前脑啡肽原基因在NIH3T3细胞的表达
发布时间：2018-12-16 12:54
【摘要】：目前,传统的镇痛药尚不能有效治疗慢性疼痛及癌痛。脑啡肽作为一种镇痛药受到关注,许多动物实验已取得成功。利用逆转录病毒载体,经过包装细胞系的包装,可将前脑啡肽原基因整合到靶细胞基因组中,以达到长期镇痛效果。本实验采用pLNCX2载体,其含有包装信号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。其包装产生的逆转录病毒,进一步转染NIH3T3细胞,达到前脑啡肽原基因的长期稳定表达。 目的 1.探讨利用RT-PCR能否获得前脑啡肽原基因; 2.探讨能否筛选出高滴度的产病毒细胞克隆; 3.探讨前脑啡肽原基因是否能够长期稳定表达,满足镇痛需要。 方法 1.RT-PCR 根据GenBank报道的大鼠前脑啡肽原基因序列(NM 017139),设计引物,其引物5′与3′端分别加上HindⅢ和ClaⅠ酶切位点,以便整和入逆转录病毒载体。提取大鼠脑组织总RNA。反转录得到大鼠脑啡肽基因的单链cDNA,PCR扩增反转录产物。 2.PCR产物测序 琼脂糖电泳,回收目的条带,同T载体连接。转化大肠杆菌JM109,
[Abstract]:At present, traditional analgesics can not effectively treat chronic pain and cancer pain. Enkephalin has attracted much attention as a kind of analgesics, and many animal experiments have been successful. The proenkephalin gene can be integrated into the target cell genome by retrovirus vector and packaged in the packaging cell line to achieve long-term analgesic effect. In this study, pLNCX2 vector was used, which contained package signal 蠄 sequence, but without gag,pol and env genes encoding viral structural proteins, it had to be packaged by packaging cell line (PT67 et al.). Thus, when transfected cells proliferate and subculture, they have little chance of forming the ability to replicate intact viruses. The retrovirus was further transfected into NIH3T3 cells to achieve long-term stable expression of proenkephalin gene. Objective 1. To investigate whether proenkephalin gene can be obtained by using RT-PCR. To explore the possibility of screening high titer of virus-producing cell clones; 3. To investigate whether proenkephalin gene can be expressed stably for a long time to meet the need of analgesia. Methods 1.RT-PCR designed primers according to the sequence of rat proenkephalin gene (NM 017139) reported by GenBank. The primers 5'and 3'were digested with Hind 鈪，
本文编号：2382381
[Abstract]:At present, traditional analgesics can not effectively treat chronic pain and cancer pain. Enkephalin has attracted much attention as a kind of analgesics, and many animal experiments have been successful. The proenkephalin gene can be integrated into the target cell genome by retrovirus vector and packaged in the packaging cell line to achieve long-term analgesic effect. In this study, pLNCX2 vector was used, which contained package signal 蠄 sequence, but without gag,pol and env genes encoding viral structural proteins, it had to be packaged by packaging cell line (PT67 et al.). Thus, when transfected cells proliferate and subculture, they have little chance of forming the ability to replicate intact viruses. The retrovirus was further transfected into NIH3T3 cells to achieve long-term stable expression of proenkephalin gene. Objective 1. To investigate whether proenkephalin gene can be obtained by using RT-PCR. To explore the possibility of screening high titer of virus-producing cell clones; 3. To investigate whether proenkephalin gene can be expressed stably for a long time to meet the need of analgesia. Methods 1.RT-PCR designed primers according to the sequence of rat proenkephalin gene (NM 017139) reported by GenBank. The primers 5'and 3'were digested with Hind 鈪，
本文编号：2382381
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