大鼠GAD65重组腺病毒载体的构建和鉴定  

Construction and identification of recombinant adenovirus vector carrying with GAD65 gene of rat

作　　者：陈盛强[1] 刘启才[1] 陈德忠[1] 林勇平[1] 孙卫文[1] 廖卫平[1] 

机构地区：[1]广州医学院第二附属医院神经科学研究所,广东广州510260

出　　处：《解剖学研究》2007年第3期163-167,共5页Anatomy Research

基　　金：广东省自然科学基金(NO.04009574)

摘　　要：目的构建大鼠GAD65重组腺病毒载体。方法将目的基因大鼠GAD65 cDNA亚克隆到穿梭质粒pShutile启动子CMV的下游,再通过I-CeuⅠ和PI-SceⅠ两个稀有酶切位点,将目的基因GAD65与腺病毒质粒DNA(pAdeno-X)进行体外连接,获得含目的基因GAD65重组腺病毒质粒DNA,后者经限制性内切酶PaeⅠ切割后,两端露出反向末端重复序列(rTR),利用脂质体转染293细胞,获得含目的基因重组腺病毒上清。PCR双引物检测含有目的基因GAD65的片段。结果在挑选的5个空斑中,有4个含GAD65 cDNA阳性重组腺病毒。GAD65重组腺病毒可感染293细胞并在293细胞内进行有效的复制。PCR双引物检测证明含有目的基因GAD65片段,表明利用体外连接法成功构建了含目的基因GAD65重组腺病毒转移载体。结论成功构建了GAD65重组腺病毒,为进一步研究GAD65重组腺病毒的功能提供了条件。Objective To explore a new efficient method for constructing adenovirus vector containing GAD65 of rat. Methods With gene recombination technique, GAD65 fragment was subcloned into pShutte vector under CMV promoter which has the unique PI-See I and I-Ceu I restriction sites. Then the new GAD65 expression cassette from the recombinant pShuttle plasmid was subcloned into the Adeno-XTM Genome and the recombinant Adeno/GAD65 was obtained. After cutting with restriction enzyme Pae 1, the reverse terminal repeat of the two ends were exposed. After transfection of 293 cell line with liposome, superuatant containing the recombinant adenovirus was obtained. Double primer PCR was used to detect the products. Results In the 5 lacunae selected, 4 of them contained the positive recombinant adenovirus which carrying GAD65 cDNA. The recombinant adenovirus could infect 293 cell line and could duplicate effectively in the cells. Double primer PCR attested that the recombinant adenovirus containing the GAD65 segment, which showed that the recombinant adenovirus vector, which contained aimed GAD65 segment, had been constructed successfully through in vitro ligation. Conclusion By a new in vitro ligation, the recombinant GAD65 mediated by adenovirus vector is constructed successfully.

关 键 词：腺病毒 DNA重组 谷氨酸脱羧酶65 载体 

分 类 号：R346[医药卫生—基础医学]