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构建了大肠杆菌一分枝杆菌穿梭质粒pBCG-2000,并将日本血吸虫26ku抗原(Sj26GST)基因在分枝杆菌中进行了克隆和表达.以质粒pBluescript sk为骨架,分别克隆入分枝杆菌质粒复制基因、Neo基因(编码卡那霉素抗性)以及人结核杆菌热休克蛋白70(heat shock protein,hsp70)的启动子,并带有质粒pBluescript的多克隆位点,构建成分枝杆菌-大肠杆菌穿梭质粒pBCG-2000.该重组质粒在分枝杆菌、大肠杆菌两个系统中均能稳定复制,并表达卡那霉素抗性.再以大肠杆菌表达质粒pGEX衍生质粒为模板,经过聚合酶链反应(polymerase chain reaction,PCR)得到编码日本血吸虫26ku抗原的全长DNA顺序.将其按正确的阅读框顺序,精确克隆到分枝杆菌hsp70的启动子下游,最终构建成能高效表达Sj26GST的大肠杆菌-分枝杆菌穿梭表达载体pBCG-Sj26.所表达的天然重组Sj26GST(rSi26GST)为可溶性蛋白,在SDS-PAGE上分子量为26ku处可见明显的表达蛋白带.提示生长缓慢的人结核杆菌hsp70的启动子能被快速生长的耻垢分枝杆菌RNA聚合酶所识别.通过薄层扫描分析,其表达效率分别占大肠杆菌、分枝杆菌菌体总蛋白的25%～37%和18%.本研究为外源基因在卡介苗(Bacille Calmette-Gu(?)rin,BCG)中的表达以及把分枝杆菌和BCG发展成为多价疫苗载体奠定了基础.
The shuttle plasmid pBCG-2000 of Mycobacterium tuberculosis was constructed and the 26 ku antigen (Sj26GST) gene of Schistosoma japonicum was cloned and expressed in Mycobacterium.Plasmid pBluescript sk was used as the backbone and cloned into mycobacterial plasmid Gene, Neo gene (encoding kanamycin resistance) and the promoter of human heat shock protein 70 (heat shock protein, hsp70), and carrying the multiple cloning site of plasmid pBluescript to construct mycobacteria-Escherichia coli Shuttle plasmid pBCG-2000.The recombinant plasmids could stably replicate and express kanamycin resistance in both mycobacteria and E.coli.After the plasmid pGEX derived from E.coli was used as a template, (PCR) was used to obtain the full-length DNA sequence encoding the 26ku antigen of Schistosoma japonicum, which was cloned into the downstream of the promoter of mycobacterium hsp70 in the correct reading frame order and finally constructed into a large intestine capable of expressing Sj26GST Bacillus-Mycobacterium shuttle expression vector pBCG-Sj26. The expressed native recombinant Sj26GST (rSi26GST) is a soluble protein with a clear mass at 26ku on SDS-PAGE Expression of protein bands.It is suggested that the slow-growing human Mycobacterium tuberculosis hsp70 promoter can be recognized by the rapid growth of M. smegmatis RNA polymerase.The expression efficiency of E.coli, Mycobacterium tuberculosis 25% -37% and 18% of the total protein.The present study laid the foundation for the expression of foreign genes in Bacille Calmette-Gu (?) Rin and BCG and the development of mycobacteria and BCG into multivalent vaccine vectors basis.