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

人肾上腺的总RNA是在患者知情同意的情况下从患者那里获得的，接受了手术。用逆转录酶合成了该基因的c DNA。然后通过聚合酶链式反应扩增前脑啡肽原(PPE)基因的上游和下游的特定寡核苷酸。聚合酶链式反应产物含有编码全长的PPE基因。将扩增片段克隆到载体(pDON-AI-2-Neo或pMEI-5 Neo[Not I-BamHI])中。纯化后用限制性内切酶酶切，琼脂糖凝胶电泳法鉴定。PDON-AI-2-Neo的感染效率较高。

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.