摘要
目的:构建真核表达IL-18-GPI融合基因的表达载体,观察融合蛋白在哺乳动物细胞中的表达动力学及生物学活性,为进一步研究细胞因子作为免疫增强剂在临床肿瘤治疗中的应用奠定基础。方法:通过RT-PCR的方法从脂多糖刺激的外周血淋巴细胞中钓取IL-18基因,通过共用限制性酶切位点与GPI相连形成pcDNA-IL-18-GPI真核表达质粒。将质粒转染CHO细胞后建立稳定表达融合蛋白的细胞株用于融合蛋白的提取。对提取纯化的蛋白进行生物学特性、蛋白质转移分析和γ-IFN诱导实验。结果:获得714bp的核酸序列并构建重组质粒pcDNA-IL-18-GPI。SDS-PAGE和West-blot显示在CHO细胞中表达的IL-18-GPI融合蛋白分子量约为27.5kD,该蛋白具有明显的蛋白转移和诱生γ-IFN的作用。结论:IL-18-GPI融合蛋白是一种潜在的肿瘤疫苗增强剂,具有良好的应用前景。
Objective：Express GPI anchored IL-18 protein in mammalian cells,observe its biological characters and activities in vitro so as to lay a foundation to finally provide clinic an immunopotentiator in tumor therapy. Methods：The human IL-18 gene was amplified by RT-PCR from human peripheral lymphocytes chased by LPS and the product was ligated to GPI gene followed by a subclone into an eukaryotic expression vector pcDNA3. Identified plasmid was transfected into CHO cell lines and GPI anchored IL-18 protein was expressed in a stable cell line. The purified protein then was identified by SDS-PAGE, western blot, protein transfer assay and γ-IFN release assay. Result：The GPI anchored IL-18 protein expressed in stable mammalian cell line showed an about 27.5 Kd band in SDS-PAGE and this band could be recognized by IL-18 specific antibody. When cocultured the GPI anchored protein with PBMC in the presence of ConA,the γ-IFN released by ceils to the supernatant increased and the protein transfer was observed when cocultured them with tumor cell line SMMC-7721. Conclusion：The GPI anchored IL-18 is a potential immune enhancer in tumor therapy which may be used as a immune adjuvant in tumor vaccine.
出处
《科技通报》
北大核心
2009年第4期445-450,455,共7页
Bulletin of Science and Technology
基金
浙江省科技计划项目
重点科研社会发展项目(2006C23024)
浙江省自然科学基金(YZ080889)
