• Proceedings of the PSK Conference
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• 2003.04a
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• pp.226.2-226.2
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• 2003

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause of urinary tract infection. To produce a possible vaccine antigen against urinary tract infection, the fimH gene was genetically linked to the Itxa2b gene, which was then cloned into the pMAL -p2E expression vector. The chimaeric construction of pMALfimH/Itxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)

• Korean Journal of Veterinary Research
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• v.53 no.4
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• pp.199-205
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• 2013

Avian pathogenic Escherichia coli (APEC) is a causative agent for a number of extra intestinal diseases and account for significant losses to the poultry industry. Since protective immunity against APEC is largely directed to virulence antigens, we have individually expressed four different viulence antigens, papA, papG, IutA, and CS31A, using an attenuated Salmonella Typhimurium and a plasmid pBB244. Following oral immunization of mice with combination of two or four of these strains, serum IgG and mucosal IgA responses were elicited against each antigen represented in the mixture. The antigen-specific mucosal IgA responses were significantly higher in the group of mice immunized with the heat-labile Escherichia coli enterotoxin B subunit (LTB) strain than those in the group of mice immunized without the LTB strain. While, there was no significant difference between these two groups in antigen-specific serum IgG responses. The results showed that LTB could act as mucosal immune adjuvant. To assess the nature of immunity, the distribution of antigen-specific IgG isotypes was analyzed. All groups promoted Th1-type immunity as determined by the IgG2a/IgG1 ratio. Thus, our findings provided evidence that immunization with a combination of several vaccine strains is one of the strategies of developing effective vaccines against APEC.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.617-618
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• 2005

• Biomolecules & Therapeutics
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• v.13 no.2
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• pp.101-106
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• 2005

The generation of secretory IgA antibodies (Abs) for specific immune protection of mucosal surfaces depends on stimulation of the mucosal immune system, but this is not effectively achieved by parenteral or even oral administration of most soluble antigens. Thus, to produce a possible vaccine antigen against urinary tract infections, the uropathogenic E. coli (UPEC) adhesin was genetically coupled to the heat-labile Escherichia coli enterotoxin A2B (ltxa2b) gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ltxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. The chimeric protein was then purified by applying the affinity chromatography. The purified chimeric protein was confirmed by SDS-PAGE and westem blotting using antibodies to the maltose binding protein (MBP) or the heat labile E. coli subunit B (LTXB), plus the N-terminal amino acid sequence was analyzedd. The orderly-assembled chimeric protein was confirmed by a modified $G_{M1}$-ganglioside ELISA using antibodies to adhesin. The results indicate that the purified chimeric protein was an Adhesin/LTXA2B protein containing UPEC adhesin and the $G_{M1}$-ganglioside binding activity of LTXB. thisstudy also demonstrate that peroral administration of this chimeric immunogen in mice elicited high level of secretory IgA (sIgA) and serum IgG Abs to the UPEC adhesin. The results suggest that the genetically linked LTXA2B acts as a useful mucosal adjuvant, and that adhesin/LTXA2A chimeric protein might be a potential antigen for oral immunization against UPEC.

• Journal of Microbiology
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• v.43 no.4
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• pp.354-360
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• 2005

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately $1.9\%$ of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.490-497
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• 2004

Although the E. coli heat-labile enterotoxin B subunit (LTB) is known to be a potent mucosal adjuvant towards co-administrated unrelated antigens and immunoregulator in T-helper 1-type-mediated autoimmune diseases, a more efficient and useful LTB is still required for prospective vaccine adjuvants. To determine whether a novel chimeric LTB subunit would produce an enhanced mucosal adjuvant activity and immune response, a number of LTB subunits were genetically fused with chimeric proteins using the epitope genes of the envelope glycoprotein E2 (gp51-54) from the classical swine fever virus (CSFV). It was found that the total serum immunoglobulin (Ig) levels of BALB/c mice orally immunized with chimeric proteins containing an N-terminal linked LTB subunit (LE1, LE2, and LE3) were higher than those of mice immunized with LTB, E2 epitope, and chimeric proteins that contained a C-terminal linked LTB subunit. In particular, immunization with LE1 markedly increased both the total serum Ig and fecal IgA level compared to immunization with LTB or the E2 epitope. Accordingly, the current results demonstrated that the LTB subunit in a chimeric protein exhibited a strong mucosal adjuvant effect as a carrier molecule, while the chimeric protein containing the LTB subunit stimulated the mucosal immune system by mediating the induction of antigen-specific serum Ig and mucosal IgA. Consequently, an LE1-mediated mucosal response may contribute to the development of effective antidiarrhea vaccine adjuvants.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.709-717
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• 2017

Mucosal tissues are the initial site through which most pathogens invade. As such, vaccines and adjuvants that modulate mucosal immune functions have emerged as important agents for disease prevention. Herein, we investigated the immunomodulatory mechanisms of the B subunit of Escherichia coli heat-labile enterotoxin type IIa ($LT-IIa-B_5$), a potent non-toxic mucosal adjuvant. Alternations in gene expression in response to $LT-IIa-B_5$ were identified using a genome-wide transcriptional microarray that focused on dendritic cells (DC), a type of cell that broadly orchestrates adaptive and innate immune responses. We found that $LT-IIa-B_5$ enhanced the homing capacity of DC into the lymph nodes and selectively regulated transcription of pro-inflammatory cytokines, chemokines, and cytokine receptors. These data are consistent with a model in which directional activation and differentiation of immune cells by $LT-IIa-B_5$ serve as a critical mechanism whereby this potent adjuvant amplifies mucosal immunity to co-administered antigens.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.04a
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• pp.81-81
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• 2005

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.135-135
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• 2005