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基于对有关肿瘤坏死因子结构和功能关系研究资料的分析,设计了一种缺失了Gln 102～Glu 107位共6个氨基酸的新型肿瘤坏死因子。首先,利用PCR技术分析扩增出Vall～Cys 101和Gly 108～Leu 157基因片段,连接后再克隆到表达载体pJLA503中,转化大肠杆菌,发酵时进行42℃诱导表达,得到了新型肿瘤坏死因子CG-hTNF的产物。经过对pCG-hTNF中克隆的基因进行DNA测序鉴定,证明其顺序完全正确。发酵获得的CG—hTNF的表达量约占总蛋白量的15％,经测定其体外细胞毒活性,计算得出的比活性比原型RL-hTNF仅提高了10倍以上,说明肿瘤坏死因子中Gln 102与Glu 107区域对TNF与其受体的结合没有多大影响。对CG—hTNF在小鼠体内的半数致死剂量(LD_(50))进行测定,以mg/kg体重表示CG-hTNF与原型肿瘤坏死因子的毒性基本一致。
Based on the analysis of data on the relationship between tumor necrosis factor structure and function, a novel tumor necrosis factor (TNF) deleted 6 amino acids at position 107 of Gln 102 Glu was designed. First, the gene fragments of Vall ~ Cys 101 and Gly 108 ~ Leu 157 were amplified by PCR and cloned into the expression vector pJLA503. The recombinant plasmid was transformed into E. coli and induced to express at 42 ° C during fermentation. A novel tumor necrosis factor Product of CG-hTNF. After DNA sequencing of the cloned gene in pCG-hTNF, the sequence was proved to be correct. The expression of CG-hTNF was about 15% of the total protein. The cytotoxic activity of CG-hTNF in vitro was only 10-fold higher than that of the original RL-hTNF, indicating that Gln The 102 and Glu 107 regions have little effect on the binding of TNF to its receptor. The median lethal dose (LD_ (50)) of CG-hTNF in mice was determined. The mg / kg body weight of CG-hTNF was consistent with that of the original tumor necrosis factor.