• 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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• 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.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• 제8권3호
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• pp.133-140
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• 2004

The purpose of the present study was to elucidate the possible involvement of the medial vestibular nucleus (MVN) and inferior vestibular nucleus (IVN) following acute hypotension in the vestibuloautonomic reflex through vestibulosolitary or vestibuloventrolateral projections. Acute hypotension-induced cFos expression was assessed in combination with retrograde cholera toxin B subunit (CTb) tract tracing. After injection of CTb into the solitary region, CTb-labeled neurons were located prominently around the lateral borders of the caudal MVN and medial border of the IVN. The superior vestibular nucleus also had a scattered distribution of CTb-labeled neurons. After injection of CTb toxin into the unilateral VLM, the distributions of CTb-labeled neurons in the MVN and IVN were similar to that observed after injection into the solitary region, although there were fewer CTb-labeled neurons. In the caudal MVN, about 38% and 13% of CTb-labeled neurons were double-labeled for cFos after injection of CTb into the solitary region and the VLM, respectively. In the IVN, 14% and 7% of CTb-labeled neurons were double-labeled for cFos after injection of CTb into the solitary region and the VLM, respectively. Therefore, the present study suggests that acute arterial hypotension may result in activation of vestibulosolitary pathways that mediate behavioral and visceral reflexes, and vestibuloventrolateral medullary pathways that indirectly mediate vestibulosympathetic responses.

• 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.

• 동의생리병리학회지
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• 제29권5호
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• pp.416-421
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• 2015

This study was performed to comparative investigate the distribution of primary sensory and motor neurons associated with Daereung(PC7) acupoints by using neural tracing technique. A total 16 SD rats were used in the present study. After anesthesia, the rats received microinjection of 6 ㎕ of cholera toxin B subunit(CTB) into the corresponding sites of the acupoints Daereung(PC7), 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 Daereung(PC7) acupoints were concluded as follows. Muscle meridian related Daereung(PC7) controlled by spinal segments of C5∼T1, C6∼T4, respectively.

• Journal of Yeungnam Medical Science
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• 제15권1호
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• pp.75-96
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• 1998

복강내장을 지배하는 감각신경세포체 및 신경섬유의 표지부위를 관찰하기 위하여 복강내장을 부위별(위, 십이지장, 공장, 회장, 맹장, 오름결장, 내림결장)로 나누어 2.5% WGA-HRP $30{\mu}l$와 0.5% CTB $20{\mu}l$를 장막과 근육층 사이의 4부위에 나누어 주입하였다. 그 후 48-96시간의 생존시간이 경과한 후 뇌줄기, 척수신경절과 미주신경절에서의 감각신경섬유와 신경세포체의 표지부위를 면역조직화학 염색법과 HRP 조직화학 기법으로 관찰한 결과는 다음과 같다. 1. WGA-HRP에 표지된 감각신경섬유는 위와 맹장에서만 관찰되었으며, CTB에 표지된 감각신경섬유는 복강내장의 모든 장기에서 관찰되었다. 2. 복강내장을 지배하는 감각신경섬유는 뇌줄기내 좌우 고립로핵의 교질부, 교질부의 등쪽내측부, 교차연결부, 내측부, 넷째뇌실벽, 맨아래구역의 앞쪽경계 및 중심관의 등쪽 정중선인 교차연결부에 국소적으로 강하게 표지되었다. 3. 척수신경절에서 위 (stomach)에 분포하는 감각신경세포체는 좌우 관계없이 $T_2$에서 $L_1$까지 여러 신경절에 표지되었으며 이 중 좌우 $T_{8-9}$부위에 가장 많이 표지되었다. 4. 십이지장에서의 척수신경절에 표지된 감각 신경세포체는 좌우 $T_6-L_2$부위에 표지되었으나 다른 장기에 비하여 표지된 감각신경세포체의 수는 적었다. 5. 공장에서의 척수신경절에 표지된 감각신경세포체는 좌우 $T_6-L_2$부위에 표지되었다. 가장 많이 표지된 부위는 좌측 $T_{12}$ 부위였으며, 우측은 $T_{13}$ 부위에 표지되었다. 6. 회장에서의 척수신경절에 표지된 감각신경세포체는 좌우 $T_6-L_2$부위였다. 가장 많이 표지된 부위는 좌측에서 $T_{11}$부위였고, 우측에서 $L_1$부위였다. 7. 맹장에서의 척수신경절에 표지된 감각신경 세포체는 좌측은 $T_7-L_2$부위였으며 우측은 $T_6-L_1$부위였다. 가장 많이 표지된 부위는 좌측은 $T_{11}$이었으며, 우측은 $T_{11-12}$에 표지되었다. 8. 오름결장에서 척수신경절에 표지된 감각신경세포체는 좌측은 $T_7-L_2$부위에 표지되었고 우측은 $T_9-L_4$부위에 표지되어 좌우측 표지부위의 차이를 보였다. 가장 많이 표지된 부위는 좌측은 $T_9$이었으며, 우측은 $T_{11}$에 표지되었다. 9. 내림결장에서 척수신경절에 표지된 감각신경 세포체는 좌측은 $T_9-L_2$부위에 표지되었고 우측은 $T_6-L_2$부위에 표지되었다. 가장 많이 표지된 부위는 좌측은 $T_{13}$이었으며, 우측은 $L_1$에 표지되었다. 10. 복강내장을 지배하는 좌우 미주신경절에 표지된 감각신경세포체는 위에서 가장 많이 표지되었으며 위를 제외한 나머지 장기에서는 표지된 감각신경세포체의 수는 위에 표지된 수보다 적었다. 이상의 결과로 흰쥐의 복강내장을 지배하는 감각신경섬유의 뇌줄기내 표지영역은 좌우 고립로 핵의 교질부, 교질부의 등쪽내측부, 교차연결부, 내측부, 넷째뇌실벽, 맨아래구역의 앞쪽경계 및 중심관의 등쪽 정중선인 교차연결부였으며, 감각신경세포체의 표지영역은 미주신경절과 척수신경절 $T_2-L_4$ 부위였음을 알 수 있었다. 위를 제외한 나머지 장기들에서는 $T_6-L_4$부위에 표지되었으나 소장에서 대장으로 갈수록 가장 많이 표지된 부위는 원위부 가슴신경절에서 근위부 허리신경절쪽으로 이동하는 경향을 보였다.