人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因的修正及修正基因在大肠杆菌中的表达

本文采用寡核苷酸介导的定位诱变技术修正了人嗜中性白细胞活化蛋白-1/白细胞介素-8合成基因中出现的合成错误,在该基因编码区的201位插入了原来缺失的G残基,从而使之恢复正确读框。经对修正基因进行DNA全序列测定,表明定位诱变的结果符合设计要求。在此基础上,利用原核高效表达载体pBV220在P_RP_L串联启动子的控制下在大肠杆菌中对该基因进行了表达,并测定了重组人嗜中性白细胞活化蛋白-1/白细胞介素-8的嗜中性白细胞趋化活性。本工作为开展人嗜中性白细胞活化蛋白-1/白细胞介素-8基因工程及蛋白质工程的研究奠定了基础。

Generation of a Revised Synthetic Gene Coding for Human Neutrophil Activating Protein-1/Interleukin-8 and Its Expression in Escherichia coli

A synthesis error occured in one of the cloned synthetic gene coding for human neutrophil activating protein-l/interleukin-8 was revised by inserting a single G residue in position 201 of the gene via oligonucleotid-mediated site-directed muta-genesis.The resulting gene was characterized by digestion with restriction enzymes and the mutation was confirmed by DNA sequencing of the complete coding region.The revised gene was then expressed efficiently in E.coli with a vector pBV220 under the control of bacteriophage **********A PRPL tandem promoters.The neutrophil chemo-tactic activity of the recombinant product was assayed using the agarose plate method.The general strategies used in the experiment were also discussed.