• Biomedical Science Letters
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• v.8 no.3
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.668-673
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• 2013

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen was the most enhanced by ALA dehydratase expression, uroporphyrin and coproporphyrin were the most enhanced by 1-hydroxymethylbilane synthase expression. The strain co-expressing coaA, hemA, maeB, and dctA produced heme of $0.49{\mu}mol/g$-DCW, which was twice as much from the strain without coaA expression. Further pathway gene amplifications for the porphyrin derivatives are discussed based on the results.

• KSBB Journal
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• v.11 no.4
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• pp.389-397
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• 1996

The growth of recombinant E. coli and formation of the by-products were investigated. Ammonium phosphate is known to affect the cell growth as well as the enzyme formation. When initial ammonium phosphate concentration was 0.5g/L, cell mass was 4.1g/L. By adding tryptone to the medium, acetic acid formation increased while lactic acid formation decreased. In cultivating recombinant E. coli, lactic acid and acetic acid turned out to be important by-products which affected cell yield and growth rate. Initial ammonium phosphate and tryptone concentration were optimized in our research and can be applied for other culture of recombinant E. coli.

• Biomolecules & Therapeutics
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• v.6 no.4
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• pp.400-405
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• 1998

The pharmacokinetics of CJ-50001 (recombinant human granulocyte-colony stimulating factor, developed by R&D center of Cheil Jedang Corp.) were investigated in rats and dogs. The serum concentrations of CJ-50001 were measured by a sandwich enzyme immunoassay. After single intravenous (iv) administration of Cf-50001 to rats at a dose of 5 $\mu$g/kg, the mean terminal half-life and area under the concentration-time curve (AUC) were 0.96 h and 124.497g . h/ml, respectively. After single subcutaneous (sc) administration at the same dose, maximum serum concentration was observed at about 2 hours after administration, and the mean terminal half-life, AUC and the bioavailability were 1.11 h,63.58$\mu$g . h/ml and 51.07%, respectively. In repeated dosing studies, CJ-50001 was administered iv and sc to rats at a daily dose of 5$\mu$g/kg for 7 days. The pharmacokinetic parameters, such as mean AUC and terminal half-life, were no significantly different from those of single administration. Following single iv and sc administration of CJ-50001 to dogs at a dose of 5 $\mu$g/kg, mean AUCs were much higher than those of rats, due to the decreased clearence (CL). After sc administration to dogs, maximum serum concentration was observed at 2~4 hours after administration and the bioavailability was 54.60%.

• BMB Reports
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• v.37 no.5
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• pp.556-564
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• 2004

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

• Journal of Microbiology and Biotechnology
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• v.19 no.6
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• pp.604-609
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• 2009

To investigate the potential use of microbial heme as an iron source, recombinant Escherichia coli coexpressing ALA synthase (HemA) as well as the NADP-dependent malic enzyme (MaeB) and dicarboxylic acid transporter (DctA) were cultured. The typical red pigment extracted from the recombinant E. coli after 38 h showed highest absorbance at 407 nm, and the amount of iron in 38.4 mg of microbial heme extract derived from 6-1 fermentation broth was 4.1 mg. To determine the commercial potential of the recombinant E.coli-synthesized iron-associated heme as an iron source, mice were fed the iron-free provender with the microbial heme extract. The average body weight reduction of mice fed non-iron provender was 2.3%, whereas no detectable weight loss was evident in mice fed microbial heme addition after 15 days. The heme content of the blood from microbial heme fed mice was 4.2 mg/ml whereas that of controls was 2.4 mg/ml, which implies that the microbial heme could be available for use as an animal iron source.

• Korean Journal of Veterinary Research
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• v.45 no.1
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• pp.63-70
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• 2005

African swine fever (ASF) is an infectious disease of domestic and wild pigs for which there is no vaccine in the world. A proper surveillance of viral activity and a timely response to ASF outbreaks depend upon the rapid diagnosis of ASF viral infection. Internationally prescribed enzyme-linked immunosorbent assay (ELISA) is a fast, sensitive test routinely used in the diagnosis of the ASF. However, inactivated whole ASF virus antigen used in this test is a tedious to prepare and has a risk of outside exposure of infectious virus by laboratory accident during the preparation. An ASF virus noninfectious recombinant antigen is a safe and easily produced alternative antigen for use in diagnostic assay. We have cloned the ORF O61R gene of the ASF virus to generate a recombinant baculovirus producing the p12 protein in insect cells under control of the polyhedrin promoter as non-fusion protein. When used in an indirect ELISA, the p12 antigen showed reactivity with all known ASF positive pig sera but not with negative pig sera. Our results indicated that the p12 protein would be one of alternative antigens for diagnosis of the ASF.

• Journal of Microbiology
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• v.37 no.4
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• pp.234-242
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• 1999

To obtain the recombinant circumsporozoite (CS) protein for the diagnosis of patients and seroepidemiology of Plasmodium vivax malaria which have been prevalent in northern part of Kyonggido, the CS protein gene was amplified by the polymerase chain reaction (PCR) from genomic DNA of the Korean vivax malaria patient. The gene consists of 1,123 nucleotides except signal peptide sequences and had an uninterrupted reading frame encoding a protein of 374 amino acids with a central region of 20 tandem repeats of the nonapeptide. The CS protein gene was expressed in Escherichia coli and purified, the molecular weight of recombinant CS protein was about 44 kDa (monomer) under denaturing purification and about 65 kDa (dimer) under native purification by SDS-PAGE. The purified recombinant CS protein which has antigenicity to malaria patients in Western blot analysis and Enzyme-linked immunosorbent assay, reacted only with the serum of P. vivax (PV210) infected malaria patients with no cross reaction to the P. falciparum malaria patient. The recombinant CS protein purified in this study will serve as a useful antigen to support the diagnosis of malaria patients and seroepidemiology.

• Parasites, Hosts and Diseases
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• v.36 no.3
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• pp.183-190
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• 1998

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected ill view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. slnensis antigen iIi 28 kDa as a if-falactosidase fusion protein produced in EscherichiG coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies or a 30 base pair repeat and an additional 320 bases. The deduced amino acid seqiLence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis, C. sinensis adult worm RNA showed a positive reaction with the cloned gene Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed higtl specificity for diagnosis of clonorchiasis.

• Journal of Animal Science and Technology
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• v.64 no.3
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• pp.588-598
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• 2022

Despite vaccination, equine influenza virus (EIV) and equine herpesvirus (EHV) infections still cause highly contagious respiratory diseases in horses. Recently, concurrent vaccination with EIV and EHV was suggested as a new approach; however, there have been no reports of concurrent vaccination with recombinant canarypox EIV and inactivated EHV vaccines. In this study, we aimed to compare the EIV-specific immune responses induced by concurrent administrations of a recombinant canarypox EIV vaccine and an inactivated bivalent EHV vaccine with those induced by a single recombinant canarypox EIV vaccine in experimental horse and mouse models. Serum and peripheral blood mononuclear cells (PBMCs) were collected from immunized animals after vaccination. EIV-specific serum antibody levels, serum hemagglutinin inhibition (HI) titers, and interferon-gamma (IFN-γ) levels were measured by enzyme-linked immunosorbent assay, HI assay, and quantitative polymerase chain reaction, respectively. Concurrent EIV and EHV vaccine administration significantly increased IFN-γ production, without compromising humoral responses. Our data demonstrate that concurrent vaccination with EIV and EHV vaccines can enhance EIV-specific cellular responses in horses.