• KSBB Journal
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• 제16권6호
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• 한국동물위생학회지
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• 제32권3호
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• pp.241-249
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• 2009

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains can cause broad spectrum of human disease, including diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic colitis (HUS). We examined 868 samples was taken from bovine feces and carcass from January to December 2008 in Seoul. Twenty two (9.5%) shiga toxin -producing Escherichia coli were isolated from the 230 of bovine feces, and two (0.31%) were isolated from the 638 of carcasses. Serotype of E. coli isolates were O157 (10, 41.6%), O26 (10, 41.6%), O111 (1, 4.2%) and UT (3, 12.6%). In PCR, the isolates displayed three different stx gene combination (stx1 [2, 8.4%]), stx2 [3, 12.6%] and stx1 and stx2 [19,87.5%]). The eaeA and hlyA gene were found in 11 (45.8%) of the 24 strains. Saa gene was present only one strains (4.2%). Toxin typing using reverse passive latex agglutination test showed the same result in VT 1. But it showed different result in VT 2. In antimicrobial susceptibility test, all isolates were sensitive to amikacin, amoxicillin/clavulanic acid, ciprofloxacin and colistin. Eighteen strains (75.0%) of 24 isolates showed the multi-resistant patterns with over 3 drugs. PFGE was performed after the genomic DNA of twenty four isolates was digested with Xba I. the 24 isolates showed 7 (A~G) PFGE type.

• BMB Reports
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• 제30권2호
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• pp.144-149
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• 1997

An expression vector was constructed containing the gene encoding diphtheria toxin A (DTA) which was placed after a T7 promoter. Cytoplasmic expression of the DTA gene resulted in the formation of an insoluble inclusion body. The inclusion body was collected after the complete lysis of the cell, and subsequent washing with 0.1% Triton X-100 released 16~30% of DTA protein from the inclusion body along with other contaminating proteins. The released DTA protein was purified by dialysis. The remaining pellet was dissolved in 8 M urea containing 5% ${\beta}-mercaptoethanol$, and the denatured DTA was renatured by the dilution-dialysis method. The total yield was 35%, and about 5 mg DTA was obtained from 1 L culture. The DTA protein has a free sulfhydryl group exposed to the protein surface, and was shown to have a tendency to dimerize through disulfide formation in the absence of ${\beta}-mercaptoethanol$. The utility of the sulfhydryl group was tested for the construction of recombinant toxins.

• 한국축산식품학회지
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• 제26권3호
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• pp.394-401
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• 2006

Forty Clostridium perfringens isolates were obtained from twelve animal products, following the examination of eighty six beef, pork, broiler chicken and salami meat products, and eleven milk powder products. There were 21 isolates from salami stored at $25^{\circ}C$, 3 isolates from pork, 4 isolates from beef, 9 isolates from broiler chicken, and 3 isolates from milk powder. Only the cpa gene encoding a toxin among the 5 toxin genes tested (cpa, cpb, etx, iap, and cpe) was detected in all forty isolates, suggesting contamination with C. perfringens type A. DNA fingerprinting analysis using PCR of the tRNA intergenic spacer (tDNA-PCR) and the 16S-23S internal transcribed spacer (ITS-PCR), and randomly amplified polymorphic DNA (RAPD) analysis were attempted to differentiate the isolates. RAPD analysis was the most discriminating method among the three PCR analyses. Isolates from the same products tended to show similar RAPD patterns. Antimicrobial susceptibility tests showed that some isolates from broiler chickens had the same antibiogram with multiple resistance to streptomycin, colistin, and ciprofloxacin. Antibiograms were similar between isolates from the same livestock products, but differed considerably between the products.

• 한국식품위생안전성학회지
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• 제35권3호
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• pp.219-224
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• 2020

본 연구는 2018년 10월에서 2019년 9월까지 부산지역 유통 판매되고 있는 872건의 다양한 식품을 대상으로 B. cereus 오염도를 분석하였다. 총 872건 중 78건(8.9%)에서 B. cereus 검출되었으며 식품별 B. cereus 검출률은 김치·절임·조림·젓갈류에서 23.9%, 조미식품 19.4%, 바로 섭취가능한 농산물 10.5%, 조리식품 7.3%, 즉석섭취식품 5.6%, 신선편의식품 5.0%, 즉석조리식품 0.0% 순으로 분포하였으며 통계적으로 유의한 차이가 있었다. B. cereus 오염 수준은 불검출에서 최대 20,000 CFU/g로 평균 48 CFU/g였으며, 식품별 B.cereus 오염도에는 차이가 없었다. 78건의 검체에서 분리된 113주의 B. cereus의 독소 유전자 확인 시험을 수행하였다. 식품에서 분리된 113주를 대상으로 B. cereus 독소유전자 확인 결과 최소 1종류에서 최대 5종류의 독소 유전자가 검출되었으며 총 18개 profile로 분류되었다. 장독소 5종(Cytk-nheA-entFM-bceT-hblC)을 모두 보유한 경우가 34주(30.1%)로 가장 많았다.

• 한국식품위생안전성학회지
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• 제36권6호
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• pp.520-527
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• 2021

본 연구에서는 보육시설 실내공기에서 분리된 식중독 균주의 독소 유전자 분포와 항생제 내성을 분석하여 보육시설 실내공기에 의한 식중독 발생을 사전 예방하고 식중독 발생 시 적절한 치료를 위한 기초자료를 제공하고자 하였다. 어린이집 실내공기에서 분리된 Staphylococcus aureus 16주, Bacillus cereus 37주를 실험대상으로 하였다. S. aureus와 B. cereus 독소 유전자는 PCR 방법으로 검출하였다. 항생제 감수성 실험은 Clinical and Laboratory Standard Institute의 디스크 확산법에 따라 실험하였다. S. aureus 16 균주 중 11 균주(68.6%)에서 seg와 sei 독소 유전자가 검출되었다. B. cereus 37 균주 모두에서 nheA와 nheB 독소 유전자가 검출되었다. B. cereus 독소 유전자 패턴은 총 12개로 나타났으며 nheA-nheB-nheC 독소 유전자가 가장 중요한 패턴으로 나타났다. S. aureus 16 균주의 항생제 감수성실험 결과 ampicillin과 penicillin 항생제에 93.8%, 87.5% 내성을 나타내었으나 methicillin resistance Staphylococcus aureus와 vacomycin resistance Staphylococcus aureus는 검출되지 않았다. B. cereus 37 균주의 항생제 감수성 실험 결과 ampicillin과 penicillin 항생제에 100% 내성을 나타냈었다. 이러한 결과를 종합하여 볼 때 보육시설 실내공기에 오염된 S. aureus와 B. cereus에 의한 식중독을 발생을 예방하기 위하여 주기적인 환기와 공기 질 관리가 필요한 것으로 판단되었다.

• 생명과학회지
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• 제10권4호
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• pp.380-387
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• 2000

Recently Vibrio sp. are the most frequently isolated microorganism, which causing food poisoning. We investigated the detection of toxin genes and effect of chitosan to toxin genes with PCR. Thirty strains of Vibrio sp. were isolated from sea water and sea products through biological and biochemical tests. Out of 30 strains, 8 were identified as V. parahemolyticus, 7 as V. mimcus, 6 as V. damsela, 5 as V. vulnificus, 4 as V. alginoyticus. In detection of ctx, tdh, and t고 as food poisoning-causing toxin genes, ctx from 7 strains, trh from 4 strains and tdh from 6 strains were detected. Among toxin genes detected strains, we selected V. vulnificus-2 V. damsela-1 and V. parahemolyticus-7. As adding chitosan solution to PCR product of 3 strains, the amplified DNA bands were not detected over 450$\mu\textrm{g}$/$m\ell$ concentrantion of chitosan. Over the result, chitosan is thought to influence the detection of toxin gene.

• 생명과학회지
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• 제26권2호
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• pp.265-274
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• 2016

Toxin-antitoxin (TA) system은 박테리아와 고세균에서 진화적으로 보존되어 흔히 발견되는 유전적 모듈이다. 기본적으로 이 시스템은 세포 내 toxin과 그들의 억제자로 작용하는 antitoxin으로 구성되어있으며, 현재 총 다섯가지 유형으로 구분된다. 공통적으로 toxin은 스트레스 조건에서 활성화됨으로써 세포 내 다양한 과정을 억제하는 활성을 가지는데 이는 결과적으로 세포 사멸 혹은 가역적인 생장 저해를 일으킨다. Toxin의 이러한 효과들은 유전자 발현의 조절, 성장 조절, programmed cell arrest, programmed cell death, persister cell의 형성, 박테리오파지 방어기작, 가동성 유전인자의 안정화, 플라스미드 유지 기작 등 다양한 생리학적 역할을 나타낸다. 그러므로 TA system은 일반적인 스트레스 반응모듈로서 여겨진다. 하지만 이를 역이용한다면 TA system으로부터 toxin을 활성화 시키는 인자를 개발하여 새로운 항균 물질로 이용할 수 있다. 그뿐만 아니라 TA system은 toxin의 세포 사멸 효과를 이용하여 원하는 타겟 유전자가 존재하는 세포만 선택적으로 살아남도록 하는 효율적인 클로닝 전략에 이용될 수 있다. 또한, toxin의 서열 특이적 리보핵산 가수분해효소 활성을 이용하여 타겟 단백질 이외의 단백질 합성을 막아 효과적인 단일 단백질 대량 생산을 위해서도 이용할 수 있다. 더 나아가 일부 TA system의 toxin은 진핵 세포에서도 세포 독성을 나타내기 때문에 암세포, 바이러스 감염 세포에서 toxin의 발현을 유도하여 세포사멸을 일으킴으로써 인간의 질병 치료로 이어질 수 있다.

• 한국미생물·생명공학회지
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• 제31권4호
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• pp.334-341
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• 2003

2001년 경북지역 콜레라 유행시 설사환자에서 V. cholerae O1 El Tor형 90주를 분리하였다. 분리균을 대상으로 ctxA, tcpA, hlyA gene에 대한 multiplex-PCR 시험에서는 분리균 모두에서 302, 223, 471 bps크기의 DNA 증폭산물 band를 확인하였다. ctxA 및 ctxB gene 부위의 유전자 염기서열 비교에서 분리균과 GenBank에 수록된 균간의 유형의 차이를 보였다. PFGE typing 실험에서는 분리균 모두에서 동일한 amplicon 단편을 나타내었다. 또한, 2002년 8월부터 10월까지 콜레라균 서식가능성 규명을 위해 경북 동해안 일대에서 plankton 시료채취 및 V. cholerae O1 및 V. cholerae O139의 rfb 및 CT gene 검출을 위한 multiplex-PCR 확인실험에서는 ctxA gene의 DNA band는 일부 검출되었으나, V. cholerae O1 및 V. cholerae O139는 분리되지 않았다. 험에서 ctxA gene DNA band는 검출되었으나, V. cholerae O1 및 V. cholerae O1는 분리되지 않았다.

• Journal of Microbiology
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• 제34권2호
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• pp.151-157
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• 1996

By PCR amplification using the sequence of the previously cloned shiga-like toxin II DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequence s included tow open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparison of the nucleotide and predicted amino acid sequences of newly cloned gene (slt-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophabe DNA of E. coli 933 derived from a patient with hemorrhagic colities. In addition, the sequence homology of SLT-II with SLT-II variant form bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E, coli hostvector, system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxycity for Vero cells than that form original strain.