醫(yī)學(xué)論文范文:編碼大鼠Nogo受體mRNA的shRNA重組腺病毒載體的構(gòu)建和鑒定

醫(yī)學(xué)論文范文:編碼大鼠Nogo受體mRNA的shRNA重組腺病毒載體的構(gòu)建和鑒定

【摘要】  目的: 構(gòu)建Nogo受體(NgR)特異性shRNA重組腺病毒載體,為應(yīng)用基因沉默技術(shù)從轉(zhuǎn)錄后水平進(jìn)行缺血性腦損傷的基因治療研究奠定基礎(chǔ). 方法: 將前期構(gòu)建的大鼠Nogo受體mRNA的shRNA特異性真核表達(dá)載體pGenesil��1�瞫hRNA的表達(dá)啟動(dòng)子U6連同shRNA亞克隆至穿梭質(zhì)粒pAdTrack,酶切及DNA測(cè)序鑒定后， 將含NgR基因的重組穿梭質(zhì)粒pAdTrack�睻6�瞫hRNA經(jīng)PmeI線性化后轉(zhuǎn)化入pAdEasy��1感受態(tài)細(xì)菌. 將pAd�睻6�瞫hRNA質(zhì)粒經(jīng)PacI線性化后轉(zhuǎn)染293細(xì)胞，包裝重組腺病毒Adeno�睻6�瞫hRNA，并進(jìn)行PCR鑒定、PCR產(chǎn)物測(cè)序及病毒滴度測(cè)定. 結(jié)果: 結(jié)果證實(shí)pAdTrack�睻6�瞫hRNA及pAd�睻6�瞫hRNA質(zhì)粒構(gòu)建正確，收獲病毒后的PCR及DNA測(cè)序結(jié)果證明Adeno�睻6�瞫hRNA包被成功. 結(jié)論: 成功構(gòu)建了大鼠Nogo受體mRNA的shRNA重組腺病毒載體Adeno�睻6�瞫hRNA，將為應(yīng)用基因沉默技術(shù)研究NgR在缺血性腦損傷后神經(jīng)再生中的作用奠定基礎(chǔ).

【關(guān)鍵詞】  Nogo受體；RNA干擾；腺病毒

Construction and identification of recombinant adenovirus expressing the shRNA of rat Nogo��66 receptor

ZHANG Qin�睱i1, QIN Xin�瞃ue1, YIN Cheng1, PENG Yan2

1Department of Neurology, First Affiliated Hospital, 2Department of Histology and Embryology, Chongqing Medical University, Chongqing 400016, China醫(yī).學(xué).全.在.線m.gydjdsj.org.cn

【Abstract】 AIM: To construct the recombinant adenovirus vector of shRNA targeted to the rat Nogo��66 receptor gene for the therapy of ischemic brain injury in post�瞭ranscriptional regulation. METHODS: The used pGenesil��1�瞫hRNA was construsted and identified in previous experiment. The U6 and shRNA of pGenesil��1�瞫hRNA were subcloned to pAdTrack. The product pAdTrack�睻6�瞫hRNA was linearized by Pme I to mediate homologous recombination with pAdEasy��1 in pAdEasy��1 host bacteria. The positive clone was identified by enzyme digestion, PCR analysis and DNA sequence analysis. After linearized by Pac I, the recombinant adenovirus DNA pAd�睻6�瞫hRNA was transfected into 293 cells for packaging and amplification of Adeno�睻6�瞫hRNA. Adeno�睻6�瞫hRNA was further identified by PCR analysis and DNA sequence analysis. RESULTS: PCR analysis, enzyme digestion and DNA sequence analysis proved the pAdTrack�睻6�瞫hRNA and the pAd�睻6�瞫hRNA had been successfully constructed. After being packaged in 293 cells, the recombinant adenovirus Adeno�睻6�瞫hRNA was identified by PCR analysis and DNA sequence analysis. CONCLUSION: We have successfully constructed recombinant adenovirus Adeno�睻6�瞫hRNA targeted against the rat Nogo��66 receptor gene. It will be helpful to use RNAi in the research of the role of NgR in neural regeneration after cerebral ischemic injury.

【Keywords】 Nogo��66 receptor; RNAi; adenoviruses

0引言

缺血性腦損傷后中樞神經(jīng)的再生是神經(jīng)康復(fù)難以逾越的障礙，近來(lái)研究發(fā)現(xiàn)，中樞神經(jīng)再生困難的原因是CNS三種軸突再生抑制蛋白的存在，它們是Nogo�睞,MAG和OMgp，與共同的受體NgR結(jié)合，激活下游信號(hào)及GTPase Rho系統(tǒng)，導(dǎo)致軸突生長(zhǎng)錐崩潰［1］. 因此，NgR成為理想的治療靶. RNA干擾(RNAi)是由雙鏈RNA介導(dǎo)的、在轉(zhuǎn)錄后mRNA水平關(guān)閉相應(yīng)基因表達(dá)過(guò)程-將與靶基因同源的21~23 bp的雙鏈RNA導(dǎo)入細(xì)胞,它在細(xì)胞中可與靶基因mRNA結(jié)合,并迅速將其降解,從而抑制該基因的表達(dá)的過(guò)程［2］.而成功地進(jìn)行RNAi的關(guān)鍵在于使siRNA導(dǎo)入細(xì)胞，我們利用前期實(shí)驗(yàn)已構(gòu)建的NgR shRNA真核表達(dá)載體，進(jìn)一步構(gòu)建腺病毒表達(dá)載體，通過(guò)病毒載體導(dǎo)入NgR siRNA沉默NgR基因，從而達(dá)到拮抗NgR基因的效應(yīng)，促進(jìn)神經(jīng)再生.

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