AIM: To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: Hpylori ureB and mou...AIM: To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS: The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully 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the National Natural Science Foundation of China, No. 30170427
文摘AIM: To construct a recombinant live attenuated Salmonella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: Hpylori ureB and mouse IL-2 gene fragments were amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified ureB and IL-2 genes was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions resulting in pIRES-ureB and pIRES-ureB-IL-2. The recombinant plasmids were used to transform competent E. co/i DH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-ureB and pIRES-ureB-IL-2 were used to transform LB5000 and the recombinant plasmids extracted from LB5000 were finally introduced into the final host SL7207. After that, recombinant strains were grown in vitro repeatedly. In order to detect the immunogenicib/of the vaccine in vitro, pIRES-ureB and pIRES-ureB-IL-2 were transfected to COS-7 cells using LipofectamineTM2000, the immunogenicity of expressed UreB and IL-2 proteins was assayed with SDS-PAGE and Western blot. C57BL/6 mice were orally immunized with 1 × 10^8 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 10^7 CFU of live Hpylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylon 4 wk post-challenge. RESULTS: The 1700 base pair ureB gene fragment amplified from the genomic DNA was consistent with the sequence of H pylori ureB by sequence analysis. The amplified 510 base pair fragment was consistent with the sequence of mouse IL-2 in gene bank. It was confirmed by PCR and restriction enzyme digestion that H pylori ureB and mouse IL-2 genes were inserted into the eukaryotic expression vector pIRES. The experiments in vitro showed that stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying ureB and IL-2 genes was successfully
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 。