新型重组IL6D24-PE40KDEL外毒素融合蛋白的理化分析

为了对构建成功的新型重组IL6D24-PE40KDEL外毒素融合蛋白的部分物化表征进行分析鉴定，采用Edman法、SDS-PAGE法、胰蛋白酶消化的MALDI—TOF—MS质谱法、蛋白印迹法和MTT法分别测定该融合蛋白的N-末端6个氨基酸、相对分子量、肽谱、抗原特异性以及靶向杀伤生物学活性。结果表明：所构建表达纯化的新型重组IL6D24-PE40KDEL外毒素融合蛋白N末端6个氨基酸序列为Met—lie—Asp—Lys—Gin—Ile-，而IL-6缺失24个氨基酸后的原序列为Ile—Asp—Lys—Gin—Ile-，由于采用了大肠杆菌表达系统，因此在N末端第1位多出了1个Met，经12％SDS-PAGE蛋白电泳和凝胶成像系统分析计算，其相对分子量为58．75kD，与理论值56．9kD相比误差在5％之内。MALTI—TOF—MS质谱测定共获得10个与理论预测值相符的肽段，经瑞士生物信息研究所的EXPASY分子生物服务网站的Peptident数据库搜索证明，在分子量为57kD左右的范围内未检索到与上述务件相符的已知蛋白，证明本融合蛋白为全新蛋白，与预测的目的蛋白相符。WB实验显示IL6D24-PE40KDEL外毒素融合蛋白能与IL-6及PEA抗体特异性结合。MTT生物活性测定表明，该融合蛋白对表达IL-6R的多发性骨髓瘤细胞系U266产生特异性杀伤，而对不表达IL6R的CEM淋巴细胞系无任何杀伤。结论：所研制成功的重组IL6D24-PE40KDL外毒素融合蛋白的确为一全新的具有靶向杀伤生物活性的蛋白质，与设计的完全一致，同时也证实了构建策略的可行性。

Biochemical and Physical Properties for a Recombinant IL6 Pseudomonas Exotoxin Fusion Protein IL6D24-PE40KDEL

The objective was to identify some biochemical and physical properties for fusion protein IL6D24 PE40KDEL . Edman degradation, SDS PAGE, peptide mass fingerprinting, Western blot and MTT were used for identification of the protein. The results showed that the sequence of N terminus is Met Ile Asp Lys Gln Ile, Met was added because of prokaryotic expression system; Western blot revealed that the purified protein could react with IL6 and PEA antibody. The purified protein IL6D24 PE40KDEL could kill the multiple myeloma cell lines U266 expressing high affinity IL6R, but it could not kill the cell lines CEM which not expressed IL6R; The molecular weight was 58.7 kD measuring by SDS PAGE; peptide mass fingerprinting (PMF) confirmed that the construction of IL6D24 PE40KDEL was correct. A novel protein by Peptident database in EXPASY web site was identified. In conclusion, IL6D24 PE40KDEL is a new targeting protein with bioactivity of specific killing effect.