• 한국임상수의학회지
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• 제27권6호
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• pp.704-712
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• 2010

Porcine circovirus-2 (PCV2) 는 돼지에서 여러 형태의 질병과 증후군의 발생과 관련이 되어있어 현재는 PCV-associated diseases (PCVAD)로 총괄적으로 분류된다. PCVAD에 의한 높은 경제적 손실 때문에 많은 양돈장들이 PCV2의 감염을 확인하기 위하여 혈청을 검사하고 있다. 하지만, 기존의 혈액채취법은 비용이 높고 많은 인력이 소요되므로 큰 규모의 병원체 검사에는 어려움이 있었다. 이에 본 연구에서는 혈액채취법을 이용한 돈군의 PCV2 검사법에 대한 대체방법으로 돈방 단위의 구강액채취법의 유용성을 실제 농장에서 평가하였다. 세 곳의 다른 양돈 농장들에서 각각 6개의 25두 규모의 돈방들을 선정하여 생후 3, 5, 8, 12, 16주에 돈방 마다 하나의 구강액과 5개의 혈청을 채취하였다. 모든 시료들은 real-time PCR을 이용하여 PCV2 DNA를 검사하였고 IgG 또는 IgA 간접형광항체 검사법및 세 가지의 ELISA 검사법 (blocking ELISA, indirect ELISA, and IgG/IgM sandwich ELISA)을 이용하여 PCV2에 대한 항체를 검사하였다. 구강액에서 PCV2 DNA는 8주까지는 간헐적으로 검출이 되다가 16주에는 모든 돈방에서 검출이 되었으며, 모체유래 PCV2 특이 IgG는 3주부터 검출이 되었고 모든 농장에서 5-8주까지 지속이 되었다. 16주에는 한 농장 (Site 1)의 모든 돈방에서 감염에 의한 IgG와 IgA가 검출되었다. 혈청에서의 PCV2 DNA와 PCV2 항체의 검출은 구강액에서의 검출과 높은 상관관계를 보였다. 따라서 구강액은 돈군의 PCV2 감염을 모니터링 하기 위해 혈청대신 사용할 수 있는 저비용, 고효율의 시료가 될 수 있을 것으로 사료된다.

• Archives of Pharmacal Research
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• 제29권11호
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• pp.1006-1017
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• 2006

A series of 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one, 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione and 2, 2'-dithiobis 3-(substituted-benylidene)-1, 3-dihydro-indole derivatives was investigated as inhibitor of $p60^{c-Src}$tyrosine kinase by performing receptor docking studies and inhibitory activity toward tyrosine phosphorylation. Some compounds were shown to be docked at the site, where the selective inhibitor PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl-amine] was embedded at the enzyme active site. Evaluation of all compounds for the interactions with the parameters of lowest binding energy levels, capability of hydrogen bond formations and superimposibility on enzyme active site by docking studies, it can be assumed that 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and thione derivatives have better interaction with enzyme active site then 2, 2'-dithiobis 3-(substituted-benzylidene)-1, 3-dihydro indole derivatives. The test results for the inhibitory activity against tyrosine kinase by Elisa method revealed that 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione derivatives have more activity then 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one derivatives.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

• 한국가축번식학회지
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• 제27권4호
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• pp.299-307
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• 2003

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

• 한국식물병리학회지
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• 제13권4호
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• pp.248-254
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• 1997

순무우 모자이크 바이러스 Ca 계통(TuMV-Ca)의 외피 단백질을 대장균 NM522 strain에서 발현시켰다. 발현된 바이러스 단백질은 한천젤 이중확산법, ELISA와 Western blotting을 이용하여 확인하였다. 외피단백질 발현 벡터(pGEX-Tu)의 구축은 IPTG induction site를 지니는 pGEX-KG에 TuMV-Ca 외피단백질 유전자를 결합하였다. 최적 단백질 발현 조건은 pGEX-Tu를 지니는 대장균을 액체 배지 1 ml당 $A_{595}$=0.1/ml의 농도로 접종한 후 2시간 뒤에 IPTG를 최종 농도를 1 mM로 조절하여 induction 시키는 경우였다. 합성된 목적 단백질은 발현 벡터의 특성상 GST (Glutathion S-Transferase) 단백질과 결합된 형태로 약 59 kDa의 단백질이었다. (uMV CP 33 kDa + GST 26 kDa.)

• Parasites, Hosts and Diseases
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• 제37권3호
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• pp.163-169
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• 1999

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophogoides pteronyssinus, were produce. Four monoclonal antibodies produced wee species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p2 in house dust. The sensitivity limit was 4ng/ml with rDer p2 and $8{\;}\mu\textrm{g}/ml$ with the d. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

• Journal of Periodontal and Implant Science
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• 제23권1호
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• pp.109-126
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• 1993

Refractory periodontitis manifest progressive attachment loss in a rapid and unrelenting manner regardless of the type or frequency of therapy applied. The purpose of this study was ta evaluate the relation between the level of cytokines in GCF and periodontopathic microflora with disease activity of refractory periodontitis. Selection of patients with refractory periodontitis (7 males, 3 females) were made by long term clinical observation including conventional clinical history and parameters. Teeth that showed pocket depth greater than 6mm were selected as sample teeth. Subjects were examined at baseline and after 3 months. Prior to baseline test, individual acrylic stent was fabricated. Reference grooves were made on each sample tooth site. Pocket depth and attachment loss were measured by Florida Probe. Gingival index was measured at 4 sites each sample teeth. Disease activity was defined as attachment loss of ${\ge}$ 2.1mm, as determined by sequential probing and tolerance method. The pattern and amount of alveolar bone resorption was observed with quantitative digital subtraction image processing radiography. Morphological analysis of subgingival bacteria was taken by phase contrast microscopy. Predominant cultivable bacterial distribution and frequency were compared between disease-active and disease-inactive site using immunofluorescence microscopy and selective microbial culturing. Levels of $interleukin-l{\beta}$, 2, 4, 6 and $TNF-{\alpha}$ in GCF and blood serum sample were quantified by ELISA. In active sites, P. intermedia was significantly increased to compare with inactive site. $IL-1{\beta}$, IL-2, IL-6 and $TNF-{\alpha}$ in GCF were increased in active sites and IL-2 in serum was increased in active patients significantly. Alveolar bone loss in active site was correlated with $IL-1{\beta}$, IL-2 in GCF. And loss of attachment in active site was correlated with IL-2 in GCF. These results demonstrate that IL-2 in serum, $IL-1{\beta}$, IL-2, IL-6 and $TNF-{\alpha}$ in GCF, P, intermedia might be used as possible predictors of disease activity in refractory periodontitis before it is clinically expressed as attachment loss and quantitative alveolar bone change.

• BMB Reports
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• 제31권1호
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• pp.58-63
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• 1998

In this study, a mouse monoclonal antibody (mAb) against gp41(584-618), the immunodominant epitope protein, was generated. For this purpose, BALB/c mice were immunized with double branched multiple antigenic peptides derived from the HIV-1 gp41(584-618) sequence, and antibody-secreting hybridoma were produced by fusion of mice splenocytes with SP2/0 myeloma cells. One clone producing an antigen specific mAb, termed KI-41(isotype IgG1) was identified, whose specific reactivity against gp41(584-618) could be confirmed by ELISA and Western blot analysis. Epitope mapping revealed the recognition site of the mAb KI-41 to be located around the sequence RILAVERYLKDQQLLG, which comprises the N-terminal region within the immunized gp41(584-618) peptied. Since this mAb recognizes this specific epitope within the HIV-1 gp41 without any cross-reactivity to other immunodominant regions in the HIV-2 gp35, KI-41 will provide some alternative possibilities in further applications such as the development of indirect or competitive ELISA for specific antibody detection in HIV-1 infection or for other basic researches regarding the role and function of HIV-1 gp41.

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.83-92
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• 2009

Individual immunochromatographic assays (ICG) for ochratoxin A (OTA) and zearalenone (ZEA) were optimized and used in the development of a one-step simultaneous immunochromatographic assay (OS-ICG) for the rapid multianalysis of two mycotoxins in corn samples. The nitrocellulose membrane of the OS-ICG was treated with OTA-bovine serum albumin (BSA), ZEA-ovalbumin (OVA), and anti-mouse IgG in the OTA test, ZEA test, and control zones, respectively. Monoclonal antibody-gold conjugates (OTA3 MAb-gold and ZEA2C5 MAb-gold) were sprayed onto the conjugate pad. The visual detection limits were 2.5 and 5 ng/ml for OTA and ZEA, respectively, and the results were obtained within 15 min after starting the analysis. An efficient, simple, and rapid extraction method using 30% MeOH/PBS was established and validated by analyzing the corn samples spiked with OTA/ZEA mixtures (0/0, 5/10, 10/20, and $20/30\;{\mu}g/kg$). The cut-off values of the OS-ICG for the spiked corn were 5 and $10\;{\mu}g/kg$ for OTA and ZEA, respectively. Natural corn samples were analyzed by OS-ICG, direct competitive enzyme-linked immunosorbent assay (DC-ELISA), and HPLC. Results of the OS-ICG were in good agreement with those obtained by DC-ELISA and HPLC. The developed OS-ICG offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site simultaneous determination of OTA and ZEA in cereals, food, and agricultural products in one analytical cycle.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.