Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is u...Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.展开更多
AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aerugi...AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.展开更多
Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118...Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.展开更多
目的观察EW S-FLI1介导的白喉毒素A链基因(d iphtheria toxin A cha in gene,DTA)在尤文肉瘤裸鼠移植瘤模型体内特异表达情况。方法采用尾静脉注射给药,将含有EW S-FLI1特异结合序列ACCGGAAGT和白喉毒素A链基因的真核表达载体pS2-DTA导...目的观察EW S-FLI1介导的白喉毒素A链基因(d iphtheria toxin A cha in gene,DTA)在尤文肉瘤裸鼠移植瘤模型体内特异表达情况。方法采用尾静脉注射给药,将含有EW S-FLI1特异结合序列ACCGGAAGT和白喉毒素A链基因的真核表达载体pS2-DTA导入尤文肉瘤裸鼠移植瘤模型体内,通过免疫组化及RT-PCR检测DTA的表达情况。结果DTA在肿瘤组织中有高表达,肝组织中有弱表达,心肌组织中表达结果为阴性。结论EW S-FLI1介导的DTA可在尤文肉瘤裸鼠移植瘤内特异表达。展开更多
基金The authors gratefully acknowledge supported by Bio-Manguinhos/FIOCRUZ,PAPESII/FIOCRUZ,FAPERJ,CNPq,CAPES,Programa de Nucleo de Excelencia(PRONEX/MCT/CNPq)We thank Fundacao Ataulfo de Paiva for the strain of BCG.
文摘Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.
文摘AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.
文摘Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.
文摘目的观察EW S-FLI1介导的白喉毒素A链基因(d iphtheria toxin A cha in gene,DTA)在尤文肉瘤裸鼠移植瘤模型体内特异表达情况。方法采用尾静脉注射给药,将含有EW S-FLI1特异结合序列ACCGGAAGT和白喉毒素A链基因的真核表达载体pS2-DTA导入尤文肉瘤裸鼠移植瘤模型体内,通过免疫组化及RT-PCR检测DTA的表达情况。结果DTA在肿瘤组织中有高表达,肝组织中有弱表达,心肌组织中表达结果为阴性。结论EW S-FLI1介导的DTA可在尤文肉瘤裸鼠移植瘤内特异表达。
