Objective: To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit(LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified...Objective: To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit(LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L.Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Results:LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3.36-10.56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the prevention of mucosa-route evading pathogen.展开更多
Human papillomavirus (HPV), mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated c...Human papillomavirus (HPV), mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated cervical cancer.展开更多
Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-...Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.展开更多
基金Supported by the National Natural Science Foundation ofChina (No. 30070848)
文摘Objective: To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit(LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L.Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Results:LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3.36-10.56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the prevention of mucosa-route evading pathogen.
文摘Human papillomavirus (HPV), mainly types 16 and 18, are the most important initiating agents of cervical cancer. Prevention of high-risk HPV infections is a potentially effective approach to control HPV associated cervical cancer.
文摘Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.