定点突变提高IL-4免疫毒素对淋巴瘤的选择性和杀伤活性  

Improvement of Selectivity and Cytotoxicity of IL-4 Immunotoxin on Lymphoma Through Site-directed Mutagenesis

作　　者：崔京霞[1] 纪剑飞 吕安国[1] 吴文芳[1] 

机构地区：[1]中国科学院沈阳应用生态研究所 [2]BeckmanResearchInstitute,CityofHopeNationalMedicalCenter

出　　处：《高等学校化学学报》2005年第5期874-879,F006,共7页Chemical Journal of Chinese Universities

基　　金：沈阳市科技局科学基金项目(批准号:20011221-03);中国科学院沈阳应用生态研究所与屹昌科技集团股份有限公司合作项目资助.

摘　　要：采用软件分析选择与IL-4分子结合与活性相关的重要位点13T,121R,通过定点突变得到IL-4突变基因cpIL4(13D121E),将其与绿脓杆菌外毒素突变基因PE38KDEL融合,成功地构建了编码免疫毒素cpIL4(13D121E)-PE38KDEL的融合基因.该基因在原核表达系统中得到了高效表达,表达量占细胞全蛋白的30%以上.表达产物经亲和色谱和阴离子交换色谱纯化后,进行细胞毒性实验,证明其对表达型IL-4受体的淋巴瘤细胞Daudi具有良好的细胞毒作用,活性是同类型IL-4免疫毒素的2倍,而对表达型IL-4受体的内皮细胞活性较低.A wide variety of human cancer cells such as glioma and lymphoma express interleukin-4 receptors(IL-4R), therefore, it may be a good option to treat IL-4R-bearing tumor with IL-4-containing immunotoxins. By software analysis, two important amino acids 13T and 121R of (IL-4) were chosen for site-directed mutation. Interleukin-4 mutein cpIL-4(13D121E) was obtained through overlapping PCR and the chimeric immunotoxin was constructed by fusion of the gene encoding cpIL-4(13D121E) to a gene encoding a modified Pseudomonas exotoxin A(PE38KDEL). The chimeric immunotoxin was expressed in E. coli with the yield of about 30% of the total bacterial protein. After being highly purified by affinity chromatography and anion exchange chromatography, the chimeric protein was tested for its cytotoxicity. The data show that cpIL-4(13D121E)-PE38KDEL had improved cytotoxicity to lymphoma cells Daudi expressing class Ⅰ IL-4R in comparison with other IL-4-containing immunotoxin and had a lower cytotoxicity to endothelial cells expressing class Ⅱ IL-4R.

关 键 词：免疫毒素 白细胞介素-4(IL-4) 绿脓杆菌外毒素 

分 类 号：Q784[生物学—分子生物学]