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Screening and construction of recombinant BCG strains expressing the Ag85B-ESAT6 fusion protein
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF TUBERCULOSIS AND RESPIRATORY DISEASES
- Issue:
- 4
- DOI:
- 10.3760/j:issn:1001-0939.2005.04.011
- Key Word:
- 分枝杆菌,结核;抗原,细菌;重组融合蛋白质类;疫苗,DNA;卡介苗
Abstract： 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基因分别克隆入大肠杆菌(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37 000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.
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