• 한국미생물·생명공학회지
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• 제27권5호
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• pp.359-363
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• 1999

To prevent appearance of resistant mosquitoes against $\delta$-endotoxin of bacillus thuringiensis subsp. israelensis (Bti) in field, a mosquitocidal Bacillus thuringiensis strain 21-2(Bt21-2) producing a new type of $\delta$-endotoxin was isolated. The strain Bt 21-2 belongs to H serotype 43, B. thuringiensis subsp. guiyangiensis (Btg). The $\delta$-endotoxins from the strain Bt 21-2 and the strain Bti were a cuboid shape morphologically, but the $\delta$-endotoxin of the strain Bt 21-2 was composed of 150, 90 and 70kDa proteins on SDS-PAGE, and the antigenicity of $\delta$-endotoxin of the strain Bt 21-2 was different from that of the strain Bti on immunoblot. The $\delta$-endotoxin gene of the strain Bt 21-2 was not amplified with specific primers of $\delta$-endotoxin gene (cry4A and cry4B) of the strain Bti on PCR.

• Journal of Microbiology and Biotechnology
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• 제6권5호
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• pp.326-330
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• 1996

To illustrate whether a hemolysin in $\delta$-endotoxins of Bacillus thuringiensis strain 73E10-2 and subsp. israelensis had immunological identity, a cyt gene of the strain 73E10-2 which encodes a hemolysin was cloned to B. subtilis (transformant 2753). The transformant 2753 containing cyt gene produced the hemolysin which lysed sheep erythrocytes after treatment of proteinase K. The hemolysin was proved also to be toxic against mosquito larvae (Aedes aegypti). The molecular weight of the hemolysin produced from the transformant 2753 was determined to be about 25 kDa by SDS-PAGE and immunoblot. The hemolysin in $\delta$-endotoxin of subsp. israelensis and subsp. kyushensis did not react on immunoblot using polyclonal anti-$\delta$-endotoxin of the strain 73E10-2, but 70-140 kDa mosquitocidal toxins in $\delta$-endotoxin of subsp. kyushuensis reacted.

• 한국미생물·생명공학회지
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• 제21권5호
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• pp.393-398
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• 1993

One strain of mosquitocidal Bacillus thuringiensis, H9B, was isolated from soil. The biochemical characteristics and flagella antigenicity of the strain H9B is similar to that of B. thuringiensis subsp. darmstadiensis. The delta-endotoxin of the strain H9B coincided with that of B. thuringiensis subsp. darmstadiensis strain 73E10-2 on agarose double immunodiffusion test. The delta-endotoxin of B. thuringiensis subsp. israelensis contains hemolysin fragment (28 kb) on SDS-PAGE when the delta-endotoxin was solubilized in alkali, while that of the strain H9B does not contain 28 kb protein.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.303-307
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• 1991

B.thuringiensis subsp. darmstadiensis 73E10-2의 내독소에 존재하는 hemolysin이 Sephadex G-100 gel filtration과 DEAE-cellulose ion exchange column chromatography에 의해 정제되었으며, 그 순도는 SDS-PAGE와 Ouchterlony test로 확인하였다. 그 결과 정제된 hemolysin의 분자량은 64KDa 의 단백질 이었으며, in vivo 상태에서는 전혀 모기유충에 독작용을 나타내지 않았다는 점이 28KDa 단백질의 차이가 있었다. 또한 정제된 hemolysin과 B.thuringiensis subsp. israelensis의 내독소를 효소항체법으로 검토해 본 결과 양단백질 사이에는 면역학적으로 전혀 상관이 없었다.

• 환경위생공학
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• 제13권2호
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• pp.40-46
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• 1998

For a biological control on a plant pathogen, Pryicularia Oryzae, and a mosquito, Aedes aegypti, Bacillus thuringiensis strain AF6 which produces parasporal inclusion, delta-endotoxin, was isolated. The B. thuringiensis strain AF6 was produced not only an antifungal substance(AFS) against P. oryzae, but also a mosquitocidal delta-endotoxin. The AFS of the strain AF6 in more stable at pH 4.0 than pH 10.0. At the mode of action, the AFS of the strain AF6 was inhibited hypha growth on potato agar plate(pH 5.0), and degraded cell walls of P. oryzae.

• 환경위생공학
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• 제12권2호
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• pp.59-64
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• 1997

For purification of a 70kDa toxic protein of mosquitocidal delta-endotoxin from B. thuringiensis strain H9B, immuno-affinity chromatography was performed. After separation of 70kDa toxic proteins from the delta-endotoxin of the strain H9B on SDS-PAGE, the 70kDa toxic protein was subcutaneously injected into rabbit for making a polyclonal antibody. A anti-70kDa toxic protein was purified by a column chromatography packed with protein A-sepharose 4B gels. The 70kDa toxic protein from delta-endotoxin of the strain H9B was also purified by an immuno-affinity chromatography packed with CNBr-activated sepharose 4B gels conjugated anti-70kDa toxic protein after elution with 1/10M citric acid-1/5M Na$_{2}$HPO$_{4}$ buffer(pH3.2) containing 0.5M NaCl. The 70kDa toxic protein was purified through only one step-separation system, was demonstrated by SDS-PAGE and immunoblot.

• 한국미생물·생명공학회지
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• 제18권3호
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• pp.301-304
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• 1990

Bti와 Btd의 내독소에 대한 면역학적인 검토를 행한 결과 양균주의 내독소에는 동일한 기원의 단백질이 혼존하고 있음을 면역확산반응 및 면역전기영동법으로 확인되었다. 또한 동일한 기원의 단백질 함량은 Bti 내독소 보다 Btd 내독소에 많이 존재함을 효소항체법으로 확인하였다.

• 환경위생공학
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• 제13권2호
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• pp.34-39
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• 1998

For more convenient and rapidly isolation of Bacillus thuringiensis subsp. israelensis(Bti), 1) heat treatment spore forming bacteria, 2) growth in enrichment culture media for Bacillus sp. and 3) selection of bacteria producing a lecithinase for Bacillus thuringiensis subsp. israelensis, were performed. Spore forming bacteria were counted 4.8 $\times $ 10$^{8}$cells/g soil on NAPGCY media, 9.2 $\times $ 10$^{7}$cells/g soil on NA media, and 3.6 $\times $ 10$^{8}$cells/g soil on NAAC media, respectively. Bacteria producing only a lecithinase were reached at 25.2% on medium contained egg york, bacteria only producing a delta-endotoxin were reached at 23.2% by phase contrast microscope, and bacteria producing a lecithinase & a delta-endotoxin simultaneously were reached at 13.7%. Bacillus thuringiensis which producing a lecithinase and a delta-endotoxin simultaneously among bacteria producing a lecithinase, were reached at 56.5%; A half of Bacillus thuringiensis was produced a delta-endotoxin, but not produced a lecithinase. Among 8 isolates of Bacillus thuringiensis, two strain of Bti which has a mosquito-cidal toxin, were detected by PCR using a specific primer of $\delta $-endotoxin gene from Bti.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.308-312
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• 1991

B.thuringiensis subsp. darmstadiensis 73E10-2의 내독소에 대한 화학적 처리에서 고농도의 중성염(4M NaBr), 유기용매(50% acetone), 변성제(4M urea) 및 중성 계면활성제(10% triton X-100)로 내독소를 처리하였을 때 모기유충에 대한 독력의 소실이 거의 나타나지 않았으나, guanidine HCL이나 $CCl_4$ 또는 양이온 및 음이온 계면활성제로 처리함으로써 그 독력이 크게 소실되었다. 또한, 내독소의 sulfhydryl기의 변형은 모기유충에 대한 독력에 영향을 나타내지 못하였으나, lysine기 변형으로 내독소의 독력이 거의 완전히 소실되었다.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.230-234
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• 2002

모기 살충성 Bacillus thuringiensis subspangiensis 21-2균주의 용혈성 내독소 단백질의 특성을 검토하고자 21-2균주의 용혈성을 가진 유전자를 Escherichia coli HBIO쎄 형질전환시켰다. 이들 중에서 형질전환 균주 47은 독소 단백질을 생산하며 2.5 kb DNA을 함유한다는 것을 효소항체법, immunoblot및 DNA전기영동법으로 확인하였다. 또한, 형질전환 균주 47-5는 2.5 kb DNA를 다시 Hind ll견 절단하여 pUC118 연결시켜서 조제하였다 그 결과형질전환 균주 47-5은 1.Bkb DNA를 함유하며, 23 kD꺼 독소 단백질을 발현하고, 발현된 독소 단백질은 Aedes aegypti모기 유충에 독성을 나타내었다. 또한 23 kDa의 내독소 단백질 그 자체로는 사람의 적혈구를 용해하지 못하였으나, proteinase K로 처리한 후에는 적혈구에 대해 용혈성을 나타내었다.