• 한국동물위생학회지
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• 제19권2호
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• pp.139-153
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• 1996

Porcine E coli infection is a disease caused by Enterotoxin produced by Enterotoxigenic Escherichia coli(ETEC). Enteric colibacillosis has become an economically important disease in pigs as a result of increasing intensification of farrowing management. The present study undertaken to obtain the antibiotic sensitivity and distribution of serogroups and pili producibility test of ETEC from E. coli isolates in Chonnam. The results obtained were as follows. 1. A total of 71 isolates identified as E, coli employing IMViC system from rectal specimens of 54 piglets with diarrhea. 2. In antibiotic sensitivity test, isolates showed high sensitivity to AN, CM, Fox, GM, but resistance to EM, NA TC. 3. The distribution of 25 Isolates of serogroups were 0141:K85(11.3%), 08:K87(8.5%), 064:K (5.6%), 0138:K8l (4.2%), 0139 :K82(2.8%), 0157:K88ac(1.4%) and 0149:K9l (1.4%). 4. MRHA of guinea pig erythrocytes was detected in 8 out of 25OK serotypes and 9 out of 46 unidentified serotypes. MRHA titers of serotypes showed from 64 to 128 in 0141: K85, 2 in 0138:K8l and no titers in 0139:K82. 5. The production of heat labile enterotoxin of ETEC was detect 39 out of 52 isolates showed $\beta$-hemolysin, 7 out of 52 isolates showed ${\gamma}$-hemolysin and 6 out of 52 isolates showed ${\gamma}$-hemolysin by $GM_1$ganglioside ELISA. The distribution of LT toxin were in 12 isolate showed $\beta$-hemolysin, 2 isolates showed ${\alpha}$-hemolysin and 3 isolates showed ${\gamma}$-hemolysin in 25 OK serotypes.

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.268-274
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• 2009

Porphyromonas gingivalis, the gram-negative anaerobic oral bacterium, initiates periodontal disease by binding to saliva-coated oral surface. The cholera toxin B subunit (CTB) genetically linked to FimA1 (1-200 aa) or FimA2 (201-337 aa) of the P. gingivalis fimbrial antigen were introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation method. The integration of CTB-FimA1 or CTB-FimA2 fusion genes were confirmed in the chromosome of transformed leaves by genomic DNA PCR amplification method. Synthesis and assembly of the CTB-FimA fusion proteins into oligomeric structures with pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding activities of CTB-FimA fusion proteins to intestinal epithelial cell membrane receptors were confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA showed that the expression levels of the CTB-FimA1 or CTB-FimA2 fusion proteins were 0.0019, 0.002% of the total soluble protein in transgenic tuber tissues, respectively The synthesis of CTB-FimA monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using edible plants for the production of enterocyte targeted fimbrial antigens that could elicit mucosal immune responses.

• Journal of Microbiology and Biotechnology
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• 제19권5호
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• pp.502-510
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• 2009

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmci reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked inmmunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active aligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

• Archives of Pharmacal Research
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• 제23권5호
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• pp.525-530
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• 2000

mST3GaIV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GaIV (CMP-NeuAc: lactosylceramide $\alpha$2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GaIV in mice, we investigated the spatial and temporal expression of mST3GaIV mRNA during the mouse embryogenesis [embryonic (E) days; 19, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from 19 and E11 were positive for mST3GaIV mRNA. On E13, mST3GaIV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3GaIV which was a quite similar to that of E13. In this stage, mST3GaIV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GaIV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.

• 생명과학회지
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• 제20권4호
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• pp.467-473
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• 2010

갱글리오시드는 포유동물 세포막의 중요한 구성요소로서 세포와 세포 혹은 세포와 단백질간의 상호작용을 포함한 다양한 면역학적 역할을 수행하고 있다. 이 연구는 NIH-미니돼지의 간과 심장을 인간에게 이식할려고 할 때 예측되어지는 거부 반응과 관련된 구성성분들 중 시알산을 함유하고 있는 스핑고당지질인 갱글리오시드에 대해 조사하였다. 얇은막크로마토그래피와 면역조직화학적분석을 실시한 결과 NIH-미니돼지의 간은 갱글리오시드의 발현이 심장보다 높게 나타났다. 갱글리오시드 GD3, GD1a, GD1b, GT1b는 간과 심장의 두 기관에서 발견되었다. 그러나 GQ1b는 간에서만 발견되었고 심장에서는 검출되지 않았다. 이러한 결과는 갱글리오시드의 발현양상은 간과 심장에서 조직특이적이라는 것을 의미한다. 한편, GM3를 포함한 다른 갱글리오 시리즈인 갱글리오시드들은 NIH-미니돼지의 간과 심장에서 검출되어지지 않았다. 이와 같은 연구결과로부터 갱글리오시드는 미니돼지의 장기중 특히, 간과 심장의 이종장기이식과 관련된 면역거부반응에서 어떤 역할을 수행하고 있다고 여겨진다.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.335-342
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• 2008

Purpose: Cytolethal distending toxin (CDT) considered as a key factor of localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis is composed of five open reading frames (ORFs). Among of them, the individual role of CdtA and CdtC is not clear; several reports presents that CDT is an AB2 toxin and they enters the host cell via clathrin-coated pits or through the interaction with GM3 ganglioside. So, CdtA, CdtC, or both seem to be required for the delivery of the CdtB protein into the host cell. Moreover, recombinant CDT was suggested as good vaccine material and antibody against CDT can be used for neutralization or for a detection kit. Materials and Methods: We constructed the pET28a-cdtC plasmid from Aggregatibacter actinomycetemcomitans Y4 by genomic DNA PCR and expressed in BL21 (DE3) Escherichia coli system. We obtained the antibody against the recombinant CdtC in mice system. Using the anti-CdtC antibody, we test the native CdtC detection by ELISA and Western Blotting and confirm the expression time of native CdtC protein during the growth phase of A. actinomycetemcomitans. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and generated the anti CdtC antibody against recombinant CdtC subunit expressed in E. coli system. Our anti CdtC antibody can be interacting with recombinant CdtC and native CDT in ELISA and Western system. Also, CDT holotoxin existed at 24h but not at 48h meaning that CDT holotoxin was assembled at specific time during the bacterial growth. Conclusion: In conclusion, we thought that our anti CdtC antibody could be used mucosal adjuvant or detection kit development, because it could interact with native CDT holotoxin.

• Journal of Periodontal and Implant Science
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• 제38권sup2호
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• pp.317-324
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis. Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease.