摘要
目的:表达融合蛋白LFn-MAGE3,探讨无毒炭疽毒素用于治疗肿瘤的可能性。方法:将LFn的编码序列导入PET21a表达载体中,构建LFn融合表达载体PET21a-LFn。将全长MAGE-3基因插LFn融合表达载体中,构建了PET21a-LFn-MAGE3融合蛋白表达载体。将该重组质粒转化大肠杆菌BL21(DE3)中,诱导表达LFn-MAGE3融合蛋白。表达产物用QsepharoseFF离子交换柱和PheHP疏水柱进行纯化。LF毒性抑制实验和细胞免疫荧光实验检测融合蛋白的生物学活性。结果:融合蛋白LFn-MAGE3在大肠杆菌BL21a获得胞内可溶性表达,经纯化后,获得一定纯度的LFn-MAGE3融合蛋白。细胞免疫荧光实验显示LFn-MAGE3能在PA(炭疽保护性抗原)协同下高效进入巨噬细胞。结论:表达纯化获得的LFn-MAGE3融合蛋白能在PA协助高效进入细胞,可以用于下一步的肿瘤免疫治疗的动物实验中。
AIM： To explore the possibility of using the nontoxic form of anthrax toxin in cancer immunotherapy by LFn-MAGE3 fusion protein expression. METHODS： A fusion expression vector named PET21 a-LFn was constructed by inserting LFn coding senquence into PET21a. PET21a- LFn-MAGE3 fusion protein expression vector was construc- ted by cloning the whole MAGE-3 gene into plasmid PET21a-LFn. Q sepharose FF and Phe HP columns were employed to purify the fusion protein. The biological activity of LFn-MAGE3 was determined by cell test of repressing the cytotoxity of LF and the tests of immunofluscence of mouse macrophage. RESULTS： The resulting plasmid expressed fusion protein LFn-MAGE3 in the soluble form in E. coil BL21, the cell tests showed that purified LFn-MAGE fusion protein was delivered into macrophage effectively with the help of PA （ anthrax protective antigen ）. CONCLUSION： The successful delivery of fusion protein into macrophages coordinated by PA may lay the foundation for its further use in cancer immunotherapy in animal experiments.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期181-184,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(No.30400545)
