摘要
目的 在大肠杆菌中表达炭疽菌保护性抗原受体结合区。方法 将大肠杆菌外膜蛋白A(OmpA)的信号肽序列融合到炭疽菌保护性抗原 (PA)受体结合区 ,即PA结构域 4 (PA D4 )基因的5′端 ,构建成分泌型表达质粒 ,在大肠杆菌中尝试PA D4的表达 ,并对重组蛋白进行纯化和鉴定。结果 重组PA D4以可溶形式表达 ,表达量约占菌体总蛋白的 10 %。经过离子交换层析和凝胶过滤纯化 ,每升诱导培养物可获得约 10mgPA D4。蛋白N端测序表明PA D4与天然序列一致。免疫印迹试验显示PA D4可与抗PA血清结合。结论 在大肠杆菌中实现了PA D4的分泌型表达 ,为进一步评估其作为新型疫苗和潜在治疗药物的可能性打下基础。
The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第10期799-801,共3页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 ( 3 0 3 0 0 0 16)
