幽门螺杆菌双价亚单位分子内佐剂疫苗的构建表达与纯化.docVIP

幽门螺杆菌双价亚单位分子内佐剂疫苗的构建表达与纯化 张卫军，郭 刚，刘开云，解庆华，邹全明*(第三军医大学临床微生物学及免疫学教研室,重庆市生物制药工程技术研究中心,重庆 400038) 摘 要：目的 克隆表达幽门螺杆菌外膜蛋白烷基过氧化氢还原酶ahpC、ureB414功能片段基因并与大肠杆菌不耐热肠毒素B亚单位构建融合基因ahpC-ureB414-ltB（aul），为制备预防幽门螺杆菌感染的疫苗奠定基础。 方法 采用重叠延伸PCR的方法，以AhpC做为融合蛋白前端，与UreB414功能片段及粘膜佐剂LTB依次串联，在片段间引入5个氨基酸的柔性linker GGGGS进行连接；将融合基因构建在原核表达载体pET-22b(+)中，经酶切及测序鉴定后转化宿主菌E.coli BL21(DE3)，IPTG诱导表达融合蛋白AhpC-UreB414-LtB (AUL)。融合蛋白经AKTA-explore 纯化仪纯化融合蛋白。 结果 PCR扩增出1 350 bp的目的基因片段aul，工程菌pET22b-aul/BL21经IPTG诱导后，SDS显示有新生的蛋白表达条带，约为50 000，与预期值一致，目的蛋白约占菌体总蛋白的28.7％，重组蛋白用Ni2+-NTA树脂提纯。纯化后的蛋白质经SDS分析可见单一条带，图软件分析表明纯度可达85％以上。Western blot显示重组蛋白质具有良好的免疫反应性。 结论 成功构建、表达融合蛋白rAUL，纯化后的融合蛋白保持各亚单位组分及佐剂的独立免疫反应性。 幽门螺杆菌；不耐热肠毒素B亚单位；融合蛋白 R392.33 文献标识码：A Expression and Purification of Fusion Protein of Helicobacter pylori AhpC-UreB414 and E.coli Heat-labile Enterotoxin B Subunit ZHANG Wei-jun，GUO Gang，LIU Kai-yun，XIE Qing-hua，ZOU Quan-ming (Laboratory of Clinic Microbiology and Immunology ，Faculty of Medical Laboratory Sciences，Third Military Medical University，Chongqing 400038，China) AbstractObjective To construct the fusion gene of H.pylori outer membrane protein AhpC,UreB414 and E.coli heat-labile enterotoxin B subunit．Methods We constructed the fusion gene aul includeing hpC、reB414 and ltB gene by SOE PCR(splicing by overlap extension),setting AhpC gene before UreB414-LTB fusion gene and a fiexible linker GGGGS was introduced between them.Then the AUL gene was constructed into the expression plasmid pET-22b(+).The fusion protein was expressed in E.coli BL21 induced by IPTG, confirmed by SDSand West-blotting tests. The inclusion body was purifyied with AKTA-explore 100 system after washing. Results The fusion gene was successfully cloned into pET-22b(+). The gene segment inserted into the recombinant vector was identified by using restriction enzyme digestion and sequencing methods and was consistent with that in Genbank．The relative molecular weight of expressed product was Mr 50000. The purity of rAUL