人源骨架蛋白FN3展示脑钠肽前体N端抗原表位研究
Study on the human scaffold protein FN3 for displaying NT-proBNP epitopes

目的 ·应用人源骨架蛋白 FN3展示脑钠肽前体 N端 (NT-proBNP)抗原表位，采用大肠杆菌表达系统高效表达具有脑钠肽前体N端抗原表位活性的结构稳定的重蛋白。方法 ·将编码脑钠肽前体 N端第12～21位和第13～20位氨基酸残基的抗原表位序列分别取代骨架蛋白 FN3 FG loop区，并克隆到大肠杆菌表达载体 pET28(a)+上，由大肠杆菌BL21(DE3) plySs表达后纯化，检测其抗原活性并进行理化性质分析。结果 ·成功构建骨架蛋白FN3展示脑钠肽前体第12～21位和第13～20位氨基酸残基抗原表位的表达载体，获得高效表达骨架蛋白FN3展示脑钠肽前体N端抗原表位的大肠杆菌表达菌株。镍柱纯化后获得高纯度重组蛋白，经Western blotting与ELISA鉴定具有脑钠肽前体N端抗原免疫活性。通过差式热量扫描法检测显示重组蛋白保持FN3骨架蛋白原有结构稳定性；血浆稳定性检测显示重组蛋白是与抗原蛋白等效的高稳定的抗原蛋白替代物，可明显延长降解周期（血浆半衰期）。结论 ·成功利用了FN3骨架蛋白展示脑钠肽N端前体，采用大肠杆菌表达系统可以简单、快速、高效制备具有与脑钠肽前体的抗原性相当的脑钠肽前体抗原替代物。
： Objective · To display NT-proBNP epitopes using human scaffold protein FN3 and to efficiently express a stable recombinant protein with active NT-proBNP epitopes via E.coli expression system. Methods · The FG loop sequence of scaffold protein FN3 was replaced by NTproBNP epitope 12-21 and 13-20 sequences at amino acid residue, respectively, and was cloned into the E.coli expression vector pET28(a)+. The recombinant FN3 proteins displaying NT-proBNP epitopes were expressed in E.coli BL21(DE3) plySs and were purified. The antigen activity was detected and physiochemical properties were analyzed. Results · The expression vector for recombinant protein FN3 displaying NT-proBNP epitope 12-21 and 13-20 sequences at amino acid residue was successfully constructed. The E.coli strain with efficient expression of scaffold protein FN3 displaying NT-proBNP epitopes was obtained. Highly purified recombinant protein with antigen immune activity against NT-proBNP was obtained with nickel column purification and was verified with Western blotting and ELISA. Differential thermal scanning method showed that the recombinant protein maintained the stability of scaffold protein FN3. Plasma stability test indicated that the recombinant protein was a highly stable substitute for antigen protein with the same effect and could significantly extend the degradation cycle (serous half-life). Conclusion · NT-proBNP was displayed successfully via scaffold protein FN3. A substitute for antigen protein of NT-proBNP with the same antigenicity can be simply, rapidly, and efficiently prepared with E.coli expression system