摘要
用 PCR（polymerase chain reaction）技术克隆了卡介苗（Bacille Calmette-Guerin,BCG）热休克蛋白 65（heat shock Protein 65, hsp65）启动子序列,构建成穿梭载体ME-65。该载体含有分枝杆菌和大肠杆菌的质粒复制基因、BCG的hsp65高效启动子序列以及在分枝杆菌和大肠杆菌中都能表达的卡那霉素抗性基因。将其转化到耻垢分枝杆菌和BCG中均可稳定复制并表达卡那霉素抗性。质粒对宿主菌的生长无明显的影响,未观察到质粒的丢失和突变。该载体具有分子量小、容量大、转化效率高、复制稳定、含有多克隆位点等优点,将外源基因插入到hsp65启动子的下游,可望在BCG中获得高效表达。为BCG和其它分枝杆菌发展成多价重组疫苗开辟了新途径。
Using polymerase chain reaction (PCR) technique, the fragment of BCG heat shock protein 65 (hsp65) promoter was cloned and a new recombinant E. coli-mycobacterial shuttle plasmid ME-65 was constructed. The ME-65 contained both E.coli and mycobacterial plasmid origin of replication, the high efficient promoter from BCG hsp65 gene and the selectable antibiotic resistance marker for kanamycin that could express in both E.coli and mycohacterial. Using electroporation, transformation of both M. Smegmatis and BCG was successfully accomplished. The shuttle plasmids were genetically stable and maintained expression of kanamycin resistance in continuous subcultures containing kanamycin. No inhibitory effect of the vector on the growth of host bacteria and no deletion or mutation of the vector were observed. The new shuttle vector had the advantages of lower molecular weight, high capacity, high transforming efficiency, stable replication and convenient multi-cloning sites that permitted the insertion of foreign protective antigen genes. The expression of foreign genes was expected with the high efficient expression promoter from BCG hsp65 gene. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vector.
出处
《同济医科大学学报》
CSCD
1998年第2期89-93,共5页
Acta Universitatis Medicinae Tongji
基金
卫生部基金(No.94-1-144)
国家自然科学基金( No.39480022)
湖北省卫生厅基金(No.W-940303007)资助项目
