• BMB Reports
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• v.37 no.5
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• pp.546-551
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• 2004

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.870-875
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• 2001

A DNA fragment, pCKB13, containing two genes encoding Coenzyme a transferase, was isolated from a genomic DNA library of S. marcescens KCTC 2172. The complete nucleotide sequence of the 2,081-bp BamHI fragment on pCKB13 was determined. Sequencing of the fragment led to the identification of two open reading frames showing high homology with two Coenzyme A (CoA) transferases, Acetoacetyl-CoA transferase (Acot) and Succinyl-CoA transferase (Scot), enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The enzyme activity of Coenzyme A transferase increased after introducing the multicopy of the cloned gene in E. coli. The recombinant protein, overexpressed by multicopy and induction with IPTG, was a polypeptide of 42 kDa, as confirmed by SDS-PAGE. The protein was purified to homogeneity through three sequential chromatographic procedures including ion-exchanged DEAE-sepharose, CM-sepharose, and Mono Q.

• Journal of Oral Medicine and Pain
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• v.24 no.2
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• pp.181-187
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• 1999

The most important progress in diagnostic sciences is the increased sensitivity and specificity in diagnostic procedures due to the development of newer micromethodologies and increasing availability of immunological and molecular biological reagents. The outcome of researches in this field has already provided DNA probes and antibodies which can be used for diagnosing various kinds of diseases including inherited ones. This development can be also applied to diagnose diseases in oral and maxillofacial regions. Technological advances have yielded highly sensitive test methodologies so that low analyte concentration and small sample volume are no longer limiting factors. Therefore, saliva can be useful test fluid for an array of analytes. Salivary constituents of diagnostic significance include steroid hormones, antibodies, drugs, and tumor markers. Of the proteins present in saliva, viral-specific immunoglobulins are of the greatest diagnostic interest. The development of conjugates and antigens by recombinant DNA technique and peptide synthesis is necessary for clinical application. Several kits developed for the purpose of blood testing should be modified to permit their application to saliva. The final practical outcome of researches in diagnostic sciences will be various diagnostic agents which can be used for detection of bacteria and viruses, screening and diagnosis of diseases, genetic screening for forensic individual identification. For these purposes, collaboration researches and development between institutions and companies are essential.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.154-159
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• 2006

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 35S promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using the Agrobacterium-mediated transformation method. The integration of the hlL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hlL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hlL-18 protein approximated $166{\mu}g/L$ in the suspension culture medium. Bioassay results from the induction of $interferon-{\gamma}$ from a KG-1 cell line indicated that the hlL-18 secreted into the suspension culture medium was bioactive.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.256-260
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• 1993

DNA subfragments, sopA, sopB and sopC which help to maintain the stability of an ori C plasmid, were derived from a mini-F plasmid DNA (EcoRI restriction fragment f5) after digestion with restriction endonuclease, and cloned in the vector plasmid pBR322. The recombinant plasmids obtained were introduced into E. coli KY7231 and E. coli CSR603 strains, and proteins specified by the mini-F fragments were analysed by SDS-PAGE. Two proteins encoded by the F fragments were detected, and their molecular weights were 41,000 and 37,000 daltons. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins had been overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of sopA and sopB proteins were 6.6 and 7.0, respectively.

• Journal of Microbiology
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• v.34 no.2
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• pp.151-157
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• 1996

By PCR amplification using the sequence of the previously cloned shiga-like toxin II DNA, a gene encoding it has been cloned from an isolate of healthy Korean native bovine feces, Escherichia coli KSC109. The nucleotide sequence s included tow open reading frames coding for 319 and 89 amino acids corresponding to A and B subunits, respectively. Comparison of the nucleotide and predicted amino acid sequences of newly cloned gene (slt-II) with those of others in the SLT-II family revealed completely identical homology with SLT-II cloned previously from bacteriophabe DNA of E. coli 933 derived from a patient with hemorrhagic colities. In addition, the sequence homology of SLT-II with SLT-II variant form bovine was more than 95% at both the nucleotide and protein levels. Overexpression of SLT-II recombinant gene by induction with IPTG using an E, coli hostvector, system was conducted and the correctly processed products with active mature form exhibited 1000-fold higher cytotoxycity for Vero cells than that form original strain.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.321-329
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• 2005

Ornithine decarboxylase (ODC) antizyme is a key regulatory protein in the control of cellular polyamines. We have isolated two distinct ODC antizyme cDNA clones (AZS and AZL) from a flounder (Paralichthys olivaceus) brain cDNA library. Their sequences revealed that both clones required translational frameshifting for expression. Taking + 1 frameshifting into account, AZS and AZL products were 221 and 218 amino acid residues long, respectively, and shared $83.3\%$ amino acid sequence identity. Comparison of the structure and nucleotide sequence of the antizyme genes showed that the genes were highly conserved in flounder, zebrafish, mouse, and human. A phylogenetic tree was constructed, based on the antizyme amino acid sequences from various species. The presence of the two types of antizyme mRNA species in brain, kidney, liver, and embryo was confirmed by using the reverse transcription­polymerase chain reaction (RT-PCR) and Northern blot analysis. Recombinant proteins of flounder ODC antizymes, containing His-Nus-S tag at the amino-terminus, were overexpressed as His-AZL and His-AZS fusion proteins in Escherichia coli BL21 (DE3) pLys by using the pET­44a(+) expression vector.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.228-234
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• 1991

- A synthetic gene coding for human interleukin-2 (IL-2) was constructed from the oligonucleotides synthesized by an automatic DNA synthesizer. The nucleotide sequence of the synthetic gene was chosen considering the preferred codons of E. coEi by not changing the amino acid sequence of IL-2 polypeptide. The synthetic gene was expressed in E. coli by placing the gene under the control of the $\lambda$ PL promoter. IL-2 was produced in the E. coli cytoplasm in the form of inclusion bodies. The recombinant IL-2 showed growth promoting activity on the IL-2 dependent cell line.

• Journal of Fisheries and Marine Sciences Education
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• v.28 no.4
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• pp.903-912
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• 2016

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.881-884
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• 1999

The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide-incorporation activity on an in vitro transcribed RNA containing the 3' end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.