人白细胞介素17基因探针制备、克隆及在冠心病中的应用

Cloning and Probe Preparation of Human Interleukin-17 Gene and It's Application for Patients with Coronary Disease

为探讨人白细胞介素 17冠心病在发生发展中的作用。我们应用逆转录多聚酶链反应技术 ,利用自行设计合成的引物 ,从植物血凝素活化的正常人外周血单个核细胞中 ,扩增出人白细胞介素 17基因 ,成功构建了人白细胞介素 17基因重组体 ,经测序确认为人白细胞介素 17基因 ,同时利用随机引物标记基因探针 ,经核酸杂交显示冠心病患者人白细胞介素 17mRNA表达明显增加 ,其相应血清标本中可溶性细胞间粘附分子 - 1含量亦增高。说明人白细胞介素 17在冠心病发病机制中起着重要作用 ,并为进一步研究人白细胞介素 17的生物功能提供可靠的物质基础。

Aim To explore human interleukin-17 (hIL-17)gene in the pathogenesis of coronary disease. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to amplified hIL-17 gene from activated peripheral blood mononuclear cells (PBMC), hIL-17 gene was confirmed by DNA sequencing. Expression of hIL-17 mRNA in PBMC was determined by dot hybridization techniques. Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) was determined by enzyme linked immunosorbent assay (ELISA) method. Results With reference to the published sequence of the human IL-17 gene, a pair of DNA primer were designed and synthesized. A 473 bp fragment encoding fall length of hIL-17 cDNA gene was successfully amplified by using RT-PCR, and then cloned into pUC18 vector. The recombinant plasmid was sequenced to confirm hIL-17gene. Meanwhile, Using random-prime synthesis of probe for hIL-17 gene. We apply for dot hybridization techniques to determine the over expression of hIL-17 mRNA in peripheral blood monocytes (PBMC) with coronary disease, and their high levels of sICAM-1. Conclusions hIL-17 gene may play an important role in the pathogenesis of coronary Disease. Our experiment also lay a profound foundation for further analysis of hIL-17 biological functions.