人前脑啡肽原基因逆转录病毒载体的构建及其表达

发布时间：2018-03-25 05:09

  本文选题：前脑啡肽原基因　切入点：逆转录病毒　出处：《郑州大学》2006年硕士论文

【摘要】：慢性疼痛，尤其是癌性疼痛和顽固性疼痛一直是医疗界的一道难题。传统的镇痛药尚不能有效治疗慢性疼痛尤其癌痛。脑啡肽作为一种内源性阿片肽类镇痛药受到关注，用于许多动物实验已证明有明显的镇痛效应。将前脑啡肽原基因做为目的基因，利用逆病毒载体，经过包装细胞系的包装，产生的病毒可将前脑啡肽原基因整合到靶细胞NIH3T3细胞的基因组中，制造出可以分泌镇痛物质脑啡肽的工程化细胞系，利用人工大量获得。本实验采用pLNCX2载体，其中5′LTR(包括病毒启动子／增强序列)可控制neo~r基因的表达，用于真核细胞中抗生素筛选。目的基因可克隆进多克隆位点，在P_(CMVIE)控制下表达。pLNCX_2含有包装信号Ψ序列，但没有gag、pol和env等编码病毒结构蛋白的基因，须通过包装细胞系(PT67等)的包装。因此，当转染细胞增殖传代时，其形成复制完整病毒能力的的机会极少。其包装产生的病毒，携带前脑啡肽源基因，，可作为疼痛基因治疗的实验研究的有力工具。 目的 研究利用RT-PCR技术，可否获得人前脑啡肽原基因，并采用pLNCX2逆转录病毒载体，将外源性脑啡肽基因导入并整合到靶细胞NIH3T3细胞的基因组中，观察是否有外源性脑啡肽基因的表达，探讨一条有效的治疗疼痛的转基因方法。 方法 1．RT-PCR 根据GenBank报道的人脑啡肽基因序列(NM_006211)，设计引物，其引物5’与3’端分别加上HindⅢ和NotⅠ酶切位点，以便目的基因插入逆转录病毒载体。提取人脑组织总RNA，然后反转录得到包含前脑啡肽原基因的单链总cDNA，
[Abstract]:Chronic pain, especially cancerous pain and intractable pain, has been a difficult problem in the medical field. Traditional analgesics have not been effective in the treatment of chronic pain, especially cancer pain. Enkephalin has attracted much attention as an endogenous opioid peptide analgesics. The proenkephalin gene was used as the target gene in many animal experiments. The virus can integrate the proenkephalin gene into the genome of the target cell NIH3T3 cells and produce an engineering cell line which can secrete the analgesic substance enkephalin. The expression of neo~r gene (including virus promoter / enhancer sequence) can be controlled by 5 LTRs for antibiotic screening in eukaryotic cells. The target gene can be cloned into polyclonal sites and expressed under the control of Pappa CMVIE.pLNCX2 contains the package signal 蠄 sequence. But without genes that encode viral structural proteins such as Gagapol and env, they have to be packaged through a packaging cell line, such as PT67, etc. Therefore, when transfected cells proliferate, they have little chance of forming the ability to replicate complete viruses. Carrying proenkephalin gene can be used as a powerful tool for experimental study of pain gene therapy. Purpose. To study whether human proenkephalin gene can be obtained by using RT-PCR technique, and to introduce exogenous enkephalin gene into the genome of target cell NIH3T3 cells by pLNCX2 retrovirus vector, and to observe whether there is exogenous enkephalin gene expression. To explore an effective transgenic method for the treatment of pain. Method. 1.RT-PCR. According to the sequence of human brain enkephalin gene reported by GenBank, NM006211A was designed and primers were designed. The primers 5 'and 3' end of the primer were digested with Hind 鈪

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