• The Korean Journal of Food And Nutrition
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• v.6 no.1
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• pp.37-46
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• 1993

Brain, heart, liver, lung, kidney and thymus etc. 12 organs were removed and homogenized from Dawley-Sprague rats after suffocation. After fractionation of the tissue cytosols, enzymatic activities of the key enzymes in metabolic inositol phosphates cycle, PLC, IPSK and Ins(1,4,5) P35-phosphatase, were measured respectively. Hybridoma monoclones producing anti-lP3K murine monoclonal antibodies were obtained by the fusion of SP2/Ag 0-14 and spleen cells of mouse immunized with purified 53KDa IPSK, screening and cloning procedures. 18 cloned hybridoma cells were obtained, background due to nonspecific binding was very low with 10 clones. These Abs were purified from ascitic fluids by using affi-gel 15, and determined subtype of Abs. When immunoreactivities for rat tissues IP3K were exercised by adding the mixed Abs of 19Gl and 19G2b, they showed an overall similarity with noncompetitive inhibition. Brain tissue has high sensitivity for anti-lP3K Ab, whereas heart tissue has very low activity. In kinetic parameters Km value was 1.58 mM and Vmx value was 5.41umol/min/ml, respectively Only one form of 40 KDa IPSK was detected in heart tissues, however rat brain contains at least three immunologically distinct IP3K (53, 51 and 40 KDa) in western blot analysis. Of them 53 KDa protein was major enzyme in enzymatic activity. Northern blot analysis with 32P-labeled CDNA probe which encodes 1.8 Kb IPSK gene was performed. These results suggest that IPSK are regulated at transcriptional level during rat tissue development.

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.197-202
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• 1993

The budding yeast S. cerevisiae contains 10-nm filament ring that lies just inside the plasma memhrane in the region of the mother-bud neck. It is possihle that CDC3. CDCIO, CDCII. CDCI2 genes encode the filaments. Recently it has been shown that the CDC3 and CDCI2 gene products arc localized to [he vicinity of the neck lilaments by immunolluorescence. However. the role of the lilament ring is not clear. In order to find out the role of filament ring. I have tried to clone the similar gene in S. pomhe to the CDC3 in S. cerevisiae. Genomic library was constructed by use of $\lambda$gtll expression vector and screened with CDC3 antibodies. From sequencing data, there were more than two introns in the newly cloned gene. There was 62% homology between the part of the predicted amino acid sequence of cloned gene and CDC3 amino acid sequence.

• Parasites, Hosts and Diseases
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• v.39 no.1
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• pp.57-66
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• 2001

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP 12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP 12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one you. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.1-7
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• 1997

Recently the production of transgenic animals to express foreign proteins in mammary glands has been a routine procedure. However, it still takes a considerable time and effort, and is faced with various technical challenges until the protein of interest is successfully made. Thus, a development of an a vitro model for mamm a ary-specific gene expression for recombinant genes was carried out in this study. To this end, bovine $\alpha$$_S1$ casein cDNA was inserted at the multiple cloning site of pMSG vector under the control of MMTV promoter. MCF$_7$ cells were tran sfected with pMSG $\alpha$$_S1$ CN by CaP0$_4$ precipitation. Transfectants were selected in HAT medium and induced with dexamethasone. The cells were analyzed with chicken anti-casein and FITC-labeled rabbit anti-chicken antibodies. The results showed that dexamethasone induced 30-40 fold increase in the MMTV- $\alpha$$_S1$ casein e expression. Therefore MCF$_7$ cells, which have multiple steroid receptors, along with pMSG vector can be used as an in vitro model for the study of mammary-specific gene expression.

• Korean Journal of Weed Science
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• v.15 no.3
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• pp.206-213
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• 1995

Glyphosate(N-[phosphonomethyl]glycine) applied to the assimilate-exporting leaves(i.e. third old leaf) of tomato(Lycopersicon esculentum Mil var. Moneymaker). Herbicide binding protein, QB protein(D1), has been immunoblotted using the antibodies raised against the hybrid-protein expressed by a part of spinach psb A gene cloned in frame with the 3'end of lac Z gene to allow expression of the ${\beta}$-galactosidase(EC 3.21.23) in Escherichia coli. Glyphosate has an effect on a turnover of D1 within photosystem II of thylakoid membrane. The dysfunction of D1 protein within light harvesting complex(LHC-II) seems to be a pleiotropic effect of glyphosate.

• Korean Journal of Veterinary Research
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• v.42 no.1
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• pp.101-108
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• 2002

The p27 coding region of the SIVmac239 isolate was amplified by PCR and cloned into an expression vector, pMAL-cri, which expressed high levels of the p27 protein from Escherichia coli. The purified p27 protein was used for detection of anti-SIV antibodies with the sera from 11 macaques and 21 marmosets by immunoblot assay of which one macaque was suspicious for the SIV infection. The optimum conditions of ELISA was studied by the check board system with the recombinant purified p27 protein. For the plate coating, 200ng/well of the purified p27 was satisfactory. The conjugate was diluted 1:1000. The sera from the 32 monkeys were negative for the anti-SIV by ELISA.

• Journal of Food Hygiene and Safety
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• v.32 no.1
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• pp.75-81
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• 2017

For people who have a food allergy the only way to manage the allergy is to avoid the food allergen. The mackerel is one of the major food allergens, but no immunoassay for the rapid and simple detection of mackerel has been reported. The objectives of this study are to develop and characterize monoclonal antibodies (MAbs) specific to mackerel using thermal stable-soluble proteins (TSSP) as an immunogen and to characterize the MAbs by indirect enzyme-linked immunosorbent assay (iELISA). The mice immunized with mackerel TSSP and showing high titer were used for cell fusion and cloning. The characterization of MAbs produced from hybridoma cells obtained was confirmed by indirect ELISA and western blot. Four MAbs were confirmed to be specific to mackerel without cross-reaction to other marine products and livestock products in the both methods. The iELISA and western blot based on the MAbs can sensitively detect 1% mackerel protein in other marine products. These results support that immunochemical methods based on the MAb produced could be used as rapid means to detect low levels of mackerel and to identify mackerel adulterated in food.

• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Radiation Oncology Journal
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• v.19 no.2
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• pp.171-180
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• 2001

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.