表达人幽门螺杆菌外膜蛋白的穿梭质粒的构建及在耻垢分枝杆菌中的表达  

Recombination of Helicobacter pylori(Hp) OMP Escherichia coli-Mycobacteria Shuttle plasmids and its expression in Mycobacterium smegmatis mc^2 155

作　　者：向廷秀[1] 谯敏[1] 姜政[1] 陶小红[1] 黄爱龙[2] 王丕龙[1] 

机构地区：[1]重庆医科大学附属第一医院消化内科,重庆400016 [2]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《中国现代医学杂志》2006年第20期3047-3051,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金资助项目(No.30371318)

摘　　要：目的建立表达人幽门螺杆菌外膜蛋白重组耻垢分枝杆菌疫苗株,为进一步应用重组耻垢分枝杆菌疫苗防治幽门螺杆菌感染打下基础。方法利用聚合酶链反应(Polymerasechainreaction,PCR)扩增的幽门螺杆菌的外膜蛋白528编码基因全长片段,克隆入pET32a(+),鉴定无误后,再亚克隆入大肠杆菌-分枝杆菌穿梭质粒pLA71,构建pLA71-omp载体,将pLA71-omp电转化耻垢分枝杆菌,经卡那霉素筛选后,抽提质粒用PCR法鉴定,Westernblot检测omp的表达及活性。结果从幽门螺杆菌基因组中扩增出约528bp的基因片段。所构建重组体阳性克隆经酶切和PCR鉴定与预期结果一致,测序结果确证了插入片段的正确性,Westernblot检测说明幽门螺杆菌的外膜蛋白528编码基因在分枝杆菌中获得了表达并具有良好的免疫原性。结论成功构建了幽门螺杆菌外膜蛋白528编码基因大肠杆菌-分枝杆菌穿梭表达质粒。该质粒能在E.coli/MS间进行穿梭,并在耻垢分枝杆菌中表达。[Objective] To develop an Mycobacterium smegmatis mc2 155（MS） vaccine strain expressing Helicobacter priori outer membrane protein（Hp-OMP）. [Methods] The Hp-OMP gene was amplified by PCR and was cloned into prokaryotic expression plasmid pET32a （＋） to construct recombinant plasmid pET32-OMP. After the pET32-OMP was performed successfully by PCR,enzyme digestion analysis and sequencing. The recombinant shutde plasmid expressing Hp-OMP protein was obtained by subcloning the Hp-OMP gene fragment from pET32-OMP to Mycobacterium shuttle plasmid pLA71, named pLA71-OMP. The vaccine strain M. smegmatis mc2155 was transformed by pLA71-OMP. After cultivation, positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again. The expression of Hp-OMP protein in the MS bacteria was proved by SDSPAGE. The antigenic of recombinant protein was analysed by Western blotting. [Results] Sequencing and homologous analysis showed that the homology between the cloned Hp-OMP gene and related genes in GenBank reached 100% for their nucleotide and deduced amino acid sequences. SDS-PAGE analysis showed that recombinant vector could be expressed in MS. Relative molecular mass（Mr） of expression product was about 1 8 000. Western blot result showed that recombinant protein could be recognized bv anti-Hp positive serum,suggesting that this protein had good antigenic effect. [Conclusion] An M. smegmatis mc 2155 vaccine strain has been established successfully. The recombinant E.coli -Mycobacterium shuttle plasmid pLA71-OMP replicated in both E. coli and M. smegmatis, and expressed HP-OMP protein in M. smegmatis, which lays the foundation for further developing HP vaccine.

关 键 词：大肠杆菌-分枝杆菌穿梭质粒 幽门螺杆菌 OMP 耻垢分枝杆菌 

分 类 号：Q784[生物学—分子生物学]