RELATION OF STRUCTURE AND FUNCTION OF HU IFN-GAMMA BY SITE-DIRECTED MUTAGENESIS

已经表明，IFN-γ的残基-1到7或9个残基的缺失， γ导致生物活性的丧失。由于局限性 化学修饰程序，我们使用的是蛋白质工程， 结构与功能关系的研究方法 IFN-γ。 已将Hu IFN-γ基因克隆到pGitem 2载体中 和PKK 223-3、PKK 233-2表达载体。 我们获得了高表达 IFN-γ的2种形式，截短形式和完整形式。 IFN- 还使用Genentech提供的W3110/PIIF-cye表达γ基因， Inc. 我们还构建了IFN-γ的T7 RNA聚合酶/启动子系统 基因表达。 在该系统中，IFN-γ基因通过 用M13噬菌体感染。 Western Blot、SDS PAGE和抗病毒活性 这些细胞经IPTG或IAA诱导后， Hu IFN-γ。 我们用单克隆抗体纯化了IFN-γ 层析和SephacrylS-200凝胶过滤。 在未来的研究中，我们将通过PCR技术制备嵌合和突变的Hu IFN-γ 或定点突变来阐明结构之间的关系 和Hu IFN-γ的功能。

It has been shown that deletion of residue -1 to 7 or 9 residues of IFN- gamma results in the loss of biological activity. Due to the limitations of chemical modification procedures, we are using a protein engineering approach to study the relationship between the structure and function of IFN-gamma. The Hu IFN-gamma gene has been cloned into a pGitem 2 vector and pkk 223-3, pkk 233-2 expression vector. We obtained high expression of 2 forms of IFN-gamma, a truncated form and an intact form. The IFN- gamma gene was also expressed using W3110/PIIF-cye provided by Genentech, Inc. We also constructed T7 RNA polymerase/promoter system for IFN-gamma gene expression. In this system, IFN-gamma gene was expressed by infection with M 13 phage. Western Blot, SDS PAGE and antiviral activity have revealed that these cells which are induced by IPTG or IAA produced Hu IFN-gamma. We have purified the IFN-gamma by monoclonal antibody chromatography and sephacryl s-200 gel filtration. In future studies, we will make chimeric and mutant Hu IFN-gammas by PCR or site-directed mutagenesis to elucidate the relationship of structure and function of Hu IFN-gamma.