• 한국동물위생학회지
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• 제35권3호
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• pp.175-182
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• 2012

Rotaviruses have been known to be a major etiological agent of gastroenteritis in both infants and young animals. Subsequently new rotaviruses, which were morphologically indistinguishable but antigenically and electrophoretically distinct with each other, were reported from several animals throughout world including Korea. These new rotaviruses were named as non-group A or group B or group C rotaviruses and so on. It has been very difficult to isolate and grow the non-group A rotaviruses in vitro, and this has greatly limited the characterizations of non-group A rotaviruses and serological studies. In this study, monoclonal antibodies (MAbs) against porcine non-group A rotavirus were produced and characterized. The VP6 gene of porcine group C rotavirus Korean isolate(#06-52-1) was cloned and expressed. For expression of VP6 gene, baculovirus expression system was applied. The VP6 gene and expressed protein in the recombinant virus were confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test and Western blot, respectively. The expressed VP6 was used for MAbs production. The MAbs produced in this study would be promising as diagnostic reagents for detection of group C rotavirus infection.

• Journal of Pharmaceutical Investigation
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• 제40권6호
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• pp.357-362
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• 2010

To enhance therapeutic effects of insulin-sensitizing adipokine, ADN gene and potent agonists, rosiglitazone for the $PPAR{\gamma}$, cationic liposomes as non-viral vectors were formulated. The particle size and zeta potential of drug loaded and unloaded cationic liposomes were investigated. The complex formation between cationic liposomes and negatively charged plasmid DNA was confirmed and the protection from DNase was observed. In vitro transfection was investigated in HepG2, HeLa, and HEK293 cells by mRNA expression of ADN. Encapsulation efficacy of rosiglitazone-loaded liposomes was determined by UV detection. Particle sizes of cationic liposomes were in the range of 110-170 nm and those of rosiglitazone-loaded cationic liposomes were in the range of 130-180 nm, respectively. Gel retardation of complexes indicated that the complex was formed at weight ratios of cationic lipid to plasmid DNA higher than 20:1. Both complexes protected plasmid DNA from DNase either drug free or drug loading. Encapsulation efficiency of rosiglitazone-loaded emulsion was increased by drug dose. The mRNA expression levels of ADN were dose-dependently increased in cells transfected with plasmid DNA. Therefore, cationic liposomes could be potential co-delivery system for drug and gene.

• The Plant Pathology Journal
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• 제26권4호
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• pp.328-339
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• 2010

A tobamovirus, Ribgrass mosaic virus (RMV), was identified newly from chinese cabbage (Brassica campestris L. pekinensis) in Korea. Virus disease incidence of RMV on chinese cabbage was 37.9% in alpine area on August in 1993. RMV induced the symptoms of necrotic ring spots, necrotic streak on midrib and malformation. RMV, Ca1 and Ca3 isolate, could infect 35 species out of 45 plants including Chenopodium amaranticolor. Physical properties of RMV Ca1 isolate were very stable as 10.8 over for dilution end point, $95^{\circ}C$ for temperature inactivation point and 18 weeks for longevity in vitro. RMV had the soil transmission rate of 75.0% for the chinese cabbages, 'Chunhawang' and 'Seoul' cultivars. The purified virions of RMV had the typical ultraviolet absorption spectrum of maximum at 260 nm and minimum at 247 nm. RMV of Ca1 isolate was related serologically with antisera of Tobacco mosaic virus (TMV)-Cym, TMV-O and Pepper mottle virus, but not related with antiserum of Odontoglossum ring spot virus. coat protein gene of RMV-Ca1, sized 473 nucleotides, encoded 158 amino acid residues. Nucleotide identity of RMV-Ca1 CP gene was 96.4% with RMV-Shanghai (GenBank accession No. of AF185272) from China and 96.0% with RMV-Impatiens (GenBank accession No. of AM040974) from Germany. Identity of amino acids between RMV-Ca1 and the two RMV isolates was 96.8%. Specific three primers were selected for rapid and easy genetic detection of RMV using Virion Captured (VC)/RT-PCR method.

• 한국작물학회지
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• 제65권4호
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• pp.496-507
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• 2020

본 연구는 사과 바이러스 ASGV, ASPV 및 ApMV를 각각 정밀하게 진단하고자 SYBR Green I 및 TaqMan probe, 두 종류의 다른 chemical dye를 사용하여 quantitative real-time PCR 검정법을 개발하고자 하였다. 1. 사과 바이러스 ASGV, ASPV 및 ApMV의 국내분리주를 바탕으로 하여 cloning 및 in vitro transcription을 수행해 10배 희석단위 표준시료를 제작하였다. 각 바이러스에 대한 SYBR Green I용 프라이머와 TaqMan probe용 프라이머 및 프로브를 디자인하였다. 2. 상기 제작된 프라이머와 프로브를 이용해 표준시료를 대상으로 real-time PCR을 수행하여 각 바이러스의 증폭곡선과 검량선을 구할 수 있었다. Real-time PCR 결과, SYBR Green I기반의 검정법은 TaqMan probe기반의 검정법 못지 않은 결과를 보여주었으며, 적은 비용에 대량 검정이 요구되는 곳에 효과적으로 응용될 수 있을 것이다. 3. 현장평가를 본 실험에서 개발된 TaqMan probe기반의 real-time PCR검정법과 기존의 RT-PCR검정법과 비교분석하였다. 그 결과 real-time PCR 검정법은 singleplex 및 multiplex RT-PCR보다 더 민감하고 정확한 결과를 내어 RT-PCR로 검출할 수 없는 농도까지 검정할 수 있음을 입증하였다. 4. 본 실험에서 개발한 real-time PCR검정법은 검역현장과 같은 대량의 검사가 요구되는 곳에서는 SYBR Green I 기반의 검정법을, 바이러스 연구분야에서는 세밀한 정량이 가능한 TaqMan probe 방식의 검정법이 활용될 것으로 기대한다.

• Investigative Magnetic Resonance Imaging
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• 제17권2호
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• pp.133-143
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• 2013

목적 : 직결장암 조직의 자기공명영상과 고주파 초음파검사를 시행한 후 종양의 탐지와 침윤 깊이에 대해 각각의 영상 소견과 병리 소견을 비교하여 진단적 정확도를 두 영상 기기 간 비교하고, 자기공명영상의 경우 종양의 침윤 깊이를 주변 정상 조직과 가장 명확히 보여주는 펄스 연쇄 (pulse sequence)에 대해 알아보았다. 대상 및 방법 : 직결장암으로 절제술을 시행한 45명의 환자에서 얻은 45예의 제거된 종양 조직을 수조에 넣고 생리 식염수에 담가서 고주파수 (5-17 MHz)의 선형 탐촉자를 이용하여 초음파영상을 얻었으며 8-channel 두경부 코일에 넣어 자기공명영상을 얻었다. 이 연구에 대하여 기관감사위원회의 공지에 입각한 동의는 면제되었다. 자기공명영상은 정- 및 탈위상 경사에코 T1 강조영상, 급속스핀에코 T2 강조영상 및 이의 지방억제 영상, fast imaging employing steady-state acquisition (FIESTA)와 이의 지방억제영상, 확산강조영상 등 일곱 가지 펄스 연쇄를 시행하였다. 각 조직의 자기공명영상과 초음파영상 소견을 각각 독립적으로 종양의 탐지와 침윤 깊이에 대하여 두 명의 영상의학과 의사가 합의 하에 평가하였고 각각의 영상 소견을 병리 조직 소견과 비교하여 두 영상 기기 간 진단적 정확도를 비교하였다. 자기공명영상의 일곱 가지 펄스 연쇄 중에 종양의 침윤 깊이를 주변 정상 조직과 구분하여 명확히 보여주는 펄스 연쇄에 대해 알아보았다. 결과 : 직결장암 조직의 종양 탐지와 침윤 깊이를 평가하는데 있어 자기공명영상과 초음파의 진단적 정확도는 각각 91.1%와 86.7%로 높게 나타났다. 조기 직결장암의 경우 초음파 검사의 정확도는 87.5%, 자기공명영상 검사의 정확도는 75.0%로 나왔다. 두 영상 기기 간에 통계적으로 유의한 차이는 없었다 (p > 0.05). MR의 펄스 연쇄 중에 종양의 침윤 깊이를 주변 정상 조직과 구분하여 명확하게 보여주는 영상은 직결장암 및 조기 직결장암 모두 급속 스핀에코 T2 강조영상이었다. 결론 : 자기공명영상과 초음파 검사는 직결장암 조직의 종양 탐지와 침윤 깊이를 평가하는데 높은 진단적 정확도를 가지고 있으며, 자기공명영상의 급속스핀에코 T2 강조영상이 직결장암 조직의 종양 침윤 깊이를 평가하는데 가장 우수하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권2호
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• pp.84-91
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• 2000

Mycopasma contamination of tissue culture cells easily evades detection and, thus, represents a continous therat to cell biologists. In case where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplacma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains ; however, cell associated mycoplasma are often protected from antibiotics at concentrations shown to be effective in vitro. Antibiotic concentrations high enough to be lethal to cell as sociated mycoplasmas frequently are also detrimentrations to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistanct even to high concentrations of the antibiotis applied. Hare, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes thens limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent, Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycopalsma while leaving the host cells unharmed. Upon successful mycoplasma eradicated, cultvation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibillity to the toxic agent. Cressation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chrosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.

• 대한본초학회지
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• 제25권3호
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• pp.65-71
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• 2010

Objectives：To develop and validate HPLC-PDA methods for simultaneous determination of seven constituents in Samchulkunbi-tang (SKT). Additionally, we investigated the cytotoxicity against BEAS-2B cell line and splenocytes of SKT. Methods：Reverse-phase chromatography using a Gemini $C_{18}$ column operating at $40^{\circ}C$, and photodiode array (PDA) detection at 230, 254 and 280 nm, were used for quantification of the three marker components of SKT. The mobile phase using a gradient flow consisted of two solvent systems. Solvent A was 1.0% (v/v) aqueous acetic acid and solvent B was acetonitrile with 1.0% (v/v) acetic acid. The cytotoxicity of SKT were measured by the CCK-8 assay method. Results：Calibration curves were acquired with $r^2$>0.9999, and the relative standard deviation (RSD) values (%) for intra- and inter-day precision were less than 6.0%. The recovery rate of each compound was in the range of 86.89-109.78%, with an RSD less than 4.0%. The contents of seven compounds in SKT were 1.39-6.84 mg/g. SKT had no cytotoxicity effect at 50-200 ${\mu}g$/mL concentrations. Conclusions：The established method will be helpful to improve quality control and in vitro efficacy study of SKT.

• The Plant Pathology Journal
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• 제19권5호
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• pp.260-265
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• 2003

Gummy stem blight caused by Didymella bryoniae occurs exclusively in cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. In this study, cultural conditions for the mass-production of pycnidiospore by Metal Halide (MH) lamp irradiation were maximized. The mycelia were cultured at $26^{\circ}C$ on PDA for 2 days under dark condition, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$. Results show that a great number of pycnidia were simultaneously formed. The pycnidiospores produced were then used as immunogen. Fusions of myeloma cell (v-653) with splenocytes from immunized mice were carried out. Two hybridoma cell lines that recognized the immunogen D. bryoniae were obtained. One monoclonal antibody (MAb), Dbl, recognized the supernatant while another MAb, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid of the two MAb, Dbl and Db15, the immunotypes of which were identified as IgG1 and IgG2b, respectively. Titers of MAb Dbl and MAb Db15 were measured and the absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with D. bryoniae but none reacted with other viral isolates, Cucumber mosaic virus and Cucumber green mottle mosaic virus. Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{-3}$/well by indirect ELISA. Characterization of the MAbs Dbl, Db15 antigen by heat and protease treatments, which suggests that the epitope recognized by these two MAbs was glycoprotein.

• 대한수의학회지
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• 제42권2호
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• pp.225-229
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• 2002

We report that mycoplasma organisms from lung tissues of slaughter pigs were identified to genes fragments with references use of nested-PCR technique(nPCR). Seven strains of mycoplasma species were isolated from 70 lung tissues. The organisms were detected by in vitro amplification of 16S rRNA and 23S rRNA genes. Nucleotide sequences of the spacer between 16S and 23S in the ribosomal RNA operons of mycoplasma were identified by the analysis of products from the nested PCR. Four common PCR primers, MhF1, MhF2 MhR1 and MhR2, were designed by analysis between these sequences by first amplified with F1, R1 and second with F2, R2, respectively. Specific amplification of the spacer region for reference strains of M. hyopneumoniae, M. hyorhinis, M. flocculare were confirmed by first round of PCR in which the traduced fragments of 690bp, 460bp, 630bp. But amplications of second round was changed to 240bp, 210bp, 230bp, respectively. Three different strains (M. hyopneumoniae:4, M. hyorhinis:2, M. flocculare:1) were detected by the nested-PCR technique. The results suggest that the detection of swine mycoplasma by n-PCR can be analyzed the nucleotide sequences between rRNA operons and homology study.

• 생명과학회지
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• 제16권5호
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• pp.729-733
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• 2006

크립토스포리디움의 활성 및 감염성 판정을 위해 Direct RT-PCR, 세포배양 후 RT-PCR 및 면역형광염색법을 비교한 결과는 다음과 같다. 1) 크립토스포리디움의 HSP70 gene에 대해 direct RT-PCR한 결과, 민감도가 매우 높아 저농도로 존재하는 환경시료에서의 크립토스포리디움 활성을 모니터링하는데 장점이 있을 것으로 보이나, 감염성의 판정은 알 수 없으며, 정량화가 안되는 단점이 있었다. 2) ${\beta}-tubulin$ gene에 대해 RT-nested PCR을 한 결과 크립 토스포리디움의 난포낭이 $1{\times}10^4$ 세포수정도 되어야 검출이 되는 것으로 나타나 HSP70 gene에 대한 RT-PCR결과와 비교할 때 10,000배 이상 민감도가 떨어지는 것으로 나타났다. 3) 세포배양 후 RT-PCR 또는 면역형광염색법을 이용할 경우에는 민감도가 direct RT-PCR보다 다소 떨어지는 단점이 있었으나 크립토스포리디움의 오염원이나 오염이 심한 지역의 감염성 조사에 적합할 것으로 나타났으나, 정량화가 필요한 경우에는 세포배양 후 면역형광염색법이 효과적일 것으로 나타났다.