• Korean Journal of Ecology and Environment
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• v.54 no.3
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• pp.190-198
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• 2021

Human Aichivirus (Aichivirus A; AiV-A) is a positive-sense single-strand RNA non-enveloped virus that has been detected worldwide in various water environments including sewage, river, surface, and ground over the past decade. To develop a method with excellent sensitivity and specificity for AiV-A diagnosis from water environments such as groundwater, a combination capable of reverse transcription (RT)-nested polymerase chain reaction (PCR) was developed based on existing reported and newly designed primers. A selective method was applied to evaluate domestic drinking groundwater samples. Thus, a procedure was devised to select and subsequently identify RT-nested PCR primer sets that can successfully detect and identify AiV-A from groundwater samples. The findings will contribute to developing a better monitoring system to detect AiV-A contamination in water environments such as groundwater.

• Microbiology and Biotechnology Letters
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• v.49 no.2
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• pp.148-156
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• 2021

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

• Journal of Ginseng Research
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• v.43 no.3
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• pp.482-487
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• 2019

Background: The mixed-cultivation of different Panax ginseng cultivars can cause adverse effects on stability of yield and quality. K-1 is a superior cultivar with good root shape and stronger disease resistance. DNA markers mined from functional genes are clearly desirable for K-1, as they may associate with major traits and can be used for marker-assisted selection to maintain the high quality of Korean ginseng. Methods: Five genes encoding pathogenesis-related (PR) proteins of P. ginseng were amplified and compared for polymorphism mining. Primary, secondary, and tertiary structures of PR5 protein were analyzed by ExPASy-ProtParam, PSSpred, and I-TASSER methods, respectively. A coding single nucleotide polymorphism (SNP)-based specific primer was designed for K-1 by introducing a destabilizing mismatch within the 3' end. Allele-specific polymerase chain reaction (PCR) and real-time allele-specific PCR assays were conducted for molecular discrimination of K-1 from other cultivars and landraces. Results: A coding SNP leading to the modification of amino acid residue from aspartic acid to asparagine was exploited in PR5 gene of K-1 cultivar. Bioinformatics analysis showed that the modification of amino acid residue changed the secondary and tertiary structures of the PR5 protein. Primer KSR was designed for specific discrimination of K-1 from other ginseng cultivars and landraces. The developed real-time allele-specific PCR assay enabled easier automation and accurate genotyping of K-1 from a large number of ginseng samples. Conclusion: The SNP marker and the developed real-time allele-specific PCR assay will be useful not only for marker-assisted selection of K-1 cultivar but also for quality control in breeding and seed programs of P. ginseng.

• Journal of Veterinary Science
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• v.22 no.4
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• pp.45.1-45.13
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• 2021

Background: Peste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in great losses in the world's agriculture industry every year. Single-domain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. Objectives: The purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. Methods: A VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. Results: The PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 10⁶ cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. Conclusions: The results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.

• Journal of Microbiology
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• v.45 no.6
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• pp.528-533
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• 2007

In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementary-determining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

• Food Science and Biotechnology
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• v.17 no.2
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• pp.357-361
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• 2008

One of the major allergenic proteins in common buckwheat (Fagopyrum elculentum) was found to be a BW10KD. In this work, allergenic BW10KD genomic DNAs from the native common buckwheat 'Pyeongchang' and Tartarian buckwheat 'Clfa47' were cloned by polymerase chain reaction (PCR), and their nucleotide sequences were determined. In addition, a novel PCR assay targeting the allergenic BW10KD gene was developed to detect and differentiate both buckwheat species in food. The nucleotide sequences of the BW10KD genomic DNA from 'Pyeongchang' and 'Clfa47' were 94% identical. Base differences in the nucleotide sequences of the BW10KD genes are probably useful as a molecular marker for species-specific identification. The 'Pyeongchang'-specific primer set 154PF/400PR and the 'Clfa47'-specific primer set 154DF/253DR generated 247 and 100 bp fragments in singleplex PCR, respectively. A duplex PCR assay with 2 species-specific primer sets simultaneously differentiated the 'Pyeongchang' and 'Clfa47' in a single reaction. The PCR assay also successfully allowed for the rapid detection of buckwheat ingredients in foods.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.9
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• pp.1241-1247
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• 2009

A large number of branded chicken products exist in Japan, and in some cases, the breed of chicken is an important factor used to attract consumer interest in the retail product. In order to establish a simple method for verifying such breed claims we applied the amplified fragment length polymorphism (AFLP) technique to nine chicken breeds (White Cornish, Red Cornish, White Plymouth Rock, New Hampshire, Rhode Island Red, Barred Plymouth Rock, Hinaidori, Tosajidori, Tsushimajidori) to search for molecular markers able to discriminate chicken breeds. Three breed-specific single nucleotide polymorphisms (SNP) were identified, one for each of Hinaidori, Tosajidori, or New Hampshire. A total of 219 individuals from the nine breeds were analyzed using a specific PCR test for each of these SNP. The PCR tests made it possible to discriminate between the breeds of chickens to identify products from these three breeds. This PCR method provides an efficient method for the routine analysis and verification of certified chicken products.

• Journal of Digital Convergence
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• v.10 no.8
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• pp.179-184
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• 2012

A car accident on a highway occurs a chain-reaction collision because of a vehicle's fast velocity. In order to prevent it, the accident vehicle should broadcast a safe message to its neighbors. If there are many neighbor nodes, a frame collision probability is high. To solve this, it was proposed for a system as a previous study to send a safe message without frame-collision using separating channels. However, the separation of multiple channels make feasibility low because of increasing hardware's development cost and complexity. In this paper, we proposes a TDMA-based MAC protocol using a single channel. As a result, we show the frame reception success rate of our protocol was almost the same as the previous protocol.

• Proceedings of the IEEK Conference
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• 2004.08c
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• pp.468-472
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• 2004

In this paper a VLSI design for the automatic magnetizing system has been presented. This is the design of a peripheral controller, which magnetizes CRTs and computers monitors and controls the automatic inspection system. We implemented a programmable peripheral interface(PPI) circuit of the control and protocol module for the magnetizer controller by using a O.8um CMOS SOG(Sea of Gate) technology of ETRI. Most of the PPI functions has been confirmed. In the conventional method, the propagation/ramp delay model was used to predict the delay of cells, but used to model on only a single cell. Later, a modified "apos;Linear delay predict model"apos; was suggested in the LODECAP(LOgic Design Capture) by adding some factors to the prior model. But this has not a full model on the delay chain. In this paper a new "apos;delay predict equationapos;" for the design of the timing control block in PPI system has been suggested. We have described the detail method on a design of delay chain block according to the extracted equation and applied this method to the timing control block design.

• Molecules and Cells
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• v.40 no.4
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• pp.307-313
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• 2017

Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.