摘要
目的:对人幽门螺杆菌(Helicobacter pylori,H.pylori)36ku(OMP36)外膜蛋白进行基因克隆表达,探索研制H.pylori疫苗的新途径。方法:培养H.pylori菌株NCTC11637,采用酚:氯仿抽提和纯化基因组DNA。设计上下游引物,并以该基因组为模板,采用聚合酶链反应(PCR)扩增目的基因片断。将目的基因和PET32a(+)同时经HindⅢ和KpnⅠ双酶切,纯化,连接后,转化含有目的基因的重组载体,酶切鉴定后进行序列分析。结果:经酶切,测序分析表明,插入的基因片断为987bp,与GenBank公布的H.pylori 26695、ATCC43504及J99序列相比较,同源性高达94%-96%,推测的氨基酸序列同源性为97%-99%,GenBank登录号059968。结论:成功克隆了H.pylori 36ku的外膜蛋白的编码基因,其表达产物OMP36有望成为新的Hp疫苗候选分子,为H.pylori疫苗的研制和试剂盒的开发奠定了基础。
Objective： To search the new protective antigens which can be used in a vaccine to prevent H. pylori infection, a gene encoding the structural 36ku outer membrane protein from H. pylori strain NCTC11637 was cloned and its sequence was analyzed. Methods： Polymerase chain reaction （PCR） was used to amplify the OMP36 gene from H. pylori chromosomal DNA. The target gene was digested by restricted endonuclease of Hind III and Kpn I, and inserted into the prokaryotic expression vector PET32a （＋） digested by corresponding restricted endonuclease. The recombinant vector was used to select and transform for nucleotide sequence analysis. Re- suits： Enzyme digestion analysis and sequencing showed that the target gene omp36 had been inserted into recombinant vector. The 987bp omp36 gene was amplified, but as compared with the sequences ofH. pylori 26695, ATCC43504 and J99 reported by GenBank, it has high nucleotide homology ranging from 94% to 96%, and high amino acid homology ranging from 96% to 99%. Conclusions： The gene coding for 36ku out membrane protein is cloned successfully. The results obtained lay the foundation for research on development ofH. pylori protein vaccine.
出处
《现代生物医学进展》
CAS
2007年第12期1835-1837,共3页
Progress in Modern Biomedicine
基金
江苏省科技厅项目(BS2004021)
江苏大学高级人才项目(JDG2004008)
