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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。
Objective To express anthrax protective antigen receptor binding region in Escherichia coli. Methods The signal peptide sequence of OmpA from E. coli was fused to the 5 ’end of the protective antigen (PA) receptor of PAH, ie PA D4 gene to construct a secretory expression Plasmids were tested for PA D4 expression in E. coli and the recombinant protein was purified and identified. Results Recombinant PA D4 was expressed in soluble form and expressed at about 10% of total bacterial proteins. After ion exchange chromatography and gel filtration purification, about 10 mgPA D4 was obtained per liter of induction culture. Protein N-terminal sequencing showed that PA D4 was identical to the native sequence. Western blotting showed that PA D4 binds to anti-PA serum. Conclusion The secretory expression of PA D4 was achieved in E. coli, laying the groundwork for further assessment of its potential as a novel vaccine and potential therapeutic.