Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine...Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.展开更多
Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in...Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL^(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.展开更多
Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and cap...Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.展开更多
To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected...To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial Gl261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.展开更多
The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple d...The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.展开更多
A live attenuated AraA autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVWL-12, pCMVmGM-CSF and PCMVhGM-CSF and was administered orally...A live attenuated AraA autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVWL-12, pCMVmGM-CSF and PCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increasedsignificantly in treated mice, so did the ratio of CD8+/CD4+,which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.展开更多
The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated ...The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1+ VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1+VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8+ cytotoxic T lymphocytes, as well as an increase in CD4+ cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.展开更多
基金This work was supported by Chinese Postdoctoral Fund (Grant No. 2004035426).
文摘Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.
文摘Objective:To develop allenualed slrains of Salmonella enterica serorar Typhi(S.typhi) for the candidate vaccine by osmolar stress.Mothods:S.typhi SS3 and SS5 strains were isolated from asymptomatic typhoid carriers in Mamakkal,Tamil Nadu.India.Both strains were grown in LB(Luria Bertani) medium supplemented with various concentration of NaCl(0.1- 0.7M) respectively.The effecl of osmolar stress was determined at molecular level by PCR using MCR 06 and MCR07 primers corresponding to ompR with chromosomal DNA of S.typhi SS3 and SS5 strains.Attenuation by osmolar stress results in deletion mutation of the.S.typhi slrains was determined by agglutination assays,precipitation method.SDS PAGE analysis and by animal models.Results:The 799 bp amplified ompR gene product from wild type S.typhi SS3 and SS5 illustrate the presence of virulent gene.Interestingly,there was only a 282 bp amplified product from S.typhi SS3 and SS5 grown in the presence of 0.5.0.6 and 0.7 M NaCl.This illustrates the occurrence of deletion mutation in ompR gene al high concentration of NaCl.Furthermore,both the wildtype and mutant S.typhi outer membrane SDS-PAGF.profile reveals the differences in the expression of ompF.ompC and ompA proteins.In mice,wild type and mutant strains lethal dose(LD_(50)) were determined.The mice died within 72 h when both the wild type strains were injected intraperitoneally with 3 log CFU-mL^(-1).When the mice were injected with the mutants in same dosage,no clinical symptoms were observed;whereas the serum antibodv litre was elicited within two weeks indicated that the mutants have the ability to induce protective humoral immune response.These results suggest that S.typhi SS3 and SS5 may bo used as good candidate strains for the development of live attenuated vaccine against salmonellosis.Conclusions:This study demonstrates that the S.typhi strains were allenualed and could be good vaccine candidates in future.
基金supported by the National Key Technology R&D Program of China(2015BAD12B05)the earmarked fund for China Agricultural Research System(CARS-43-8)+1 种基金the Integration and Demonstration of Key Technologies for Duck Industry in Sichuan Province,China(2014NZ0030)the Sichuan Province Research Programs,China(2014-002)
文摘Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.
文摘To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207. Mouse models of intracranial Gl261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene. The survival period was recorded and vessel density was observed by immunofluorescence. CTLs activity was measured by MTT assay. Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice. The flk-1 specific CTLs activity was increased obviously after the vaccination. Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.
基金supported by the National KeyR&DProgramof China(2022YFF0710500)the National Natural Science Foundation of China(32172853 and 32373013)the Central Public-interest Scientific Institution Basal Research Fund,China(1610302022001).
文摘The Salmonella pathogenicity islands(SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs(SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants(SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant(SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates(17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover,the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups.In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic(including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.
文摘A live attenuated AraA autotrophic mutant of Salmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVWL-12, pCMVmGM-CSF and PCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increasedsignificantly in treated mice, so did the ratio of CD8+/CD4+,which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.
基金Supported by the National Natural Science Foundation of Yunnan Province (Grant No. 2005C0073M)the Inaugurate Colony Research Fund of Kunming Medical College (Grant No. KMC2005DG01)
文摘The purposes of this research were to study the stable expression of exogenous gene encoding therapeutic protein in attenuated Salmonella typhimurium, observe the metabolism of oral gene vaccine carried by attenuated Salmonella typhimurium in BALB/c mouse, and investigate the feasibility of prevention and treatment of tumors by the recombinant bacteria. Recombinant plasmid pcDNA3.1+ VEGFR2(n1-7) was transformed into competent attenuated Salmonella typhimurium SL3261 to develop oral DNA vaccine SL3261-pcDNA3.1+VEGFR2(n1-7). To observe whether the exogenous gene can be expressed in the recombinant bacteria, PCR was performed to amplify the CMV promoter of the eukaryotic expression vector as the proof of stable expression of exogenous protein; transmission elec- tron microscopy (TEM) was applied to observe the morphology of the recombinant bacteria to confirm that the exogenous gene has no impact on the growth of the bacteria, and then BALB/c mice were immunized with the gene vaccine. After inoculation of the gene vaccine, the recombinant bacteria SL3261 could be detected in the tissues such as small intestine, colon, liver and spleen. And then, mice in each group were challenged with tumor cells. The results of animal experiment showed that tumor growth of the mice in experimental group was inhibited and survival time of immunized mice was prolonged compared with control groups. A higher lymphocyte infiltration in tumors from animals treated with DNA vaccine was observed. Immunohistochemical analysis of tumor samples revealed an en- hanced accumulation of CD8+ cytotoxic T lymphocytes, as well as an increase in CD4+ cells in the tumors of animals treated with the oral gene vaccine compared to tumors from control group mice. Ultrastructure of the tumor tissue showed that tumor cells in the samples of the immunized mice were well-differentiated. Our research confirmed that the exogenous gene can be stably expressed in the attenuated Salmonella typhimurium and has no impact on the growth of the recombinant bacteria; the exogenous gene can de delivered to the host by attenuated Salmonella typhimurium to produce anti-tumor effect with no obvious cytotoxity to the host. In this study, it is established that attenuated Salmonella typhimurium could be used as a vector for oral gene vaccine, and our study provided a theoretical basis for the body distribution and the metabolism of the recombinant bacteria. This strategy may provide a simple, safe and effective way for the prevention and treatment of tumors.
