RELATIONSHIP OF STRUCTURE & FUNCTION OF HU IFN-GAMMA BY SITE-DIRECTED MUTAGENESIS

已有研究表明，rhu-1~7或9个残基的缺失 -伽马导致生物活性丧失(H.Hogrefe等人参考J. 比奥尔。化学。21,12179-86年，1989年)。由于材料的数量有限， 可以通过酶修饰来分离，我们使用的是一种蛋白质 研究结构与结构之间关系的工程方法 人干扰素-γ的功能。人干扰素-γ基因已克隆到 PGEM-2载体和PKK223-3干扰素-γ基因表达载体 建造的。免疫沉淀法和十二烷基硫酸钠凝胶电泳法显示细胞 在IPTG诱导下，在E.ColiJM105中产生人干扰素-γ。 利用聚合酶链式反应和寡核苷酸定向突变，几个突变基因 在N端包含截断或替换的部分将被获得。一些人 将突变基因克隆到表达载体中，并对其进行鉴定。

It has been shown that deletion of residue -1 to 7 or 9 residues of rhu IFN -gamma results in the loss of biological activity (H. Hogrefe et al ref. J. Biol. Chem. 21, 12179-86, 1989). Due to the limited amount of material that can be isolated by enzymatic modification, we are using a protein engineering approach to study the relationship between the structure and function of human IFN-gamma. The human IFN-gamma gene has been cloned into PGEM 2 vector and PKK223-3 IFN-gamma gene expressing vector has been constructed. Immunoprecipitation assay and SDS PAGE revealed that cells induced by IPTG produced human IFN-gamma in E. coli JM105. Using PCR and oligonucleotide directed mutagenesis, several mutant genes which contain truncate or replacement at N-terminal will be obtained. Some mutant genes will be cloned into the expression vector, and characterized.