• Journal of Plant Biotechnology
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• v.36 no.3
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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.

• Korean Journal of Veterinary Service
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• v.19 no.2
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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 Microbiology and Biotechnology
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• v.19 no.5
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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.

• BMB Reports
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• v.38 no.6
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• pp.717-724
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• 2005

The nontoxic B subunit of cholera toxin (CTB) can significantly increase the ability of proteins to induce immunological tolerance after oral administration, when it was conjugated to various proteins. Recombinant CTB offers great potential for treatment of autoimmune disease. Here we firstly investigated the feasibility of silkworm baculovirus expression vector system for the cost-effective production of CTB under the control of a strong polyhedrin promoter. Higher expression was achieved via introducing the partial non-coding and coding sequences (ATAAAT and ATGCCGAAT) of polyhedrin to the 5' end of the native CTB gene, with the maximal accumulation being approximately 54.4 mg/L of hemolymph. The silkworm bioreactor produced this protein vaccine as the glycoslated pentameric form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB. Further studies revealed that mixing with silkworm-derived CTB increases the tolerogenic potential of insulin. In the nonconjugated form, an insulin : CTB ratio of 100 : 1 was optimal for the prominent reduction in pancreatic islet inflammation. The data presented here demonstrate that the silkworm bioreactor is an ideal production and delivery system for an oral protein vaccine designed to develop immunological tolerance against autoimmune diabetes and CTB functions as an effective mucosal adjuvant for oral tolerance induction.

• Journal of Life Science
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• v.20 no.4
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• pp.467-473
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• 2010

Gangliosides are a major component of the plasma membrane of mammalian cells, which are directly involved in a variety of immunological events, including cell-to cell or cell-to-protein interactions. In this study, we investigated whether gangliosides, sialic acid-containing glycosphingolipids, are related to rejection during the xenotransplantation of NIH-miniature pig livers and hearts to humans. Both high performance thin-layer chromatography and immunohistochemistry analyses revealed that the expression of gangliosides in the liver tissue of NIH-miniature pigs was higher than that in the heart. Gangliosides GD3, GD1a, GD1b, GT1b and GQ1b were observed in both the liver and heart, whereas GQ1b was detected only in the liver, indicating that the ganglioside expression profiles are tissue specific. Moreover, other ganglio-series gangliosides, including GM3, were not detected in the livers and hearts of NIH-miniature pigs. Taken together, these results suggest that gangliosides may play important roles in immune responses in clinical xenotransplants of pig livers and hearts.