang-Hyuk;Yu, Tai-Hyun
    • Journal of Life Science
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    • v.5 no.2
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    • pp.7-7
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    • 1995
  • Cockroach antigen have been known as a cause of allergic disease. German ockroach(Blattella germanica L.) was chosen because it has the highest distribution range and poulation density. To identify the common and specific antigens of adult and larval stage of german cockroach, we made monoclonal antibodies which were confirmen by SDS-PAGE and EITB. Anti-B. germanica antibody producing hybridomas were 24 among the total 960wells. Only 4 hybridomas did not have cross reaction to other species of cockroach and hluse dust mites(Dermatophagodies farinae and D. pteronyssius). SDS-PAGE revealed about 20 bands from 90Kd to 15Kd to 15Kd. ETB showed specific antigens a6 60, 72 and 82Kd which were experimented by the culture supernatant of 4 selected hybridomas. Especially 60Kd coincided with a band of immunized mouse sera.

Differentiation of Three Lactobacillus rhamnosus Strains (E/N, Oxy, and Pen) by SDS-PAGE and Two-Dimensional Electrophoresis of Surface-Associated Proteins

  • Jarocki, P.;Podlesny, M.;Wasko, A.;Siuda, A.;Targonski, Z.
    • Journal of Microbiology and Biotechnology
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    • v.20 no.3
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    • pp.558-562
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    • 2010
  • SDS-PAGE of extracted surface-associated proteins of Lactobacillus rhamnosus strains E/N, Oxy, and Pen, was performed. The obtained protein patterns allowed differentiation of the examined strains, which was not accomplished by the commonly used RAPD genotypic method. The differentiation by the SDS-PAGE method proved to be a useful tool for strain-specific identification, which was further confirmed by 2DE analysis. Therefore, it can be used as an alternative or complementary method for both conventional and genotypic identification procedures, especially when closely related lactobacilli isolates are identified.

Subunits of Sweet Potato $\beta$-Amylase (고구마 $\beta$-아밀라제의 subunit)

  • 안용근
    • The Korean Journal of Food And Nutrition
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    • v.1 no.2
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    • pp.50-55
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    • 1988
  • B-Amylase was obtained from sweet potato extract in a crystalline state by dialysis against water after precipitated with acetone according to the method reported previously followed by DEAE Sephadex A-50 ion exchange chromatography plus gel chromatography of Sephadex G-200. The purified enzyme was homogeneous by SDS PAGE. The efforts had done to remove the miner bands in SDS PAGE by Sephadex G-200 gel chromatography, DATE Sephadex A-50 ion exchange chromatography. isoelectrophocusing, affinity chromatography, hydroxyapatite chromatography, recrystallizstion and HPLC on a column of TSK gel SW 3000 but have given any result. But, N -terminal amino acid of the enzyme was revealed mainly alanine and trace of glycine and glutamic acid. Therefore, it seems that the miner bands in SDS PAGE have a role of subunit.

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Effect of Gamma Irradiation on Egg White Protein (卵白단백질에 대한 감마선 조사의 영향)

  • 변명우;임성일;육홍선;윤혜현;김영지
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.27 no.2
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    • pp.291-295
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    • 1998
  • The ovalbumins obtained from hen's and duck's egg white were irradiated with up to 100 kGy at room temperature. The purified proteins were evaluated for their in vitro digestibility by incubating successively with pepsin and pancreatin conjugate. Amino acid compositions and SDS-PAGE pattern in these proteins were also analyzed. The obtained results indicated that gamma irradiation within the tested dose range(up to 100kGy) produced no statistically significant changes in the in vitro digestibility an amino acid compositions. Analysis of gamma-irradiated ovalbumins by SDS-PAGE revealed radiolysis of ovalbumin into proteins or peptides of low molecular weights.

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Comparison of Biochemical Characteristics of Myofibrillar Protein from Fresh Water Fish and Sea Water Fish (담수어와 해수어의 근원섬유단백질의 특성 비교)

  • 신완철;송재철;홍상필;김영호
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.28 no.2
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    • pp.292-298
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    • 1999
  • Myofibril and actomyosin were prepared from red muscle and white muscle of fresh water fish and sea water fish, and their biochemical characteristics and SDS PAGE patterns of myofibril were compared. SDS PAGE analysis showed that electrophoretic patterns of myofibril were similar be tween white muscle and red muscle, while difference of 30kDa component of myofibril was detected between fresh water fish and sea water fish. When myofibril were treated with trypsin, difference in hydrolysis of heavy chain was observed between white muscle and red muscle. In activities of Ca ATPase, Mg ATPase, EDTA ATPase and ATPase activity pH curve, myofibrillar protein from fresh water fish showed higher specific activity than those from sea water fish.

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Characterization of extracellular proteases of Aeromonas hydrophila isolated from the intestine of carp(Cyprinus carpio) (잉어(Cyprinus carpio)로부터 분리된 Aeromonas hydrophila의 extracelluar proteases 연구)

  • Lee, Jong-Kyu;Kim, Jong-Pil;Choi, Tae-Jin;Song, Young-Hwan
    • Journal of fish pathology
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    • v.10 no.1
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    • pp.31-38
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    • 1997
  • Aeromonas hydrophila isolated from the intestine of carp produced several kinds of proteases into the medium. Inhibitor assay with the culture supernatant of A. hydrophila showed that there were major metalloproteases and minor serine proteases. Gelatin SDS-PAGE showed two proteolytic bands. One broad protease band was inhibited by metalloprotease specific inhibitor, EDTA, indicating a metalloprotease. The other was inhibited by serine protease specific inhibitor, PMSF, suggesting a serine protease. The proteolytic activities of both extracellular proteases remained on Gelatin SDS-PAGE after heating at $70^{\circ}C$ for 30 min. However, the major metalloprotease was separated into two proteolytic bands on Gelatin PAGE by gel filtration chromatography on Sephadex G-75.

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Adiponectin Gene Cloning and Its Expression in Insect Cell Expression System

  • Yuh, In Suh;Sheffield, Lewis G.
    • Reproductive and Developmental Biology
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    • v.36 no.3
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    • pp.193-198
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    • 2012
  • This study was to examine expression of the recombinant full-length adiponectin (recombinant adiponectin) in insect ovarian cell culture system and to characterize structural properties of the recombinant adiponectin secreted in medium. Gene construct encoding the recombinant adiponectin contained N-terminal collagen-like domain (110 Amino Acids, AAs), C-terminal globular domain (137 AAs) and C-terminal peptides for detection with V5 antibody (26 AAs included adaptor peptide) and purification using the 6xHis tag (6 AAs). The approximate molecular weight of the product (monomer) was 35 kDa. Molecular mass species of the expressed recombinant adiponectin were monomer (~35 kDa), dimer (~70 kDa), trimer (~105 kDa) and hexamer (~210 kDa). The major secreted species were the LMW forms, such as monomer, dimer, and trimer. There was MMW of hexamer as minor form. HMW multimers (~300 kDa) were shown as a tracer or not detected on the SDS-PAGE in several experiments (data not shown). The multimer forms in this study were not compatible to those in animal or human serum and adipose tissue by other researcher's study in which the major multimer forms were HMW. By protein denaturing experiments with reducing reagent (${\beta}$-MeOH), anionic detergent (SDS) and heat ($95^{\circ}C$) on the SDS-PAGE, not all adiponectin multimers seemed to have disulfide bond linked structure to form multimers. The recombinant adiponectin which expressed in insect ovarian cell culture system seemed to have the limitation as full physiological regulator for the application to animal and human study.

Effect of Gamma Irradiation on the Conformational Changes of Garlic (Allium sativum) Protein, Allivin (감마선 조사가 마늘(Allium sativum) 단백질 Allivin의 구조적 변화에 미치는 영향)

  • 김미리;이주운;변명우;서지현;육홍선;김미정
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.31 no.5
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    • pp.723-727
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    • 2002
  • Conformational changes of allivin, a major allergen protein in garlic, by gamma irradiation were observed. Allium solution (3.0 mg/mL in 0.01 M PBS, pH 7.4) and raw garlic were irradiated in a gamma irradiation facility to be absorbed with doses of 2.5, 5.0, 7.5 and 10.0 kGy. After irradiation, a half of irradiated allivin solution was filtered through 0.22 ${\mu}{\textrm}{m}$ filtration kit. UV spectrum and SDS-PAGE were used for observing the conformational changes of allivin by gamma irradiation. Turbidity of allivin solution was increased by optical observation, and the result was validated by the increase of absorbance at 660 m. Turbidity of irradiated and filtered allivin solution significantly (p<0.05) decreased, and the concentration of soluble protein in the solution decreased by increase of the dose. Aggregation among allivin molecules was observed by SDS-PAGE, but irradiated and filtered samples showed decrease of allivin with reducing aggregation trend. SDS-PAGE profiles of irradiated raw garlic were not different. Results indicate that gamma irradiation can decrease the allivin in solution state which may impact on allergenic reaction of garlic.