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
Background： Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural s...Background： Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural subunits of urease, was expressed in E. coil BL21 （DE3） strain. The objective of this study was to evaluate the effects of He/icobacter py/ori urease B subunit on the immune responses in mice by subcutaneous immunization. Methods： The mice were immunized and boosted with Helicobacter pylori urease B subunit antigen subcutaneously three times with 2-wk intervals between the immunizations and boosters. The mice in the control group were immunized with PBS. The adjuvant group received PBS containing complete/incomplete freund＇s adjuvant identical to antigen group without Helicobocter pylori urease B subunit antigen. Four weeks after the final booster, all the mice were sacrificed. Blood was collected on d 0, 14, 28 and 56 before immunization, booster and sacrifice, respectively. Immediately after sacrifice, gastric liquid and spleen were collected for antibody and cytokine analyses. Results： Urease B subunit increased the concentrations of serum and gastric anti-urease B antigen specific IgG, and the levels of interteukin-4 and interferon-y in splenocytes of the mice （P 〈 0.05）. Conclusions： This study demonstrated that recombinant responses in mice by subcutaneous immunization, which against Helicobocter pylori. urease B subunit can induce systemic and local immune might be used as the effective component of vaccine展开更多
目的 :构建含幽门螺杆菌 (H elicobacter pylori,Hp)尿素酶 B亚单位 (Ure B)基因的核酸疫苗。方法 :抽提 Hp标准菌株 CCUG1 7874基因组 DNA,应用 PCR技术从基因组 DNA扩增 U re B基因 ,克隆入 PUCm T载体 ,检测 U re B基因序列。经过一...目的 :构建含幽门螺杆菌 (H elicobacter pylori,Hp)尿素酶 B亚单位 (Ure B)基因的核酸疫苗。方法 :抽提 Hp标准菌株 CCUG1 7874基因组 DNA,应用 PCR技术从基因组 DNA扩增 U re B基因 ,克隆入 PUCm T载体 ,检测 U re B基因序列。经过一系列酶切、连接反应将其克隆入真核表达载体 p IRES,转入感受态大肠杆菌 DH5 α,筛选阳性克隆 ,通过 PCR和酶切反应进行鉴定。通过脂质体法将构建好的重组载体 p IRES- Ure B转染 COS- 7细胞 ,Western印迹分析检测 p IRES- U re B表达 Ure B蛋白的免疫原性。 结果 :扩增出长约 1 70 0 bp的 U re B基因 ,与基因库 Hp U re B序列一致 ,PCR和酶切鉴定结果证实成功构建了含 U re B基因的 Hp核酸疫苗 p IRES- U re B,并且 Western印迹分析检测到特异性的蛋白条带。结论 :构建了具有免疫反应性 Ure B基因的 Hp核酸疫苗 。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.
基金financially supported by National Natural Science Foundation of China(31201824)the Agricultural Science and Technology Innovation Program(ASTIP-IAS07)Research Program of the State Key Laboratory of Animal Nutrition(2004DA125184G1103)
文摘Background： Helicobacter pylori, a gram-negative bacterial pathogen that expresses a strong urease activity, is associated with the development of gastroduodenal disease. Urease B subunit, one of the two structural subunits of urease, was expressed in E. coil BL21 （DE3） strain. The objective of this study was to evaluate the effects of He/icobacter py/ori urease B subunit on the immune responses in mice by subcutaneous immunization. Methods： The mice were immunized and boosted with Helicobacter pylori urease B subunit antigen subcutaneously three times with 2-wk intervals between the immunizations and boosters. The mice in the control group were immunized with PBS. The adjuvant group received PBS containing complete/incomplete freund＇s adjuvant identical to antigen group without Helicobocter pylori urease B subunit antigen. Four weeks after the final booster, all the mice were sacrificed. Blood was collected on d 0, 14, 28 and 56 before immunization, booster and sacrifice, respectively. Immediately after sacrifice, gastric liquid and spleen were collected for antibody and cytokine analyses. Results： Urease B subunit increased the concentrations of serum and gastric anti-urease B antigen specific IgG, and the levels of interteukin-4 and interferon-y in splenocytes of the mice （P 〈 0.05）. Conclusions： This study demonstrated that recombinant responses in mice by subcutaneous immunization, which against Helicobocter pylori. urease B subunit can induce systemic and local immune might be used as the effective component of vaccine
文摘目的 :构建含幽门螺杆菌 (H elicobacter pylori,Hp)尿素酶 B亚单位 (Ure B)基因的核酸疫苗。方法 :抽提 Hp标准菌株 CCUG1 7874基因组 DNA,应用 PCR技术从基因组 DNA扩增 U re B基因 ,克隆入 PUCm T载体 ,检测 U re B基因序列。经过一系列酶切、连接反应将其克隆入真核表达载体 p IRES,转入感受态大肠杆菌 DH5 α,筛选阳性克隆 ,通过 PCR和酶切反应进行鉴定。通过脂质体法将构建好的重组载体 p IRES- Ure B转染 COS- 7细胞 ,Western印迹分析检测 p IRES- U re B表达 Ure B蛋白的免疫原性。 结果 :扩增出长约 1 70 0 bp的 U re B基因 ,与基因库 Hp U re B序列一致 ,PCR和酶切鉴定结果证实成功构建了含 U re B基因的 Hp核酸疫苗 p IRES- U re B,并且 Western印迹分析检测到特异性的蛋白条带。结论 :构建了具有免疫反应性 Ure B基因的 Hp核酸疫苗 。
