分泌表达Ag85B与ESAT6融合蛋白的重组卡介苗菌株的筛选

目的筛选获得分泌表达Ag85B与ESAT6融合蛋白的重组卡介苗(BCG)菌株。方法采用多聚合酶链反应(PCR)方法从结核分枝杆菌毒株H37Rv中扩增出热休克蛋白60(Hsp60)基因及其信号肽序列α-ss，将测序正确的hsp60和α-ss基因分别克隆人大肠杆菌(E.coli．)-BCG穿梭载体pOLYG中，构建分枝杆菌分泌表达载体pDE22，按相应的酶切位点将ag85b和esat6按照不同的连接方式联合克隆入pDE22载体，分别命名为pDE22-Ag85B-ESAT6和pDE22-ESAT6-Ag85B，将纯化的重组质粒电穿入BCG，经潮霉素抗性筛选和PCR的方法鉴定出含目的基因的重组BCG阳性克隆。收集重组BCG的培养上清液，经浓缩和透析后，进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和固相化蛋白质免疫学测定(Western-blot法)分析。结果两株重组：BCG菌株的培养上清液分泌表达相对分子质量约37000的蛋白，且分别可与抗Ag85B和抗ESAT6蛋白免疫小鼠的血清有相应的结合条带。结论分泌表达Ag85B与ESAT6融合蛋白的重组BCG菌株构建成功，有望为结核病的预防提供有效的疫苗。

Screening and construction of recombinant BCG strains expressing the Ag85B-ESAT6 fusion protein

OBJECTIVE: To screen and construct recombinant BCG strains which express the Ag85B-ESAT6 fusion protein. METHODS: The heat shock protein 60 (Hsp60) and the alpha-ss signal peptide encoding sequence were amplified by PCR from Mycobacterium tuberculosis H(37)Rv and cloned into E. coli/Mycobacteria shuttle vector-pOLYG. The resulting expression vector was named pDE22, and then ag85b and esat6 genes were cloned into pDE22 at different sites. The resulting recombinant plasmids Ag85B-ESAT6 and ESAT6-Ag85 were electropotated into BCG. Positive clones were screened by hygromycin resistance and confirmed by PCR. Recombinant BCG culture supernatants were collected and analyzed by SDS-PAGE and Western blot. RESULTS: Two recombinant BCG strains were obtained, which secreted the 37,000 fusion protein in their culture supernatant, which was confirmed by Western blot with specific immune serum against Ag85B and ESAT6. CONCLUSIONS: Recombinant BCG strains expressing Ag85B and ESAT6 fusion proteins of Mycobacterium tuberculosis were constructed. They may serve as new vaccine candidates for preventing tuberculosis.