• 한국임상수의학회지
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• 제28권2호
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• pp.196-203
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• 2011

포도알구균은 아토피 피부염 환자의 피부병변에서 가장 많이 발견되는 세균으로, 이들 균집락의 정도는 사람 아토피 피부염의 임상증상 악화요인으로 알려져있다. 이에 본 연구에서는 개 아토피 피부염 환자의 피부에서 포도알구균의 존재를 확인하고, 이들 균주가 생산하는 외독소 유형을 분석하여 개 아토피 피부염 환자의 임상증상과의 연관성을 알아보았다. 79마리의 개 아토피 피부염 환자 중 91.1%인 72마리에서 포도알구균이 검출되었으며, 이 중 62마리에서 Staphylococcus pseudintermedius가 가장 높은 빈도로 확인되었다. 외독소 유전자 분석에서는 69.4%인 50마리에서 1가지 이상의 외독소 유전자를 포함하였고, 이들 중 56%인 28마리에서 2가지 이상의 다른 외독소 유전자를 가지고 있는 것으로 나타났다. 개 아토피 피부염 환자를 포도알구균의 존재 유무에 따라 분류하였을 때, 임상증상 점수의 차이에 통계적인 의미는 없었지만 (P=0.598), 외독소 유무에 따라 임상증상 점수를 비교하였을 때는 의미있는 차이를 보였다 (P=0.028). 또한 외독소 유형에 따라 분류하였을 때 외독소 중 SED와 exfoliative toxins에서 임상증상에 의미있는 차이를 보였으며 (P<0.05), 외독소 유무에 따라 분류하였을 때는 임상증상 점수 중 특히 발적과 구진/농포에서 의미있는 차이를 보였다 (P<0.05). 이와 같은 결과를 통해 개 아토피 피부염에서 포도얄구균이 생산하는 외독소가 개 아토피 피부염의 증상악화와 관련이 있는 것으로 사료된다.

• Applied Biological Chemistry
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• 제19권4호
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• pp.189-201
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• 1976

공업폐수를 이용하여 미생물 살충제를 생산하기 위하여 배양방법, 배양조건 및 생물학적 검정을 행한 결과는 다음과 같다. (1) B. thuringiensis avr. thuringiensis는 특히 나비목 해충에 유효한 ${\delta}-endotoxin$과 파리목에 유효한 fly-toxin을 생산하는 세균이다. (2) 제사공장에서 폐기되는 자견수를 영양원으로서 본 세균배양에 이용할 목적으로 분석한 결과 많은 량의 무기물과 단백질이 용으로부터 추출되어 나왔음을 알았다. (3) GYS 배지와 citrate salts배지를 만들때 물대신 자견수를 사용하면 가장 경제적으로 많은 량의 ${\delta}-endotoxin$을 얻을 있다. (4) GYS 배지에 8gr/l의 glucose 첨가시 균체와 crystal의 혼합물 생산량이 가장 많았다. (5) 아미노산중 leucine + isoleucine _ valine을 $1.25{\times}10^{-3}M$ 농도로 citrate salts 배지에 첨가시 공시세균의 증식이 훨씬 좋았다. (6) Na-glutamate를 citrate salts배지에 $2.5{\times}10^{-3}M$ 첨가시 가장 좋은 증식을 보였다. (7) 공시 나무목 해충중에서 total body extract의 pH가 가장 높은 Pieris rapae Linne (pH 8.4)에서 가장 좋은 살충효과를 보이고 Dendrolimus spectabilis Bulter와 Bombyx mori Linne에서는 효과가 낮았다. (9) 본 세균의 배양 상등액은 초파리에 대하여 높은 살충률을 보여 열에 안정한 exotoxin의 존재를 확인하였다.

• BMB Reports
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• 제52권8호
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• 환경위생공학
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• 제14권2호
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• pp.32-39
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• 1999

The following is experimental result of selecting soil bacteria showing toxicity against Root-knot nematode (Meloidogyne hapla). Out of 286 strains isolated from soil, one(NC67) showing toxicity against M.hapla is selected The selected strain(NC67) is identified of B. thuringiensis subsp. indiana. It proved out that the toxic maerial against M. hapla produce by NC67 strain is an exotoxin. The result of examining the existence of the extercellular toxicity product by the toxic strain(NC67) by usign activated carbon column chromatography, Dowex 50W column chromatography and TLC of silical gel etc. proved out that it is a single material.

• 대한수의학회지
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• 제43권2호
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• pp.247-253
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• 2003

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

• Journal of Microbiology and Biotechnology
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• 제18권8호
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• pp.1475-1481
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• 2008

B3 antibody specifically binds the $Lewis^Y$-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, $(Fab-PE38fl)_2$. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences hi, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers $(Fabh1-PE38fl)_2$, $(Fabh2-PE38fl)_2$, and $(Fabh3-PE38fl)_2$. The refolding yields of these dimers were 5-16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment $(Fabh2-PE38)_2$ [8]. Our data suggest that the steric repulsion between the two PE38s in $(Fabh1-PE38)_2$ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

• 생명과학회지
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• 제9권1호
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• pp.106-110
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• 1999

감염성 Vibrio vulnificus에 의한 패혈증과 같은 질병의 원인을 알아보기 위하여 Vibrio vulnificus로 부터 세포벽 lipopolysaccharide를 추출한 후 지방산 조성을 분석하고 limulus gelation activity와 lethal toxicity를 측정하였다. 이 결과들을 비감염성 Escherichia coli LPS와 감염성 Salmonella typhimurium LPS의 것들과 비교하였다. V. vulnificus LPS의 주 지방산은 myristic acid 이었고 E. coli LPS는 lauric acid, S. typhimurium LPS는 capric acid 이었다. 세가지 LPS의 Limulus gelation activity는 같았으며(0.1ng/ml), V. vulnificus LPS의 lethal toxicity는 E. coli LPS와 S. typhimurium LPS의 것과 비슷하였다. LPS 이외에도 exotoxin과 같은 인자들도 V. vulnificus에 의한 세포 독성의 원인으로 고려해야 할 것이다

• 생명과학회지
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• 제18권3호
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• pp.298-303
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• 2008

펩타이드 문고 기술을 이용하여 흑색종 세포주인 B16FI0에 결합하는 펩타이드 리간드를 검색하였다. 먼저 세포 내부로 들어간 파지들을 선택하는 방법으로 두 번 검색한 후 표면에 결합한 파지들 가운데 트랜스페린 단백질을 이용하여 트랜스페린 수용체에 결합한 파지들만을 선별적으로 용출시키는 방법으로 세 번 검색하였다. 다음으로 이 두 가지 방법을 통해 선별된 파지들에 표현된 펩타이드들을 Pseudomonas exotoxin의 전이 영역과 촉매 영역에 융합시킨 재조합 독소들을 만들었다. B16FI0 세포에 대한 각 재조합 독소의 활성을 측정하여 일곱 개의 클론을 선택한 후 염기서열을 분석하였다. 그 결과 그 가운데 한 클론에서 표현하는 펩타이드의 아미노산 서열이 사람의 트랜스페린과 유사한 서열을 갖는 것으로 확인되었다. 그 펩타이드를 화학적으로 합성한 후 항암제를 포함하는 리포솜에 붙여 실험한 결과 트랜스페린 수용체를 통해 치료물질을 전달할 수 있는 가능성을 지닌 것으로 평가되었다.

• Bulletin of the Korean Chemical Society
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• 제22권12호
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• pp.1361-1365
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• 2001

Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudo monas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.

• Molecules and Cells
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• 제46권12호
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• pp.764-777
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• 2023

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC₅₀ values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.