摘要
以人粒细胞-巨噬细胞集落刺激因子(GM-CSF)受体(GM-CSFR)为靶向的白喉毒素(DT)与GM-CSF免疫毒素DT386-GMCSF为急性髓系白血病提供了一种新的替代治疗途径,但该蛋白在E.coli中的表达量很低,难以进行工业化生产。为探索造成其低表达的关键影响因素,对DT386-GMCSF中的GM-CSF进行了C端的截短表达,发现GM-CSF中L114编码序列可明显影响融合蛋白的表达量。在此基础上,构建了一系列突变体,发现保留GM-CSF1-123位氨基酸且将L114L115V116突变为G114V115T116的突变体DF123GVT的表达量高于DT386-GMCSF,且对来源于高表达GM-CSF受体的HL60细胞的肿瘤单细胞具有相似的细胞毒作用。DF123GVT突变体的获得为GM-CSFR靶向的免疫毒素的开发应用打下了基础。
The development of immunotoxin DT386-GMCSF, a usion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage cclony-stimulating factor （ GM-CSF） and targets the GM-CSF receptor （GM-CSFR） , has provided a promising alternative therapy to the acute myeloid leukemia （AML）. However, the poor expression of the protein in E. coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E. coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF Whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT356 -GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第6期38-45,共8页
China Biotechnology
基金
国家自然科学基金资助项目(30300412)
关键词
免疫毒素
白喉毒素
粒细胞-巨噬细胞集落刺激因子
原核表达
突变体
Immunotoxin Diphtheria toxin Granulocyte-macrophage colony-stimulating factor Prokaryotic expression Mutant
