Evaluation and application of activities of anti-HIV・1 ribozyme

抗HIV·1核酶活性评价及应用

• 批准号: 07557078
• 负责人: HATTORI Toshio
• 金额: $ 9.6万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557078/
• 关键词: HIV; Ribozyme; RNA

The ribozyme (Rz) was syntheisizes which recognizes relatively conserved sequences in neutralizing epitope (V3 loop) of envelope protein of HIV-1. Wild tye Rz and phosphorothioate DNA-RNA chimeric ribozyme (thio-Rz) were synthesized. Rz was labeled with fluorescence isithiocyanate and was introduced to cells with transfectam. After delivery into the cells, both wild type and thio-Rz were able to be readily detected. The positive signals faded within 12 hr in the former case, while they lasted 24 hr in the latter case. The thio-Rz as well as wild type ribozyme cleaved RNA transcripts (414 base) of V3 region, and produced the expected products (236 base and 178 base). We have developed a new assay system for envelope medaited fusion assay in which DiI labeled SupT1 cells were co-cultured with gp120 expressing cells (CHO-WT). In this assay, the fusion was associated with a dye transfer to CHO cells. The inhibitory effect by thio-Rz on cell fusion between envelope expressing CHO cells and CD4^+ cells appeared to be time-dependent, and about 70% of inhibition was seen at 72 hr. The mechanisms for inhibition of fusion ishould be clarified.

合成了识别HIV-1包膜蛋白中和表位（V3环）中相对保守序列的核酶（Rz）。合成野生型Rz和硫代DNA-RNA嵌合核酶（thio-Rz）。Rz用荧光isithiocyanate标记，用转染tam导入细胞。在进入细胞后，野生型和硫代rz都能很容易地被检测到。前者的阳性信号在12小时内消退，后者的阳性信号持续24小时。硫代rz和野生型核酶切割V3区414碱基的RNA转录物，产生预期产物（236碱基和178碱基）。我们开发了一种新的检测系统用于包膜介导融合检测，其中DiI标记的SupT1细胞与表达gp120的细胞（CHO-WT）共培养。在这个实验中，融合与染料转移到CHO细胞有关。硫代rz对表达CHO的包膜细胞与CD4^+细胞融合的抑制作用具有时间依赖性，约70%的抑制作用发生在72小时。抑制融合的机制有待澄清。

项目成果

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Xu Younong: "characterization of V3 loop binding proteins of Holt-4 and U937 cells" AIDS Rps.Hum.Retrovir.11. 563-570 (1995)

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