重组铜绿假单胞菌外毒素PE38KDEL的克隆、表达与活性检测  

Cloning, expression and cytotoxicity of Pseudomonas aeruginosa exotoxin PE38KDEL with high content of GC

作　　者：崔京霞[1] 纪剑飞 倪剑锋[1] 吴文芳[1] 

机构地区：[1]中国科学院沈阳应用生态研究所微生物工程室,中国科学院研究生院,沈阳110016 [2]Beckman Research Institute,City of Hope National Medical Center,California USA

出　　处：《中华微生物学和免疫学杂志》2004年第12期985-990,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的对铜绿假单胞菌外毒素A的编码序列进行改造,以获得相对分子质量小、活性强的重组毒素PE38KDEL。方法基于铜绿假单胞菌外毒素A的编码序列的高GC含量,通过PCR反应和酶切技术,构建重组毒素PE38KDEL基因,并于大肠杆菌表达,用Westernblot分析表达产物。为检测其生物活性,将其与靶向分子IL4突变体cpIL4(13D)融合,构建融合蛋白表达载体,并于大肠杆菌表达,表达产物经亲和色谱和阴离子交换色谱纯化后,用MTT法检测其对表达IL4受体的黑色素瘤细胞LiBr的细胞毒性。结果成功构建了PE38KDEL基因,并在大肠杆菌BL21(DE3)中得到了高效表达,表达量占细胞全蛋白的21%左右;构建了融合蛋白cpIL4(13D)PE38KDEL,并于大肠杆菌AD494(DE3)中得到高效表达,纯化后融合蛋白的纯度达95%以上,细胞毒实验证明其对黑色素瘤细胞LiBr具有良好的杀伤作用,这说明改造后的PE38KDEL是具有细胞毒效应的。结论重组毒素PE38KDEL的构建,为各种导向药物的构建奠定了基础。Objective To obtain a modified exotoxin PE38KDEL with low molecular mass and high activity. Methods PCR and enzyme digestion technology were used to construct the recombinant exotoxin PE38KDEL. The resulting gene encoding PE38KDEL was expressed in E.coli and the expression product was identified by Western blot assay. In order to test the biological activity of PE38KDEL, a fusion protein was constructed by genetically fusing PE38KDEL to interleukin-4 mutant cpIL4(13D) and was expressed in E.coli. After purificatioin, the fusion protein was tested for its cytotoxicity to melanoma cell line LiBr that expresses IL-4R using MTT technology. Results The gene of PE38KDEL was successfully constructed and expressed in E.coli BL21(DE3). Fusion protein cpIL4(13D)-PE38KDEL was constructed and expressed in E.coli AD494(DE3). The purity of the fusion protein was above 95%. cpIL4(13D)-PE38KDEL showed significant cytotoxicity to IL4R-bearing tumor cell line LiBr. Conclusion The construction of PE38KDEL laid a solid foundation for the development of immunotoxins for potential tumor therapy.

关 键 词：PE38 铜绿假单胞菌 大肠杆菌表达 外毒素A IL-4 重组毒素 融合蛋白 高效表达 GC含量 PCR反应 

分 类 号：R346[医药卫生—基础医学]