新型重组IL6D24PE40KDEL外毒素融合蛋白的理化分析  

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

作　　者：崔建武[1] 郭斯启[1] 孙玉英[1] 刘楠[1] 梁飞[1] 奚永志[1] 

机构地区：[1]军事医学科学院附属医院免疫学研究室,国家生物医学分析中心免疫学研究室,北京100039

出　　处：《中国实验血液学杂志》2004年第6期825-828,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助课题;编号 395 0 0 0 62及 39870 875

摘　　要：为了对构建成功的新型重组IL6D2 4 PE4 0KDEL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IL6D2 4 PE4 0KDEL外毒素融合蛋白N末端 6个氨基酸序列为Met Ile Asp Lys Gln Ile ,而IL 6缺失 2 4个氨基酸后的原序列为Ile Asp Lys Gln 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 5 8.75kD ,与理论值 5 6.9kD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 5 7kD左右的范围内未检索到与上述条件相符的已知蛋白 ,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IL6D2 4 PE4 0KDEL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U2 66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.

关 键 词：外毒素融合蛋白 IL6D24-PE40KDEL 纯化鉴定 

分 类 号：R996[医药卫生—毒理学]