LFn-MAGE3融合蛋白表达及生物学功能初步研究

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。

Preliminary study of LFn-MAGE3 fusion protein expression and its biological function

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 PET21a-LFn was constructed by inserting LFn coding senquence into PET21a. PET21a-LFn-MAGE3 fusion protein expression vector was constructed 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.coli 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.