【摘要】：目前,傳統(tǒng)的鎮(zhèn)痛藥尚不能有效治療慢性疼痛及癌痛。腦啡肽作為一種鎮(zhèn)痛藥受到關(guān)注,許多動物實驗已取得成功。利用逆轉(zhuǎn)錄病毒載體,經(jīng)過包裝細(xì)胞系的包裝,可將前腦啡肽原基因整合到靶細(xì)胞基因組中,以達到長期鎮(zhèn)痛效果。本實驗采用pLNCX2載體,其含有包裝信號ψ序列,但沒有g(shù)ag、pol和env等編碼病毒結(jié)構(gòu)蛋白的基因,須通過包裝細(xì)胞系(PT67等)的包裝。因此,當(dāng)轉(zhuǎn)染細(xì)胞增殖傳代時,其形成復(fù)制完整病毒能力的的機會極少。其包裝產(chǎn)生的逆轉(zhuǎn)錄病毒,進一步轉(zhuǎn)染NIH3T3細(xì)胞,達到前腦啡肽原基因的長期穩(wěn)定表達。 目的 1.探討利用RT-PCR能否獲得前腦啡肽原基因; 2.探討能否篩選出高滴度的產(chǎn)病毒細(xì)胞克隆; 3.探討前腦啡肽原基因是否能夠長期穩(wěn)定表達,滿足鎮(zhèn)痛需要。 方法 1.RT-PCR 根據(jù)GenBank報道的大鼠前腦啡肽原基因序列(NM 017139),設(shè)計引物,其引物5′與3′端分別加上HindⅢ和ClaⅠ酶切位點,以便整和入逆轉(zhuǎn)錄病毒載體。提取大鼠腦組織總RNA。反轉(zhuǎn)錄得到大鼠腦啡肽基因的單鏈cDNA,PCR擴增反轉(zhuǎn)錄產(chǎn)物。 2.PCR產(chǎn)物測序 瓊脂糖電泳,回收目的條帶,同T載體連接。轉(zhuǎn)化大腸桿菌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，

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