• Korean Journal of Veterinary Research
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• v.38 no.3
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• pp.553-558
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• 1998

Using the previous established multiplex PCR (mPCR), the presence of six kinds of staphylococcal superantigen (SAg) genes was investigated for nineteen Staphylococcus aureus isolates from the commercialized meat sources. As a result, only one isolate from pork among 19 S aureus isolates (5.3%) was confirmed as a potential SAg producer and harbored sec gene. The results in this study suggest that meat may not be major contagion of staphylococcal food poisoning (SFP) in Korea and that staphylococcal enterotoxin type C may be associated with the disease. Also, the mPCR method in this study can be a useful genotypic method which can overcome the typical disadvantages of conventional antibody-based methods due to antigenic homology, and furthur survey on food-borne S aureus isolates can provide the important epidemiological data for SFP in Korea.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1323-1329
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• 2021

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.1
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• pp.101-108
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• 1998

We investigated the effect of ${\alpha}-subunit$ of the stimulatory GTP-binding protein ($G{\alpha}_s$) gene mutation on the expression of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) gene in GH3 cells and in growth hormone (GH)-secreting adenomas of acromegalic patients. In the presence of cyclohexicmide, forskolin and isobutylmethylxanthine, cholera toxin, and GH-releasing hormone (GHRH) decreased rat TRH-R (rTRH-R) gene expression by about 39%, 43.7%, and 46.7%, respectively. Transient expression of a vector expressing mutant-type $G{\alpha}_s$ decreased the rTRH-R gene expression by about 50% at 24 h of transfection, whereas a wild-type $G{\alpha}_s$ expression vector did not. The transcript of human TRH-R (hTRH-R) gene was detected in 6 of 8 (75%) tumors. Three of them (50%) showed the paradoxical GH response to TRH and the other three patients did not show the response. The relative expression of hTRH-R mRNA in the tumors from patients with the paradoxical response of GH to TRH did not differ from that in the tumors from patients without the paradoxical response. Direct PCR sequencing of $G{\alpha}_s$ gene disclosed a mutant allele and a normal allele only at codon 201 in 4 of 8 tumors. The paradoxical response to TRH was observed in 2 of 4 patients without the mutation, and 2 of 4 patients with the mutation. The hTRH-R gene expression of pituitaty adenomsa did not differ between the tumors without the mutation and those with mutation. The present study suggests that the expression of TRH-R gene is not likely to be a main determinant for the paradoxical response of GH to TRH, and that $G{\alpha}_s$ mutation may suppress the gene expression of TRH-R in GH-secreting adenoma. However, a certain predisposing factor(s) may play an important role in determining the expression of TRH-R.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.305-311
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• 1997

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

• Horticultural Science & Technology
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• v.18 no.3
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• pp.342-347
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• 2000

The objectives of this study were to investigate the genetic and phenotypic features of male sterile transformants by pollen-specific expression of diphtheria toxin gene and to find out inheritance patterns of transgene to the next generation. When backcrossed (BC) progenies were tested for expression of kanamycin resistance ($Km^R$), 9 lines out of 13 lines, except 4 lines ($BC_{1}5-13,\;BC_{1}5-23,\;BC_{1}5-28,\;BC_{1}5-32$), showed the ratio of $Km^R$ to kanamycin sensitive ($Km^S$), from 1:30 to all $Km^S$. As a result, they were much lower than Mendelian segregation of a dominant gene. To determine whether male sterility is a heritable and stable trait, 5 male sterile plants ($BC_{1}5-13,\;BC_{1}5-14,\;BC_{1}5-23,\;BC_{1}5-32,\;BC_{1}5-33$ lines) which had different transgene copy numbers were backcrossed as female parents with pollens from wild type. To confirm the existence of the DTx-A gene in the genome of the progenies, PCR was conducted using specific primers of the DTx-A coding region. A PCR band of 428 bp was obtained from each generation, which is the predicted size of the DTx-A gene fragment. Trangenes were inherited to the next $BC_4T_0$ progenies and showed male sterility, however, based on the copy numbers of DTx-A gene male sterile plants did not show predicted ratio. When male sterile plants were backcrossed with fertile plants, fruit capsule sizes and seed settings were relatively reduced from those of selfing wild type plants. The fruit sizes and seed settings were reduced in proportion to the increase in the copy number of DTx-A gene.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.82-87
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• 2016

Enterotoxigenic Bacteroides fragilis (ETBF) produces enterotoxins known to be a virulence factor. Three isotypes of the B. fragilis toxin (BFT) gene have been identified: bft-1, bft-2, and bft-3. We investigated the presence of bft isotypes in clinical B. fragilis isolates and the antimicrobial resistance of BFT-negative and BFT-positive isolates. Overall, 537 B. fragilis isolates were collected from extraintestinal specimens over 8 years (2006~2013) from a university hospital in Korea. Samples were analyzed by multiplex PCR to identify the bft gene isotypes. Additionally, the antimicrobial susceptibility of 107 B. fragilis isolates (74 BFT-negative and 33 BFT-positive) was examined by the CLSI agar dilution method. PCR revealed a total bft gene detection rate of 30%, while 33% and 29% of blood and other extraintestinal isolates contained the gene, respectively. Among ETBF isolates, the most common isotype was bft-1 gene, followed by bft-2 and bft-3 (bft-1 77%, bft-2 14%, bft-3 9%). Resistance rates (%) for BFT-negative and positive isolates differed in response to various antimicrobial agents, with 3%, 5%, 1% and 38% of BFT-negative isolates and 3%, 6%, 3% an 42% of BFT-positive isolates being resistant to piperacillin-tazobactam, cefoxitin, imipenem, and clindamycin, respectively. Interestingly, neither BFT-negative nor positive isolates showed antimicrobial resistance to chloramphenicol and metronidazole. Overall, the proportion of ETBF from blood was similar to that of other extraintestinal sites and the bft-1 gene was the predominant isotype. Higher antimicrobial resistance rates were found in BFT-positive isolates than BFT-negative isolates, but these differences were not statistically significant.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.10a
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• pp.114-115
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• 2003

A Bacillus thuringiensis isolate, Bt 2385-1, which showed toxicity to lepidopteran, was isolated from Korean soil sample and characterized. PCR-RFLP showed that this isolate contains two novel cryl-type crystal protein genes. In this study, we designed cryl-type specific primer set (ATG1-F and N400-R) to clone the toxic domain of the all cryl-type genes. The two novel rlyl-type toxin genes in addition to crylJal gene were cloned and sequenced. (omitted)

• Journal of environmental and Sanitary engineering
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• v.13 no.2
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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.

• Journal of the Korea Institute of Military Science and Technology
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• v.14 no.4
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• pp.710-718
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• 2011

Edema factor(EF) is a portion of anthrax toxin which produces edema when combined with protective antigen. This paper describes about technique for cloning, expression, purification and activity test of EF. Using the E. coli expression system, we could make recombinant EF protein although it's origin is Bacillus anthracis. And also we could culture massively and purify highly pure protein. Finally we confirm a enzyme activity of purified EF to increase intracellular cAMP level. Through establishing this technique, it can be possible to research about EF in depth and apply to expression and purification of many other protein in biology.