• BMB Reports
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• 제38권6호
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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 Microbiology and Biotechnology
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• 제13권4호
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• pp.552-559
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• 2003

The FimH subunit of type 1-fimbriated Escherichiu coli (E. coli) has been determined as a major cause for urinary tract infections. Thus, to produce a possible vaccine antigen against urinary tract infections, the fimIH gene was genetically coupled to the ctxa2b gene and cloned into a pMAL-p2E expression vector. The chimeric construction of pMALfimH/ctxa2b was then transformed into E. coli K-12 TB1 and its nucleotide sequence was verified. A fusion protein, based on fusing adhesin to the cholera toxin subunit A2B (CTXA2B), was induced with 0.01 mM isopropyl-${\beta}-D-thiogalactoside$ (IPTG) for 4 h at $37^{\circ}C$ to yield a soluble fusion protein. The fusion protein was then purified by affinity chromatography. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting using antibodies to the maltose binding protein (MBP) or the cholera toxin subunit B (CTXB), plus the N-terminal amino acid sequence was also analyzed. The orderly-assembled fusion protein was confirmed by a modified $G_{Ml}-ganglioside$ ELISA, using antibodies to adhesin. The results indicated that the purified fusion protein was an adhesin/CTXA2B protein containing E. coli adhesin and the $G_{Ml}-ganglioside$ binding activity of CTXB. Accordingly, this adhesin/CTXA2B protein may be a potential antigen for oral immunization against uropathogenic E. coli.

• Journal of Microbiology and Biotechnology
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• 제10권1호
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• pp.56-62
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• 2000

The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Excherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderly assembled fusion protein was confirmed by modified Gm1-ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and $G_{m1}4-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process, Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.

• 한국미생물·생명공학회지
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• 제31권4호
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• pp.334-341
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• 2003

2001년 경북지역 콜레라 유행시 설사환자에서 V. cholerae O1 El Tor형 90주를 분리하였다. 분리균을 대상으로 ctxA, tcpA, hlyA gene에 대한 multiplex-PCR 시험에서는 분리균 모두에서 302, 223, 471 bps크기의 DNA 증폭산물 band를 확인하였다. ctxA 및 ctxB gene 부위의 유전자 염기서열 비교에서 분리균과 GenBank에 수록된 균간의 유형의 차이를 보였다. PFGE typing 실험에서는 분리균 모두에서 동일한 amplicon 단편을 나타내었다. 또한, 2002년 8월부터 10월까지 콜레라균 서식가능성 규명을 위해 경북 동해안 일대에서 plankton 시료채취 및 V. cholerae O1 및 V. cholerae O139의 rfb 및 CT gene 검출을 위한 multiplex-PCR 확인실험에서는 ctxA gene의 DNA band는 일부 검출되었으나, V. cholerae O1 및 V. cholerae O139는 분리되지 않았다. 험에서 ctxA gene DNA band는 검출되었으나, V. cholerae O1 및 V. cholerae O1는 분리되지 않았다.

• 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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• 제33권6호
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.

• Biomolecules & Therapeutics
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• 제12권3호
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• pp.179-184
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• 2004

The FimH subunit of type 1-fimbriated Escherichia coli has been determined as a major cause for urinary tract infections. In our previous study, the Adhesin/CTXA2B was expressed as soluble recombinant chimaeric protein derived from the uropathogenic Escherichia coli adhesin genetically coupled to cholera toxin A2B (CTXA2B) subunit in Escherichia coli. Since it is very important to optimize IPTG concentration and culture temperature to maximize cell growth and productivity, These optimal culture factors were determined to increase the productivity of the expressed Adhesin/CTXA2B chimaeric protein in Escherichia coli TB1 carrying pMALfimH/ctxa2b. Our data demonstrate that optimal concentration of IPTG for increased production of chimaeric protein was 0.5 mM. Additionally, culture time was 10 hours and temperature, 37${\circ}C$.

• 동의생리병리학회지
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• 제32권1호
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• pp.30-34
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• 2018

This research was practiced to comparative investigate the distribution of sensory and motor neuron linkaged with Yangji(TE4) by using neural-tracer technology. A total 16 S-D rats were used in the present research. After anesthesia, the rats received micro-injection of $6{\mu}{\ell}$ of cholera toxin B subunit(CTB) into the relation positions of the Yangji(TE4), in the human body for observing the distribution of the linkaged sensory neurons in dorsal root ganglia(DRGs) and motor neurons in the spinal cord(C3~T4) and sympathetic ganglia. 3 days after the micro injection, the rats were anesthetized and transcardially perfused saline and 4% paraformaldehyde, followed by routine section of the DRGs, sympathetic chain ganglia(SCGs) and spinal cord. Marked neurons and nerve fibers were detected by immunohistochemical method and observed by light microscope. The marked neurons were recorded and counted. From this study the distribution of primary sensory and motor neurons linkaged with Yangji(TE4) were concluded as follows. Yangji(TE4) dominated by spinal segments of C5~T1, C6~T4, individually.

• Korean Journal of Acupuncture
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• 제32권3호
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• pp.136-143
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• 2015

This study was performed to comparative investigate the distribution of primary sensory and motor neurons associated with Cheonji(PC1) acupoint by using neural tracing technique. A total 4 SD rats were used in the present study. After anesthesia, the rats received microinjection of $6{\mu}l$ of cholera toxin B subunit(CTB) into the corresponding sites of the acupoints Cheonji(PC1) in the human body for observing the distribution of the related primary sensory neurons in dorsal root ganglia(DRGs) and motor neurons in the spinal cord(C3~T4) and sympathetic ganglia. Three days after the microinjection, the rats were anesthetized and transcardially perfused saline and 4% paraformaldehyde, followed by routine section of the DRGs, sympathetic chain ganglia(SCGs) and spinal cord. Labeled neurons and nerve fibers were detected by immunohistochemical method and observed by light microscope equipped with a digital camera. The labeled neurons were recorded and counted. From this research, the distribution of primary sensory and motor neurons associated with Cheonji(PC1) acupoints were concluded as follows. Muscle meridian related Cheonji(PC1) are controlled by spinal segments of C5~T1, C6~T4, respectively.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.309.2-309.2
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• 2002

The FimH subunit of type l-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 coupled to the ctxa2b gene, which was then cloned into pMAL -p2E expression vector. The chimaeric construction of pMALfimH/ctxa2b was transformed into Escherichia coli TB1 and its N-terminal amino acid sequence was analyzed. (omitted)