人前脑啡肽原基因真核表达载体的构建

Construction of recombinant pCDNA3.1 （ ＋ ） - PENK vector

目的 构建人前脑啡肽原基因真核表达载体，为进一步研究内源性脑啡肽的镇痛活性及相关药物研发奠定基础。方法 取新鲜的人脑颞叶皮质组织，用提取RNA的试剂盒提取总RNA，逆转录成cDNA。设计两对引物，用巢式PCR的方法获得人脑啡肽原基因，并将其插入到克隆载体pMD18-T中，克隆扩增。双酶切真核表达质粒pCDNA3．1（＋）及T载体中的目的片段，胶回收重组构建成pCDNA3．1（＋）-PENK表达载体。最后限制性酶切鉴定该表达载体。结果 采用巢式RT—PCR技术可以获得人前脑啡肽原基因，经测序证明其碱基序列为编码目的基因的正确序列。凝胶电泳结果证明已将此片段克隆到pCDNA3.1（＋）内。结论 成功构建了pCDNA3．1（＋）-PENK真核表达质粒。可作为疼痛基因治疗实验研究的有力工具。

Objective To construct the recombinant pCDNA3. 1 （ ＋ ） - PENK vector encoding human enkephalin gene. Methods Human Enk gene was cloned by RT - PCR and inserted into pCDNA3.1 （ ＋ ） vector, Two pairs of primers were designed in which the restriction sites were compatible with the clone sites of the pCDNA3.1 （ ＋ ） vector. Using these primers Homosapiens proenkephalin （PENK）was amplified by polymerse chain reaction （PCR）. Then it was inserted into the vector pcdna3.1 （ ＋ ）. Results The recombinant retrovirus vector was constructed. Conclusion This recombinant plasmid may be a useful tool for gene therapy of pain and makes it possible to carry out further research in gene therapy of analgesic.