构建lt B- ure B融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T- A克隆法从幽门螺杆菌(H elicobacter pylori,Hp)临床菌株Y0 6和大肠杆菌4 4 85 1株DNA中获得了ure B和lt B全长基因扩增片段及其克隆,并构建...构建lt B- ure B融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T- A克隆法从幽门螺杆菌(H elicobacter pylori,Hp)临床菌株Y0 6和大肠杆菌4 4 85 1株DNA中获得了ure B和lt B全长基因扩增片段及其克隆,并构建了lt B- ure B融合基因及其原核表达系统p ET32 a- lt B- ure B- E.coli BL2 1DE3.在E.coli BL2 1DE3宿主菌中用不同浓度的IPTG诱导表达,并用Hp全菌抗体的Western blot、EL ISA以及GM1 -EL ISA分别证实了目的重组蛋白(r L TB- Ure B)的免疫性和佐剂活性.与报道的相关序列比较,所克隆的ure B和lt B核苷酸序列同源性分别为96 .88%～97.82 %和99.12 %～99.71% ,氨基酸序列同源性为99.6 5 %～99.82 %和97.5 8%～99.19% . 0 .1～1.0 mm ol/ L 的IPTG均能有效地诱导目的重组蛋白r L TB- Ure B的表达,该蛋白主要以包涵体形式存在,其产量约为细菌总蛋白的35 % .Western blot结果证实r L TB- U re B不仅能与商品化的Hp全菌抗体结合,免疫家兔后也能产生特异性抗体,表明r L TB- U re B有良好的免疫反应性及抗原性.GM1 - EL ISA结果显示r L TB- Ure B能与牛GM1 结合,表明r L TB- Ure B有佐剂活性.以兔抗r L TB- U re B为一抗,发现所检测的10 9株Hp临床分离菌株均表达Ure B;以r L TB- U re B为包被抗原,发现所检测的12 5例Hp感染者血清中均存在U re B抗体;表明U re B广泛存在于不同的Hp菌株中,并有很强的抗原性,也提示r L TB- Ure B确有自然表达U re B的抗原特异性.本文成功地构建了L TB- Ure B融合基因原核高效表达系统,所表达的L TB- U re B融合蛋白有良好的免疫性和佐剂活性,为Hp基因工程疫苗的产业化奠定了坚实的基础.展开更多
AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori ureB and mou...AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori 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. coli 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 immunogenicity 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 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 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 consistentwith 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 constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P < 0.01). CONCLUSION: Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.展开更多
文摘构建lt B- ure B融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T- A克隆法从幽门螺杆菌(H elicobacter pylori,Hp)临床菌株Y0 6和大肠杆菌4 4 85 1株DNA中获得了ure B和lt B全长基因扩增片段及其克隆,并构建了lt B- ure B融合基因及其原核表达系统p ET32 a- lt B- ure B- E.coli BL2 1DE3.在E.coli BL2 1DE3宿主菌中用不同浓度的IPTG诱导表达,并用Hp全菌抗体的Western blot、EL ISA以及GM1 -EL ISA分别证实了目的重组蛋白(r L TB- Ure B)的免疫性和佐剂活性.与报道的相关序列比较,所克隆的ure B和lt B核苷酸序列同源性分别为96 .88%～97.82 %和99.12 %～99.71% ,氨基酸序列同源性为99.6 5 %～99.82 %和97.5 8%～99.19% . 0 .1～1.0 mm ol/ L 的IPTG均能有效地诱导目的重组蛋白r L TB- Ure B的表达,该蛋白主要以包涵体形式存在,其产量约为细菌总蛋白的35 % .Western blot结果证实r L TB- U re B不仅能与商品化的Hp全菌抗体结合,免疫家兔后也能产生特异性抗体,表明r L TB- U re B有良好的免疫反应性及抗原性.GM1 - EL ISA结果显示r L TB- Ure B能与牛GM1 结合,表明r L TB- Ure B有佐剂活性.以兔抗r L TB- U re B为一抗,发现所检测的10 9株Hp临床分离菌株均表达Ure B;以r L TB- U re B为包被抗原,发现所检测的12 5例Hp感染者血清中均存在U re B抗体;表明U re B广泛存在于不同的Hp菌株中,并有很强的抗原性,也提示r L TB- Ure B确有自然表达U re B的抗原特异性.本文成功地构建了L TB- Ure B融合基因原核高效表达系统,所表达的L TB- U re B融合蛋白有良好的免疫性和佐剂活性,为Hp基因工程疫苗的产业化奠定了坚实的基础.
基金the National Natural Science Foundation of China, No. 30170427
文摘AIM: To construct a recombinant live attenuated Salm-onella typhimurium DNA vaccine encoding H pylori ureB gene and mouse IL-2 gene and to detect its immunogenicity in vitro and in vivo. METHODS: H pylori 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. coli 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 immunogenicity 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 × 108 recombinant attenuated Salmonella typhimurium DNA vaccine. Four weeks after vaccination, mice were challenged with 1 × 107 CFU of live H pylori SS1. Mice were sacrificed and the stomach was isolated for examination of H pylori 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 consistentwith 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 constructed and the specific strips of UreB and IL-2 expressed by recombinant plasmids were detected through Western blot. Study in vivo showed that the positive rate of rapid urease test of the immunized group including ureB and ureB-IL-2 was 37.5% and 12.5% respectively, and was significantly lower than that (100%) in the control group (P < 0.01). CONCLUSION: Recombinant attenuated Salmonella typhimurium DNA vaccine expressing UreB protein and IL-2 protein with immunogenicity can be constructed. It can protect mice against H pylori infection, which may help the development of a human-use H pylori DNA vaccine.
