• Korean Journal of Microbiology
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• v.40 no.4
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• pp.348-354
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• 2004

The expression and antibacterial. activity of recombinant human lactoferrin (hLf) was studied from meth­ylotrophic yeast Pichia pastoris. The gene encoding hLf, isolated from human breast cDNA library, was subcloned into the expression vector, pPIC3.5K under the control of AOX1 promoter. The gene was integrated into the host chromosome and was identified by Southern blotting. The expression of the integrated gene was investigated by RT-PCR, Northern blotting, SDS-PAGE and Western blotting. Discrete band corresponding to hLf was detected from the SDS-PAGE, which was confirmed by Western blotting. The expression was also confirmed by RT-PCR and Northern blotting. The antibacterial activity of the recombinant hLf (rhLf) was investigated using Staphy­lococcus aureus ATCC 6538P and Micrococcus flavus ATCC 10240 as test organisms. The rhLf showed strong antibacterial activities against the bacteria. Furthermore, many Gram-negative animal pathogens such as E.coli ATCC8739, 25922, and Salmonella typhimurium 114 and 115, Pseudomonas fluorescens ID 963 I, P. aeruginosa KCCM 11802, and Gram-positive bacteria Bacillus mesentericus were also inhibited in their growth by the rhLf.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1047-1054
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• 2013

Witches' broom of lime is a disease caused by Candidatus Phytoplasma aurantifolia, which represents the most significant global threat to the production of lime trees (Citrus aurantifolia). Conventional disease management strategies have shown little success, and new approaches based on genetic engineering need to be considered. The expression of recombinant antibodies and fragments thereof in plant cells is a powerful approach that can be used to suppress plant pathogens. We have developed a single-chain variable fragment antibody (scFvIMP6) against the immunodominant membrane protein (IMP) of witches' broom phytoplasma and expressed it in different plant cell compartments. We isolated scFvIMP6 from a naïve scFv phage display library and expressed it in bacteria to demonstrate its binding activity against both recombinant IMP and intact phytoplasma cells. The expression of scFvIMP6 in plants was evaluated by transferring the scFvIMP6 cDNA to plant expression vectors featuring constitutive or phloem specific promoters in cassettes with or without secretion signals, therefore causing the protein to accumulate either in the cytosol or apoplast. All constructs were transiently expressed in Nicotiana benthamiana by agroinfiltration, and antibodies of the anticipated size were detected by immunoblotting. Plant-derived scFvIMP6 was purified by affinity chromatography, and specific binding to recombinant IMP was demonstrated by enzyme-linked immunosorbent assay. Our results indicate that scFvIMP6 binds with high activity and can be used for the detection of Ca. Phytoplasma aurantifolia and is also a suitable candidate for stable expression in lime trees to suppress witches' broom of lime.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1173-1181
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• 2016

Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wild-type SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpL-specific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPV-OmpL-vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpL-vaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection.

• Korean Journal of Ichthyology
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• v.19 no.2
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• pp.83-87
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• 2007

Edwardsiella tarda, a gram (-) pathogen causing edwardsiellosis in farmed fish, was transformed via electroporation with a plasmid expression vector driving the PhiX174 E-lysis gene under the transcriptional control by lambda PR regulatory sequence. The persistent maintenance of the plasmid vector in recombinant E. tarda was found in numerous subculture procedures over up to 6 months without any adverse effect on the original copy number of plasmids. Comparative examination based on semi-quantitative RT-PCR analysis on transcriptional efficiency of E-lysis gene between recombinant E. coli and E. tarda indicated that promoter strength and induction capacity of bacterial ghosts would be retarded in E. tarda as compared to the E. coli. However, the completeness of induction for bacterial ghosts in E. tarda was the same with E. coli, in which at least 99.99% of induction rate was possible and further the viability of recombinant bacteria was completely eliminated by a post-induction procedure including washing and freeze drying lyophilization.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.10a
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• pp.17-17
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• 2010

Rhizobacteria are a diverse group of free-living soil bacteria that live in plant rhizosphere and colonized the root system. Plant growth-promoting rhizobacteria (PGPR) possessing ACC deaminase (ACCD) can reduce ACC and ethylene in plant tissue and mediated the growth of plants under various stresses including salt stress. ACCD decrease ethylene levels in plant tissue that produce high levels of ethylene in tissue via elevated levels of ACC under salt stress. We selected strains of Pseudomonas sp. possessing ACCD activity for their ability to promote plant growth under salt stress from soil sample collected at Byeonsan, Jeonbuk, South Korea. The Pseudomonas strains possessing ACCD increased the rate of the seedling and growth of chinese cabbage seeds under salt stress. We cloned ACCD gene from P.fluorescens and expressed recombinant protein in Escherichia coli. The active form of recombinant ACCD converted ACC to a-ketobutyrate. The in vivo treatment of recombinant ACCD itself increase the rate of the seedling and growth of Chinese cabbage seeds under salt stress. The polyclonal P.fluorescens anti-ACCD antibody specifically reacted with ACCD originated from Pseudomonas. This indicates that the antibody might act as an important indicator for ACCD driven from Pseudomonas exhibiting plant growth-promoting activity. This study will be useful for identification of newly isolated PGPR containing ACCD and exploioting the ACCD activity from PGPR against various biotic and abiotic stresses.

• The Korean Journal of Ecology
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• v.18 no.1
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• pp.109-120
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• 1995

As the genetically modified microorganisms (GMMs) and their recombinant plasmid DNAs could be released into natural environments, their stabilities and impacts to indigenous microorganisls have become very importhant research subjects concerning with environmental and ecological aspects. In this study, the genetically modified E. coli CU103 and its recombinant pCU103 plasmid DNA, in which pcbCD genes involving in degradation of biphenyl and 4-chlorobiphenyl were cloned, were studied for their survival and stability in several different waters established under laboratory conditions. E. coli CU103 and its host E. coli XL1-Blue survived longer in sterile distilled water (SDW) and filtered autoclaved river water (FAW) than in filtered river water (FW). A lot of extracellular DNAs were released from E. coli CU103 by lytic action of phages in FW and the released DNAs were degraded by DNase dissolved in the water. Such effects of the factors in FW on stability of the recombinant pCU103 plasmid were also observed in the results of gel electrophoresis, quantitative analysis with bisbenzimide, and transformation assay. Therefore, the recombinant plasmids of pCU103 were found to be readily liberated from the genetically modified E. coli CU103 into waters by normal metabolic processes and lysis of cells. And the plasmid DNAs were quite stable in waters, but their stabilities could be affected by physicoKDICical and biological factors in non-sterile natural waters.

• IMMUNE NETWORK
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• v.22 no.3
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• pp.26.1-26.18
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• 2022

IL-22, a pleiotropic cytokine, is known to have a profound effect on the regeneration of damaged intestinal barriers. The tissue-protective properties of IL-22 are expected to be potentially exploited in the attenuation and treatment of colitis. However, because of the disease-promoting role of IL-22 in chronic inflammation, a comprehensive evaluation is required to translate IL-22 into the clinical domain. Here, we present the effective production of soluble human IL-22 in bacteria to prove whether recombinant IL-22 has the ability to ameliorate colitis and inflammation. IL-22 was expressed in the form of a biologically active monomer and non-functional oligomers. Monomeric IL-22 (mIL-22) was highly purified through a series of 3 separate chromatographic methods and an enzymatic reaction. We reveal that the resulting mIL-22 is correctly folded and is able to phosphorylate STAT3 in HT-29 cells. Subsequently, we demonstrate that mIL-22 enables the attenuation of dextran sodium sulfate-induced acute colitis in mice, as well as the suppression of pro-inflammatory cytokine production. Collectively, our results suggest that the recombinant mIL-22 is suitable to study the biological roles of endogenous IL-22 in immune responses and can be developed as a biological agent associated with inflammatory disorders.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.59-62
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• 1999

The recombinant bacteria strain DPD2540, containing a fabA::luxCDABE fusion, was used to detect the toxicity of various chemicals in this study. Membrane damaging agents such as phenol, ethanol, and cerulenin induced a rapid bioluminescent response from this strain. Other toxic agents, such as DNA-damaging or oxidative-damaging chemicals, showed a delayed bioluminescent response in which the maximum peak appeared over 150 min after induction. This strain was also tested for measurement of toxicity in field samples such as wastewater and river water effluents.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.4
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• pp.567-586
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• 2001

Gastrointestinal tract of ruminants as well as monogastric animals are colonised by a variety of microorganisms including bacteria, fungi and protozoa. Gastrointestinal ecosystem, especially the rumen is emerging as an important source for enrichment and natural selection of microbes adapted to specific conditions. It represents a virtually untapped source of novel products (e.g. enzymes, antibiotics, bacteriocins, detoxificants and aromatic compounds) for industrial and therapeutic applications. Several gastrointestinal bacteria and fungi implicated in detoxification of anti-nutritional factors (ANFs) can be modified and manipulated into promising system for detoxifying feed stuffs and enhancing fibre fermentation both naturally by adaptation or through genetic engineering techniques. Intestinal lactobacilli, bifidobacteria and butyrivibrios are being thoroughly investigated and widely recommended as probiotics. Restriction endonucleases and native plasmids, as stable vectors and efficient DNA delivery systems of ruminal and intestinal bacteria, are increasingly recognised as promising tools for genetic manipulation and development of industrially useful recombinant microbes. Enzymes can improve the nutrient availability from feed stuffs, lower feed costs and reduce release of wastes into the environment. Characterization of genes encoding a variety of commercially important enzymes such as cellulases, xylanases, $\beta$-glucanases, pectinases, amylases and phytases will foster the development of more efficacious and viable enzyme supplements and enzyme expression systems for enhancing livestock production.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.117-123
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• 2010

BT toxin is produced by a soil bacterium Bacillus thuringiensis and has long been used as a biological insecticide without any competition. Recently, Photorhabdus, a symbiotic bacterium from entomopathogenic nematodes, family Heterorhabditae, has been researched and discussed as alternatives to B. thuringiensis. Photorhabdus, which lives in the gut of entomopathogenic nematodes, is a highly virulent pathogen of a wide range of insect larvae. When an insect is infected by the nematodes, the bacteria are released into the cadaver, and produce a number of insecticidal toxins. The biological role of the different Photorhabdus toxins in the infection process is still unclear. Photorhabdus toxin complex (Tc) is highly secreted gut-active toxin and has been characterized as a potent three-component (A, B and C) insecticidal protein complex. These components are necessary for full oral activity against insect larvae. The Photorhabdus PirAB binary toxins exhibit a potent injectable activity for Galleria mellonella larvae, and have oral toxicity against mosquitoes and caterpillar pest Plutella xylostella. Other toxin, 'makes caterpillars floppy' (Mcf) showed injectable activity on caterpillars. Recombinant Mcf triggers apoptosis in both insect hemocytes and the midgut epithelium and carries a BH3 domain. In this review, the relationship between the Photorhabdus and the nematode is discussed and recent important insecticidal toxins from Photorhabdus are described.