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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。
Objective: To express the fusion protein LFn-MAGE3 and explore the possibility of using non-toxic anthrax toxin in the treatment of tumor. Methods: The coding sequence of LFn was introduced into PET21a expression vector to construct the LFn fusion expression vector PET21a-LFn. The full-length MAGE-3 gene was inserted into the LFn fusion expression vector to construct a PET21a-LFn-MAGE3 fusion protein expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) to induce the expression of LFn-MAGE3 fusion protein. The expression product was purified using a QsepharoseFF ion exchange column and a PheHP hydrophobic column. LF toxicity test and cell immunofluorescence assay to detect the biological activity of the fusion protein. Results: The fusion protein LFn-MAGE3 was expressed in Escherichia coli BL21a intracellular soluble. After purification, the LFn-MAGE3 fusion protein was obtained. Immunofluorescence experiments showed that LFn-MAGE3 efficiently enters macrophages under the cooperation of PA (anthrax protective antigen). CONCLUSION: The expressed and purified LFn-MAGE3 fusion protein can efficiently enter cells in PA and can be used in the animal experiment of tumor immunotherapy in the next step.