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目的利用大肠杆菌表达系统对人肿瘤坏死因子 (hTNFα)的寡聚组氨酸 (6×His)融合蛋白进行表达和纯化研究。 方法采用大肠杆菌表达系统的表达载体 pET - 2 8a(+)和 pET - 2 2b(+)表达了hTNFα 的 6×His融合蛋白 ,其 6×His分别位于融合蛋白的N和C端 ,并利用寡聚组氨酸与过渡态金属离子的高亲和力性质 ,经Ni2 +-IDASepharose 6B亲和柱对表达产物进行了纯化。 结果其表达量分别为菌体总蛋白的 45 %和 8%。前者在胞内以不溶性包涵体存在 ,未对表达产物作进一步纯化 ;后者在胞质空间以可溶性存在 ,经亲和纯化的hTNFα- 6×His纯度可达 90 %以上 ,得率为 0 .4mg/ 10 0ml,并对L92 9细胞具有杀伤的功能 ,其活力为 5 .42× 10 4 U/mg。 结论表达产物经纯化后具有细胞毒的活性。
Objective To express and purify the oligomeric histidine (6 × His) fusion protein of human tumor necrosis factor (hTNFα) by using E. coli expression system. Methods The 6 × His fusion protein of hTNFα was expressed in E. coli expression vector pET - 2 8a (+) and pET - 2b (+). The 6 × His was located on the N and C terminus of the fusion protein respectively. The high affinity of oligohistidine with transition metal ions was confirmed by Ni2 + -IDASepharose 6B affinity column. The results of the expression of the total bacterial protein 45% and 8%. The former existed as insoluble inclusion bodies in the intracellular and did not further purify the expressed product. The latter was soluble in the cytoplasm. The purity of the purified hTNFα-6 × His was over 90% with a yield of 0. 4mg / 10ml, and the killing of L92 9 cells function, the viability of 5. 42 × 10 4 U / mg. Conclusion The expressed product has cytotoxic activity after purification.