结核分支杆菌38 000蛋白质抗原在大肠杆菌中高效表达

目的通过结核分支杆菌３８０００蛋白质抗原编码基因在大肠杆菌中稳定表达，以获得大量纯化的３８０００蛋白质抗原，进而研究其免疫学特性。方法采用ＤＮＡ重组技术构建结核分支杆菌３８０００蛋白质抗原表达载体，双酶切和聚合酶链反应（ＰＣＲ）鉴定转化子，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（ＳＤＳＰＡＧＥ）和免疫印迹法鉴定３８０００蛋白质抗原在大肠杆菌中的表达；对染色的凝胶扫描，以测定目的蛋白质表达水平。诱导的工程菌抽提包涵体，以确定３８０００蛋白质抗原在大肠杆菌中的表达形式。结果菌体蛋白经ＳＤＳＰＡＧＥ，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３６％～４０％。肺结核患者的血清和结核分支杆菌免疫的羊血清与重组３８０００蛋白质均呈阳性反应。３８０００蛋白质抗原在大肠杆菌中表达方式主要以包涵体形式。结论构建的大肠杆菌重组体能高效表达结核分支杆菌３８０００蛋白质抗原，包涵体的表达形式有利于蛋白质纯化和蛋白质稳定，蛋白质必须经过正确折叠才具有生物学活性

The 38 000 protein of Mycobacterium tuberculosis is overexpressed in Escherichia coli

OBJECTIVE: To obtain recombinant 38,000 protein in large quantities and to study its immunologic characteristics by stable expression of the gene encoding for 38,000 antigen of Mycobacterium tuberculosis in E. coli. METHODS: Expression plasmid was constructed with DNA recombinant technique. Positive clones were screened using double digestion and polymerase chain reaction. Recombinant plasmid was transformed into E. coli. Then E. coli carrying recombinant plasmid were induced. The expression of 38,000 antigen was identified by SDS-polyacrylamid gel electrophoresis (PAGE) and immunoblotting. Stained gel was scanned to detect expression level of recombinant antigen. RESULTS: Gel stained with coomassie blue G-250 showed that the induced E. coli carrying recombinant plasmid can produce 38,000 protein at high level. Gel scan showed that 38,000 antigen expression in E. coli was about 36% - 40% of total cellular protein. The recombinant 38,000 antigen existed mostly in inclusion bodies. The recombinant antigen can react with antibodies: serum of pulmonary tuberculosis patients and the goats immuned with Mycobacterium tuberculosis. CONCLUSIONS: Constructed recombinant E. coli can overproduce 38,000 antigen of Mycobacterium tuberculosis. Inclusion bodies are easy to purify and can protect the recombinant antigen from protease, on the other hand, it has not biological activity unless the denatured protein is accurately folded.