半合成人肿瘤坏死因子cDNA的构建及其在大肠杆菌中的高表达

本文报道从人基因文库中分离淋巴毒素(LT)基因的同时,克隆了肿瘤坏死因子(TNF)基因,这两个基因相距1.2kb.TNF基因有4个外显子,第4外显子编码TNF成熟蛋白157个氨基酸中的140个.将第4外显子切出一部分,再人工合成编码其余氨基酸的DNA片段,两者连接构成重组的人TNF(rhTNF)cDNA,并克隆在大肠杆菌表达载体中成功地得到表达.5 l罐发酵得菌体约20g/l,以L929为靶细胞测定细胞毒活性为10~6-10~7单位/ml.高压液相色谱仪分离纯化rhTNF,冻干后得白色粉剂.测定了这种rhTNF的氨基端的10个氨基酸序列,证明与天然的人TNF完全相同.纯度约为95％.     

EXPRESSION OF A PARTIAL SYNTHETIC HUMAN TNF cDNA IN E. coli

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exou codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fer1entator was used to produce rhTNF. About 20g (wet weight) of bacterial pellet per liter medium and 106—10~7 units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer, rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.