Basic study of gene therapy for severe pain by using virus vector

病毒载体治疗剧烈疼痛基因治疗的基础研究

• 批准号: 17591612
• 负责人: INOMATA Shinichi
• 金额: $ 2.57万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591612/
• 关键词: severe pain; enkephalin; virus vector; gene therapy; ウイルスベクター; ペインクリニック; 疼痛

Total RNA of a human adrenal gland was obtained from patients, received surgical operation, with their informed consents. The cDNA was synthesized by reverse transcriptase using a primer. The pre-pro-enkephalin (PPE) gene was then amplified by polymerase chain reaction with specific upstream and downstream oligoneucleotides. The polymerase chain reaction product contained the full-length PPE coding cDNA. The amplified fragment was cloned into a vector (pDON-AI-2-Neo or pMEI-5 Neo [Not I-BamHI]). The plasmid DNA was purified and digested with restriction enzymes and electrophoresed on agarose gel. The higher efficacy of infection was observed in pDON-AI-2-Neo.

人类肾上腺的总RNA是从接受外科手术的患者身上获得的，并获得了患者的知情同意。使用引物通过逆转录酶合成cDNA。然后通过聚合酶链反应与特定的上游和下游寡核苷酸扩增前脑啡肽原（PPE）基因。聚合酶链式反应产物含有全长PPE编码cDNA。将扩增的片段克隆到载体（pDON-AI-2-Neo 或 pMEI-5 Neo [Not I-BamHI]）中。纯化质粒DNA并用限制性酶消化并在琼脂糖凝胶上进行电泳。在 pDON-AI-2-Neo 中观察到更高的感染效率。