장독성 대장균 eKT - 53균주의 내열성 장독소의 성질

도대홍(Dae-Hong Do),김교창(Kyo-Chang Kim),김도영(Do-Young Kim) 한국식품영양과학회 1991 한국식품영양과학회지 Vol.20 No.6

형질전환균주 eKT-53(ST^+ LT^-)를 사용하여 succinate salts 배지에 배양한 배양상층액으로 부터 분리 정제한 내열성장독소(ST ; heat-stable enterotoxin)의 몇가지 성질에 대하여 조사하였다. 분리 정제된 ST의 내열성은 100℃에서 30분 이상 열처리하였을 때 ST 활성이 소실되었다. pH에 대한 안정성은 pH 2~10에서는 대단히 안정하였지만 pH 1과 pH 12에서는 불안정하였다. 또한 α-amylase(Bacillus subtilis) 와 pepsin(porcine)에 대해서 매우 안정하여 활성변화가 없었으나 disulfide 환원제를 처리할 경우 쉽게 활성이 소실되었다. 이러한 ST의 분자량은 약 4,200이었고 pI값은 4.0이였다. Heat-stable enterotoxin(ST) from enterotoxigenic E. coli eKT-53(ST+ LT-, transformant from isolate KM-7) that was produced in succinate salts medium. The culture supernatant(crude ST) was purifed by multipled steps and investigated some characterization of the ST. The heat-stability of purified ST activity was completely lost by treating at 100℃ for 30minutes. ST activity was lost by treatment at pH 1 and 12 conditions, while the activity was not reduced by treatment at pH 2~10, and then the α-amylase and pepsin was not decreased activity but disulfide reducing agents was lost the activity. The molecular weight of the purified ST was approximately 4, 200, the isoelectric point was about 4.0.

쥐의 심장근육 세포로부터 mRNA 분리 정렬 및 cDNA 합성

도대홍, 김교창 忠北大學校 遺傳工學硏究所 1988 遺傳工學硏究 Vol.2 No.1

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돼지에서 由來한 耐熱性 腸毒素生産 大陽菌의 分離 및 判定

都大洪,金敎昌,金道榮 충북대학교 농업과학기술연구소 1987 農業科學硏究 Vol.5 No.2

Enterotoxigenic E.coli is the one of major causative occured to the infantile swine diarrhea. A total of 189 isolated idem assumed E. coli, Salmonella and Shigella was isolated from swine in Kyong-Gi and Chung-Cheong province. When enterotoxigenic E. coli was infected to infant swine, these adhered to the mucosa of small intestine with pili and excrete enterotoxin, which caused the diarrhea. The detection of a heat-stable enterotoxin(ST) produced of isolated E.coli was porformed by infant mouse assay, and was identified pili by the aggutination test of rbc suspention. The plasmid DNA profile was investigated by the agarose gel electrophoresis. The results of assay to enterotoxigenic E.cofi summarized as follow. Isolation ratio of enterotoxigenic E.coli among total of 45 isolated strains was taken 56.52% during summer. Four strains(KS-4, KM-7, KM-12, CM-7) of high produced ST were showed the G/B ratio above 1.10 by infant mouse assay. Four strains of high produced enterotoxigenic E.coli was isolated from swine with diarrhea, and 3 strains (KS-4, KM-7, KM-12) were proved to possess K88 and K99 antigens, or K88 antigen. The patters of plasmid DNA was contained 4.5kb plasmids encoded for ST. The antibiotics sensitivity patters of cultured enterotoxigenic E.coli were showed a tendency to be resistant against tetracycline and streptomycin.

장독성 대장균 eKT - 53 균주의 내열성 장독소 정제

도대홍(Dea-Hong Do),김교창(Kyo-Chang Kim),김도영(Do-Young Kim) 한국식품영양과학회 1992 한국식품영양과학회지 Vol.21 No.1

장독성대장균은 영유아설사 및 여행자설사의 주요한 원인균의 하나로서 이들이 생산하는 내열성장독소는 설사 유발 원인물질로 이들 생산균주는 비병원성 대장균과 비교하여 검정할 수 있다. 본 실험에서는 이러한 내열성장독소를 순수정제하였다. 저자들이 보고한 바 있는 형질전환균주 eKT-53을 사용하여 10L의 succinate salts 배지에 배양한 배양액을 30,000g로 원심분리하여 조 ST 용액을 사용하여 몇 단계의 정제과정을 통하여 정제하였다. 즉 Amberlite XAD-2 chromatography, DEAE-Sephacel anion exchange chromatography, Bio-Gel P-6 gel filtration, Polyacrylamide slab gel 전기영동을 통하여 정제하고 장독소의 정제도 및 장독소의 생물학적 최소단위량을 조사하였다. 최종적으로 정제되어진 ST는 조 ST 용액에 비해 113배 더 정제되었고 회수율은 11%였다. 또한 생물학적 최소단위량이 2.8ng이였고 SDS-polyacrylamide disc gel상에서 단일 band를 나타내어 거의 순수정제된 것으로 보이며 면역을 위한 항원으로 이용할 수 있을 것으로 생각된다. Enterotoxigenic E. coli is one of the major causative agents of the infantile diarrhea and traveler's diarrhea. The heat-stable enterotoxin (ST) is thought to be a virulence factor in the pathogenesis of the diarrhea and to be a maker for identification of the enterotoxigenic E. coli from non pathogenic E. coli. ST producing E. coli KM-7 strain was isolated from the swine and molecular cloning of ST gene of KM-7 strain. Transformant eKT-53 (ST^+, LT^-) was selected by infant mouse assay (IMA). The culture supernatant of eKT-53 strain was performed purification by multipled steps. The culture supernatant (crude ST) was purified by seqaentially applying batch adsorption chromatography on Amberlite XAD.2 resin, ion exchange chromatography on DEAE-Sephacel anion exchanger, gel filtration chromatography on Bio-Gel P-6 and preparative polyacrylamide slab gel electrophoresis. About 113-fold purification was achieved with a yield of about 11% of crude ST and the minimum effective dose(MED) of this purified ST was about 2.8ng in IMA. Homogeneity of purified ST was demonstrated by showing a single band in analytical SDS polyacrylamide disc gel electrophoresis.

쥐의 내장근육 세포로부터 mRNA 분리 정제 및 cDNA 합성

도대홍, 김교창 충북대학교 농업과학기술연구소 1987 農業科學硏究 Vol.5 No.2

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Aspergillus fumigatus가 생산하는 cellulase에 관한 연구

도대홍 충북대학 대학원 1981 大學院論文集 Vol.7 No.-

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쥐의 心臟筋肉 細胞로부터 mRNA 分離 精製및 cDNA 合成

都大洪,金敎昌 충북대학교 농업과학기술연구소 1987 農業科學硏究 Vol.5 No.2

mRNA from Atrial muscle of rat heart has been purified poly(A)+RNA, and its physical properties have been examined. Isolation was achieved by the use of guanidinium extraction method to isolate cellular RNA. Purification was achieved by the use of oligo(dT)-cellulose chromatography to separate poly(A) +RNA from ribosomal RNA, Poly(A) +RNA was translation in a cell-free reticulocyte system. Purified poly(A) +RNA was synthesized a double strand DNA by using reverse transciptase, polymerase arid T4 ligase. The extracted mRNA was obtained approximately 5mg against 20g sample tissue (rat heart). The purification fold was 40-fold, and final purified poly(A) +RNA in orig(dT)-cellulose chromatography was taken 2.4% of the initial mRNA. When 1.0㎍ of poly(A) +RNA was added to a cell-free system in 25㎕ reaction volumn, the complex protein synthesizing ability was maximized. After purifying the poly(A):RNA it was synthesized to a single and then a double strand DNA. Its conversion rate was figured 0.38% for a single strand, and 0.356% for a double strand DNA.

돼지에서 유래 내열성 장독소생산 대장균의 분리 및 판정

도대홍, 김교창, 김도영 충북대학교 농업과학기술연구소 1987 農業科學硏究 Vol.5 No.2

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방향족 화합물 분해균주의 분리 및 동정

정준영, 김교창, 김도영, 도대홍 忠北大學校 農業科學硏究所 1994 農業科學硏究 Vol.12 No.2

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채소연부병균 Erwinia herbicola의 생육억제균 분리 및 특성

김교창,도대홍,김도영 한국식품영양학회 1996 韓國食品營養學會誌 Vol.9 No.3

Erwinia herbicola(EH) 채소 연부병 방제를 위하여 충북 청원군, 진천군 및 충남 연기군 일원의 채소 경작지 토양으로부터 형광성을 나타내는 Pseudomonas속 1,196균주를 선발하고, 연부병균 Erwinia herbicola에 대한 생육억제력과 생물학적 검정으로 발병억제력이 강한 2균주를 선발하여 Pseudomonas fluorescens S4(PS4)와 Pseudomonas fluorescens S65 (PS65)로 동정하였다. 배춧잎을 이용하여 분리길항균의 연부병균에 대한 생물학적 억제력을 확인한 결과 상당한 발병억제력을 확인하였다. 이들 두 균주가 가장 높은 길항력을 나타내는 조건은 배양 초기배지의 pH를 7.0으로하여 25℃에서 3일간 배양했을 때였다. 화학농약 및 항생물질에 대해 2균주 모두 benomyl, propamocab hydrochloride, fosethyl-Al-folpet, vancomycin, penicillin 및 lincomycin에 내성을 나타냈고, PS4균주는 erythromycin에 대해서도 내성을 나타냈다. For the selection of powerful antagonistic bacterium for biological control of Erwinia sp. causing vegetable soft rot, two excellent strains(S4, S65) were selected from 1,196 strains of bacteria which were isolated from rhizospere in vegetable root rot suppressive soil. Selected 2 strains were identified to be a species to Pseudomonas fluorescens S4(PS4) and Pseudomonas fluorescens S65 (PS65). The highest of inhibitory activity was produced in 523 synthetic broth medium at pH 7.0 and 25℃ during 3 day culture. The isolate strains were resistant to the agricultural chemicals such as benomyl, proamocab hydrochloride and fosethyl-Al-folpet, and the antibiotics such as vancomycin, penicillin and lincomycin, only PS4 was resistant to erythromycin.