摘要
目的 :克隆幽门螺杆菌 (Hp) ure B基因并构建其原核表达系统。方法 :采用高保真 PCR从幽门螺杆菌临床分离菌株 Y0 6中扩增 ure B基因 ,T- A克隆后测定核苷酸序列 ,构建 p ET32 a的 ure B表达载体 ,在 E.coliBL2 1DE3宿主菌中用不同浓度的 IPTG诱导表达 ,采用 Hp全菌抗体的 Western blot和兔抗融合蛋白血清的免疫扩散试验鉴定其免疫性。结果 :所克隆的 ure B基因与报道的相应核苷酸序列同源性为 96.88%～ 97.82 % ,氨基酸序列同源性高达 99.65 %～ 99.82 %。p ET32 a- ure B- BL2 1DE3系统的 Ure B融合蛋白表达量高达细菌总蛋白的4 0 %左右。Ure B融合蛋白能与 Hp全菌抗体发生结合反应 ,免疫家兔能获得高效价抗体。结论 :本研究成功地构建了 Hp ure B高效表达系统 ,所表达的 Ure B融合蛋白有良好的免疫原性和免疫反应性 ,可作为 Hp疫苗的抗原。
Objective: To clone Helicobacter pylori ureB gene, to construct prokaryotic expression system of the gene and to identify immunogenicity of the fusion protein. Methods: The ureB gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted ureB gene was constructed. ureB fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. Results: In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned ureB gene was from 96.88%~97.82%, while the homology of its putative amino acid sequence was as high as 99.65%~99.82%. The expression output of UreB protein in pET32a-ureB-BL21DE3 system was approximately 40% of the total bacterial proteins. UreB protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to produce high titer antibody after the animal was immunized with the protein. Conclusion: An expression system with high efficiency of H.pylori ureB gene has been established successfully. The expressed UreB protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2003年第1期4-8,共5页
Journal of Zhejiang University(Medical Sciences)
基金
国家教育部优秀年轻教师基金
浙江省科技厅项目(0 0 1110 438)
