• Journal of Microbiology and Biotechnology
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• 제25권4호
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• pp.526-536
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• 2015

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4⁺ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 춘계학술발표대회
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• pp.523-526
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• 2000

A novel cell surface display system was developed by employing the Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Poly-histidine (poly-His) peptides of 19, 32, 45, 84, and 162 amino acids (aa) could be successfully displayed by inserting them into the seventh exposed loop(L7) of OmpC. Recombinant cells displaying poly-His of 19, 32, 45, and 84 aa could absorb 18.9, 23.9, 26.1, and 32.0 ${\mu}mol$ of $Cd^{2+}$ per gram cell dry weight, respectively and therefore, would be useful as the biosorbents of heavy metals.

• 한국동물위생학회지
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• 제33권3호
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• pp.223-231
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• 2010

A total of 191 samples collected from the commercial swine farms located in Chungnam province were investigated by PCR to estimate the prevalence of Lawsonia (L.) intracellularis infection. In the group of the pigs with proliferative enteritis, 14 (93.3%) of 15 intestinal samples and 12 (80.0%) of 15 feces were positive in PCR. In contrast, a relatively low positive rate (18.0%, 29 of 161 samples) was determined in the group of normal healthy pigs. The group of pigs over 120 days showed the highest positive rates (26.8%, 15 of 56 samples). In the comparison of the sequences of 210bp for species specific fragments and 301bp for outer membrane protein, the isolates (L1. L2) showed almost 100% identity with the reference L. intracellularis (L08049, USA). For the sequences of partial 16s rDNA, the homologies among the 5 isolates (L1-L5) were 97.4% to 99.3%, and those of 5 sequences (L1-L5) versus 5 overseas reference strains of L. intracellularis ranged from 98.6% to 99.8%. In the comparison of the nucleotide sequences among 5 isolates and other species in Desulfovibrionales showed 82.4 to 99.5% identities. The 5 isolates shared relatively low identities (76.9% to 84.4%) with the species of alpha-proteobacteria. In phylogenetic analysis based on the 16s rDNA sequences, all of the 5 isolates (L1-L5) were located in the same branch with the strains of L. intracellularis that were previously isolated from the pigs in USA and China. Seven strains of Desulfovibrio sp. were clustered in the neighboring branches, whereas alpha and gamma Proteobacteria showed distant relationship with L. intracellularis strains. The present findings suggest that L. intracellularis infection is endemic in the swine farms in the regions, and that the domestic isolates maintained very limited genetic variation.

• 생명과학회지
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• 제18권2호
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• pp.241-248
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• 2008

축산물을 이용하여 생산된 특이항체는 세균성감염에 의한 설사병 치료와 충치예방에 효과가 있고, 특히 계란을 이용한 IgY (Immunoglobulin Yolk)는 비교적 산과 열에 안정하다는 실험결과가 보고되어[22] 있다. 식품분야에 특이항체를 식품소재로 산업화에 활용한 빈도는 아직 미미한 상태에 있으나, 최근 면역학, 단백질공학, 생명공학 등의 발전과 기술 향상으로 식품 소제로써의 활용성이 점차 높아질 것으로 기대된다. 본 연구에서는 H. pylori의 감염을 예방하고 치료보조제로 사용할 목적으로 포유동물을 통한 피동면역용 항체를 생산하고자 하였다. H. pylori로부터 항원을 분리하고, 분리된 항원에 대한 항체생산 및 H. pylori의 응집정도를 알아보았다. H. pylori로부터 분리된 주요 항원 단백질은 WC, OMP, crude urease, LPS 각각 12개, 7개, 3개, 1개 종류의 band를 확인할 수 있었다. 분리된 항원의 IgG 항체 생성은 동일한 항원농도인 $20{\mu}g/100{\mu}l$에서 각각 WC (L) $77.9{\pm}6.4{\mu}g/ml$, OMP $84.9{\pm}6.4{\mu}g/ml$, crude urease $123.8{\pm}2.9{\mu}g/ml$로 crude urease 항원이 가장 많은 것으로 나타났다. 그리고 IgA 항체 생성은 WC (L) $2.5{\pm}0.32{\mu}g/ml$, OMP $2.0{\pm}0.43{\mu}g/ml$, crude urease $1.3{\pm}0.25{\mu}g/ml$로 IgA 항체 생성은 WC (L) 항원이 가장 많은 것으로 나타났다 Western blotting을 통하여 항원 단백질의 면역원성 알아본 결과 WC 10종류, OMP 6종류, crude urease 3종류의 주요 항원성 물질을 확인할 수 있었다. 항체의 H. pylori에 대한 응집정도를 나타내는 응집가는 anti-WC (H), anti-WC (L), anti-OMP, anti-crude urease 항혈청에서 각각 $2^5,\;2^5,\;2^6\;and\;2^7$으로 나타났으며, 상대적으로 anti-crude urease 항혈청이 가장 높은 응집가를 나타내었다. 각 항원에 의해 생성된 항혈청의 urease활성 억제에 대한 흡광도(OD=550 nm)는 WC (H) $0.14{\pm}0.01$, WC (L) $0.16{\pm}0.01$, OMP $0.18{\pm}0.03$, Urease $0.18{\pm}0.04$로 대조구 $0.26{\pm}0.02$와 비교할 때 유의적인 억제효과가 있는 것으로 나타났다. 이상의 결과를 종합해 볼 때 H. pylori 항원의 분리와 분리된 항원의 항체 생성능, H. pylori의 응집가, urease 활성억제측면에서 WC 및 crude urease항원 모두 높은 항체 역가를 나타내었다.

• Journal of Applied Biological Chemistry
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• 제58권3호
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• pp.227-232
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• 2015

A microarray study has been employed to understand changes of gene expression in E. coli KD43162 resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefazolin, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, fosfomycin, and trimethoprim-sulfamethoxazole except for amikacin using disk diffusion assay. Using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses, 36 kDa of outer membrane proteins (OMPs) was found to be deleted in the multidrug resistant E. coli KD 43162. Microarray analysis was used to determine up- and down-regulated genes in relation to multidrug resistant E. coli KD43162. Among the up-regulated genes, these genes were corresponded to express the proteins as penicillin-binding proteins (PBPs), tartronate semialdehyde reductase, ethanolamine utilization protein, shikimate kinase I, allantoinase, predicted SAM-dependent methyltransferase, L-glutamine: D-fructose-6-phosphate aminotransferase (GFAT), phospho-glucosamine mutase, predicted N-acetylmannosamine kinase, and predicted N-acetylmannosamine-6-P epimerase. Up-regulation of PBPs, one of primary target sites of antibiotics, might be responsible for the multidrug resistance in E. coli with increasing amount of target sites. Up-regulation of GFAT enzyme may be related to the up-regulation of PBPs because GFAT produces N-acetylglucosamine, a precursor of peptidoglycans. One of GFAT inhibitors, azaserine, showed a potent inhibition on the growth of E. coli KD43162. In conclusion, up-regulation of PBPs and GFATs with the loss of 36 kDa OMP refers the multidrug resistance in E. coli KD 43162.

• Applied Microscopy
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• 제24권3호
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• pp.55-66
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• 1994

This study was carried out to investigate the histological changes of sperm cells in testis, obtained from 100 of 3-year-old male rainbow trout (Oncorhynchus mykiss) collected and analysed from March in 1992 to February in 1993. Especially, the ultrastructural changes of spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa were examined to describe the reproductive cycles of this species. The results obtained in this study were as follows: The ultrastructures of the gonadotrophs largely parallel the cyclical changes in the testes. Each nest of cells belongs to one spermatogenetic stage, although nests at different stages can be found within the one lobule. At first keterochromatin is dispersed and then is condensed. In mature gamete, the nucleus is dense and homogeneous. The nuclear membrane appeared at the beginning of differentiation. In spermatogonia, Sertoli cells are located at the periphery of their cytoplasm. In the primary spermatocytes, the small mitochondria are abundant over the outer cytoplasm. During cell differentiation, the cytoplasm decreases and the nucleus increases. In spermatids, the protein masses moved towards the posterior part of the nucleus. In late spermatids, the two large mitochondria are located over the cytoplasm. In spermatozoa, two spheroidal mitochondria (about 145nm long) are situated in parallel between the nucleus and the axoneme. Spermatozoa mitochondria are assembled into an organized sheath surrounding the outer dense fibres and axoneme of the flagellar midpiece. The two centrioles are quite separate and the central pair and sheath complex of the flagellum is inserted into the base of the distal centriole.

• Journal of Veterinary Science
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• 제22권2호
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• pp.15.1-15.13
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• 2021

Background: Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. Objectives: The study aimed to determine the protective efficacy of attenuated Salmonella strain expressing highly conserved Brucella immunogens in goats. Methods: Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 10⁹ CFU/mL and challenge of all groups was done at 6 weeks after vaccination. Results: Among these vaccines inoculated at 5 × 10⁹ CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Real-time polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the non-vaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. Conclusions: These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• 한국축산식품학회지
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• 제33권5호
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• pp.610-616
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• 2013

본 연구에서는 영유아에게 치명적인 감염을 일으키는 C. sakazakii에 대하여 LAMP 검출법을 개발하였다. LAMP법에 의한 C. sakazakii의 검출율은 100%였으며 13개의 음성 지표군에 대해서는 모두 음성 반응을 보여 특이도가 매우 높은 것으로 판단되었다. 또한, HhaI과 NruI 두 개의 제한 효소를 LAMP product에 반응시킨 결과, 유전자의 특정 염기서열이 절단되는 것을 확인하였으며, 이를 통해 LAMP 검출법에 의해 증폭된 DNA가 C. sakazakii-specific ompA임을 확인하였다. 조제분유에 오염 된 C. sakazakii를 LAMP법으로 검출 시 검출한계는 $10^0$ CFU/mL이었으며 이는 기존의 PCR법이나 real-time PCR법에 비해 100-10,000배 높은 수준으로 민감도가 매우 높은 것으로 판단되었다. 이와 같이 높은 특이도와 민감도를 가진 LAMP 검출법은 C. sakazakii와 같은 급성 기회 감염균이나 병원성 미생물에 의한 식중독 발생시 현장에서 병원체를 간편하고 신속하게 검출할 수 있는 기술로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.