应用基因工程方法制备新型重组人肿瘤坏死因子-α

Preparation of a novel recombinant human tumor necrosis factor-α by gene engineering technology

目的 应用基因工程技术 ,制备一种新型的重组人肿瘤坏死因子 α (novelrecombinanthumantumornecrosisfactor α ,nrhTNF α) ,并对其生物学活性、理化性质进行鉴定 ,为进入临床前研究提供基础。方法 应用PCR技术 ,将hTNF α基因的 5′端 17个氨基酸的编码序列删除 ,基因中Pro8Ser9Asp10 的编码序列用Arg Lys Arg的编码序列取代 ,同时Leu157的密码子被Phe的密码子所取代。将hTNF α突变基因 ,插入原核高效表达载体pBV2 2 0中 ,构建高表达工程菌株。纯化表达产物 ,对连续 3批制备的新型rhTNF α,按人用《重组DNA制品质量控制要点》检定要求进行鉴定。结果DNA序列分析和蛋白质N末端、C末端部分氨基酸序列分析表明 ,nrhTNF α与天然的hTNF α相比较 ,N末端缺失了 7个氨基酸 ,13位氨基酸为Arg Lys Arg ,其后为天然hTNF α 11位以后的氨基酸。 15 7位Leu的密码子被Phe的密码子所取代。产物表达量占菌体蛋白的 6 7.4 %。经 (NH4) 2 SO4沉淀、Q SepharoseF .F .及S SepharoseF .F .柱层析分离纯化后 ,产品的纯度达 99% ,比活性达 1× 10 9IU/mg蛋白。结论成功地制备了nrhTNF ,对连续 3批制备的nrhTNF α按人用《重组DNA制品质量控制要点》检定要求进行鉴定 。

Aim To prepare a novel recombinant tumor necrosis factor α(nrhTNF) by gene engineering technology. Methods hTNF α gene was mutated by deleting 7 amino acids at the N terminus, replacing Pro 8 Ser 9 Asp 10 by Arg Lys Arg, and substituting Leu 157 with Phe. The mutant rhTNF α gene (nrhTNF) was inserted into pBV220 and then E.coli DH5α was transformed for protein expression. Results The DNA sequence of the nrhTNF α gene and amino acid sequence of the expression protein were correct. The expression level of nrhTNF α was 67.4%. With a simple purification procedure, purity and specific activity of the novel rhTNF α achieved 99% and 1×10 9 Unit/mg protein, respectively. Conclusion The nrhTNF α has been obtained successfully. nrhTNF α is analyzed according to “the Quality Control Procedure of Recombinant DNA Products”, and all items are qualified.