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

在患者知情同意的情况下，从接受外科手术的患者处获得人肾上腺的总RNA。利用引物逆转录酶合成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-前脑啡肽（PPE）基因。聚合酶链反应产物含有全长PPE编码cDNA。将扩增片段克隆到载体pDON-AI-2-Neo或pMEI-5 Neo [Not I-BamHI]中。质粒DNA经酶切纯化，琼脂糖凝胶电泳。pDON-AI-2-Neo治疗感染的疗效更高。

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.