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％.     

YAC介导的天蚕丝素基因向家蚕的转移——Ⅰ.天蚕YAC基因库的构建与丝素基因克隆的鉴定

夭蚕(Antheraea yamamai)是一种经济价值极高的绢丝昆虫,其丝心蛋白基因的表达调控也是研究发育和分化控制的极好模型,对其丝素基因及其相关基因的研究具有重要的理论和实际意义。以夭蚕五龄期幼虫的后丝腺为材料,pYACA为载体构建了插入片段平均长度为570kb的夭蚕YAC基因库,并以多聚酶链式反应(PCR)方法对该基因库进行筛选,得到了一组天蚕丝素基因的YAC克隆。其中Afy-1全长为440kB,覆盖了整个丝素基因的编码区及上、下游调控序列,并在50代后仍然保持稳定。     

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.