结核分支杆菌Ag85B基因在大肠杆菌中的高效表达

Over Expression of the Ag85B Gene of Mycobacterium tuberculosis in Escherichia coli

目的通过结核分支杆菌Ag85B蛋白编码基因在大肠杆菌中稳定表达,以获得大量纯化的Ag85B蛋白。方法采用DNA重组技术构建结核分支杆菌Ag85B基因的表达载体,双酶切和聚合酶链反应(PCR)鉴定重组子,阳性重组子转化大肠杆菌,并诱导表达外源蛋白。十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)鉴定Ag85B蛋白抗原在大肠杆菌中的表达,对染色的凝胶扫描,以测定目的蛋白表达水平。结果菌体蛋白经SDSPAGE,含阳性重组质粒的菌体蛋白中出现一条新蛋白带,表达量占菌体总蛋白的33%～38%。Ag85B蛋白抗原在大肠杆菌中表达方式主要是包涵体形式。结论构建的大肠杆菌重组体能高效表达结核分支杆菌Ag85B蛋白抗原。

Objective To obtain recombinant Ag85B protein in large quantities by stable expression of the gene encoding for Ag85B antigen of Mycobacterium tub erculosis in E.coli. Methods Expression plasmid was constructed with DNA recombi nant technique. Positive clones were screened using doubled digestion and polyme rase chain reaction. Recombinant plasmid was transformed into E.coli. Then E.col i carrying recombinant plasmid were induced to express Ag85B. The expression of Ag85B antigen was identified by SDS-polyacrylamid gel electrophoresis (PAGE). S tained gel was scanned to detect expression level of recombinant antigen. Result s Gel stained with Coomassie blue G 250 showed that the induced E.coli carrying recombinant plasmid could produce Ag85B protein at high level, about 33%to 38% of total cellular proteins. The combinant Ag85B antigen existed mostly in inclus ion bodies. Conclusion Recombinant Ag85B antigen of Mycobacterium tuberculosis w as espressed in high level as inclusion bodies in E.coli.