Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines ...Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.展开更多
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 amp...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. Resuits： 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 preven- tion of mucosaroute evading pathogen.展开更多
In this study,the interaction of exopolysaccharides from Leuconostoc mesenteroides P35(EPS-LM)with Escherichia coli heat-labile enterotoxin B-pentamer(LTB)was investigated at different concentrations and temperatures ...In this study,the interaction of exopolysaccharides from Leuconostoc mesenteroides P35(EPS-LM)with Escherichia coli heat-labile enterotoxin B-pentamer(LTB)was investigated at different concentrations and temperatures by using surface plasmon resonance(SPR)and molecular docking approaches.FT-IR spectral analysis together with HPTLC analysis revealing that glucose is the only constitutive monosaccharide of EPS-LM suggests that its structure is composed of dextran withα-D(1→6)glycosidic linkages.SPR analysis revealed the high affinity of EPS-LM for immobilized LTB toxin(KA=(2.05±0.04)×106 mol.L−1 at 37°C).The binding process was spontaneous(ΔG<0),endothermic(ΔH>0),and entropy-driven(ΔS>0)with an increase of KA with temperature.This suggests that EPS-LM-LTB interaction is dominated by hydrophobic forces.The binding affinity of EPS-LM to LTB had negligible dependence on enthalpy(ΔH=0.084 kJ mol−1).Further,molecular docking results suggested the presence of some binding sites of EPS-LM on the LTB through hydrophobic forces(Lys,Asp,Arg,Glu)and also hydrogen bonding(Glu)in the hydrophobic core of LTB.Besides autodock studies,Schiffer-Edmundson helical wheel diagrams of LTB inα-helix domain suggested that LTB hydrophobic core is a highly effective region,which was able to form favorable non-polar interactions of the protein's binding surface(with amino acids residues such as Tyr,Leu,Ile)with EPS-LM.This study provided thus further insights into the interactions between EPS-LM and LTB,suggesting that EPS produced by some LAB,such as EPS produced by Ln.mesenteroides P35 strain are good candidates to inhibit E.coli toxin activity.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
Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to...Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to tumor site provides a promising option for reducing the systemic toxicity.Here,we constructed an iRGD peptide(H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH_(2))modified nanoparticle(iDPP)to deliver plasmids encoding SEA for melanoma treatment.The iDPP/SEA nanocomplexes efficiently mediated SEA expression in B16-F10 cells in vivo and in vitro and induced the activation of lymphocytes and maturation of murine bone marrow-derived dendritic cells(BMDCs)in vitro.In the subcutaneous B16-F10 melanoma model,the iDPP/SEA nanocomplexes could effectively enhance immune response and T lymphocytes infiltration in tumor site after intravenous administration,thereby considerably decreased melanoma growth.Meanwhile,no obvious adverse effect was observed after intravenous administration of the iDPP/SEA nanocomplexes in vivo.Our findings demonstrated that gene therapy of SEA is a potential candidate for melanoma treatment.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
文摘Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.
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. Resuits： 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 preven- tion of mucosaroute evading pathogen.
基金the Minist`ere des Affaires Etrang`eres(France)and the Ministry of Research,Science and Technology(Iran)for financing double PhD scholarship joint-supervision program between France and Iran for Mojtaba AZARI-ANPARThe authors are indebted to Conseil D´epartemental de l’Ain and Bourg en Bresse Agglom´eration for the financial support of BioDyMIA research unit activities.
文摘In this study,the interaction of exopolysaccharides from Leuconostoc mesenteroides P35(EPS-LM)with Escherichia coli heat-labile enterotoxin B-pentamer(LTB)was investigated at different concentrations and temperatures by using surface plasmon resonance(SPR)and molecular docking approaches.FT-IR spectral analysis together with HPTLC analysis revealing that glucose is the only constitutive monosaccharide of EPS-LM suggests that its structure is composed of dextran withα-D(1→6)glycosidic linkages.SPR analysis revealed the high affinity of EPS-LM for immobilized LTB toxin(KA=(2.05±0.04)×106 mol.L−1 at 37°C).The binding process was spontaneous(ΔG<0),endothermic(ΔH>0),and entropy-driven(ΔS>0)with an increase of KA with temperature.This suggests that EPS-LM-LTB interaction is dominated by hydrophobic forces.The binding affinity of EPS-LM to LTB had negligible dependence on enthalpy(ΔH=0.084 kJ mol−1).Further,molecular docking results suggested the presence of some binding sites of EPS-LM on the LTB through hydrophobic forces(Lys,Asp,Arg,Glu)and also hydrogen bonding(Glu)in the hydrophobic core of LTB.Besides autodock studies,Schiffer-Edmundson helical wheel diagrams of LTB inα-helix domain suggested that LTB hydrophobic core is a highly effective region,which was able to form favorable non-polar interactions of the protein's binding surface(with amino acids residues such as Tyr,Leu,Ile)with EPS-LM.This study provided thus further insights into the interactions between EPS-LM and LTB,suggesting that EPS produced by some LAB,such as EPS produced by Ln.mesenteroides P35 strain are good candidates to inhibit E.coli toxin activity.
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.
基金supported by the National Natural Science Foundation(No.82073363)the Sichuan Science and Technology Program(Nos.2020YFQ0059,2022YFQ0004)the Natural Science Foundation of Sichuan Province(No.2022NSFSC1304).
文摘Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to tumor site provides a promising option for reducing the systemic toxicity.Here,we constructed an iRGD peptide(H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH_(2))modified nanoparticle(iDPP)to deliver plasmids encoding SEA for melanoma treatment.The iDPP/SEA nanocomplexes efficiently mediated SEA expression in B16-F10 cells in vivo and in vitro and induced the activation of lymphocytes and maturation of murine bone marrow-derived dendritic cells(BMDCs)in vitro.In the subcutaneous B16-F10 melanoma model,the iDPP/SEA nanocomplexes could effectively enhance immune response and T lymphocytes infiltration in tumor site after intravenous administration,thereby considerably decreased melanoma growth.Meanwhile,no obvious adverse effect was observed after intravenous administration of the iDPP/SEA nanocomplexes in vivo.Our findings demonstrated that gene therapy of SEA is a potential candidate for melanoma treatment.
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.
