幽门螺杆菌47kDa微孔蛋白基因的克隆表达及特性鉴定

Cloning and characterization of 47KDa porin from Helicobacter pylori

目的对人幽门螺杆菌（H．pylori）编码47kDa外膜微孔蛋白基因omp47克隆表达及特性鉴定，探索研制H．pylori疫苗的新途径。方法培养和收集H．pylori标准菌株NTTCll637及临床菌株，采用酚：氯仿抽提、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omp47基因片断。将目的基因和与克隆载体PGEM-T连接后，转化至EcoliDH5a及BL21，碱裂解提取质粒经BamHl和Xhol双酶切鉴定并进行序列分析。重组蛋白采用IPTG诱导表达后，经SDS-聚丙烯酰胺凝胶电泳（PAGE）鉴定、镍亲和层析纯化检测抗原活性。结果经酶切、测序分析表明，插入的基因片断为1284bp，编码427个氨基酸。与GenBank公布的H．pylori标准株26695、43504及J99序列比对，核苷酸同源性高达94%～96％，编码氨基酸同源性96%～99%。经SDS-PAGE检测，电泳图谱上显示一条相对分子量为47kda的新生蛋白带。Westernblot检测表明重组蛋白有较好的抗原性。结论成功克隆了外膜微孔蛋白OMP47的编码基因，为H．pylori疫苗的研制和试剂盒的开发奠定了良好的基础。

To search the new protective antigens which can be used in a vaccine to prevent H. pylori infection, a gene encoding the structural 47kDa outer membrane protein from H. pylori strain NCTC11637 was cloned and its sequence was analysised. Polymerase chain reaction （PCR） was used to amplify the omp47 gene from H. pylori chromosomal DNA. Then the target gene was digested by restricted endonuclease enzyme of BamHl and Xhol, and inserted into the vector PGEM-T and PET32a digested by corresponding restricted endonuclease enzyme. The recombinant vcctors were used to select and transform for nucleotide sequence analysis. Enzyme digestion analysis and sequencing showed that the target gene had been inserted into recombinant vector, but as compared with the genes of H. pylori 26695, ATCCA3504 and j99 reported by GenBank, homology were reached to highly 94 % -96 %, homology of anino acid residues were reached to highly 96 % - 99 %. The DNA sequence a- nalysis showed the sequence of the target DNA was almost same as that of the published by GenBank. The SDS-PAGE analysis showed that relative molecule mass （Mr） of expressed product was 47 ）〈 103. The Western-blot results showed that recombinant fusion protein have good antigenicity. The gene coding for 47kDa porin is cloned successfully. The results obtained lay the foundation for research on development of H. pylori protein vaccine.