[1]钟诚,姜振东,张学渊.大鼠促红细胞生成素重组腺病毒载体的构建及其在大鼠SGNs中的表达[J].第三军医大学学报,2012,34(18):1818-1821.
 Zhong Cheng,Jiang Zhendong,Zhang Xueyuan.Construction and expression of recombinant adenovirus vector of erythropoietin in rat spiral ganglion neurons[J].J Third Mil Med Univ,2012,34(18):1818-1821.
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大鼠促红细胞生成素重组腺病毒载体的构建及其在大鼠SGNs中的表达(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
34卷
期数:
2012年第18期
页码:
1818-1821
栏目:
论著
出版日期:
2012-09-30

文章信息/Info

Title:
Construction and expression of recombinant adenovirus vector of erythropoietin in rat spiral ganglion neurons
作者:
钟诚姜振东张学渊
第三军医大学西南医院耳鼻咽喉头颈外科
Author(s):
Zhong Cheng Jiang Zhendong Zhang Xueyuan
Department of Otolaryngology-Head and Neck Surgery, Southwest Hospital,Third Military Medical University, Chongqing, 400038, China
关键词:
促红细胞生成素腺病毒构建
Keywords:
erythropoietin adenovirus construction
分类号:
R339.16;R394-33;R394.3
文献标志码:
A
摘要:
目的      构建大鼠促红细胞生成素(erythropoietin,EPO)重组腺病毒载体,并转染于体外培养的大鼠螺旋神经元(spiral ganglion neurons, SGNs)。      方法      全基因合成大鼠EPO基因,亚克隆至穿梭质粒pAdTrack-CMV,构建重组穿梭质粒pAdTrack-CMV-EPO。经酶切、PCR及测序鉴定,再经PacⅠ酶切线性化后电转化感受态细胞,获得重组腺病毒载体pAdTrack-CMV-EPO,经PacⅠ酶切后转染至293细胞进行包装,制备重组腺病毒,扩增后进行病毒滴度测定及RT-PCR和Western blot鉴定。并转染至体外培养的螺旋神经元,免疫荧光检测及蛋白水平检测螺旋神经元中EPO表达。      结果      经KpnⅠ、HindⅢ酶切与测序鉴定显示,腺病毒载体pAdTrack-CMV-EPO构建成功,重组腺病毒AdEPO在293细胞中成功包装,扩增后病毒滴度为1.17×1011 U/ml;经RT-PCR和Western blot检测EPO在293细胞中成功表达。重组腺病毒载体AdEPO经鉴定均构建正确;免疫荧光及Western blot检测转染腺病毒后螺旋神经元中EPO表达显著增强。      结论      成功构建了EPO重组腺病毒载体,并成功转染螺旋神经元,可显著增强螺旋神经元中EPO表达水平。
Abstract:
Objective      To construct a recombinant adenovirus vector of rat erythropoietin (EPO) and observe its expression in rat spiral ganglion neurons (SGNs).        Methods      EPO gene fragment was amplified and subcloned into shuttle plasmid pAdTrack-CMV-EPO. The constructed recombinant shuttle plasmid pAdTrack-CMV-EPO was identified by restriction analysis, PCR and sequencing, then linearilized with PacⅠ and transformed to competent AdEasy systems. The obtained recombinant adenovirus vector AdEPO was digested with PacⅠ and transfected to 293 cells for packaging, and the prepared recombinant adenovirus was amplified for titration and identification by RT-PCR and Western blotting. The vector had been transfected into the cultured SGNs, and EPO expressions were detected by immunofluorescence and Western blot analysis respectively.       Results      After the incision enzymes of KpnⅠ, HindⅢ and sequencing, pAdTrack-CMV-EPO was accomplished. Recombinant adenovirus AdEPO was successfully packaged in 293 cells with a titer of 1.17×1011 IU/ml after amplification,and successfully expressed in 293 cells. Both recombinant shuttle plasmid and recombinant adenovirus vector AdEPO were constructed correctly. EPO level was increased obviously after the virus transfection in SGNs either with immunofluorescence or Western blot assays.       Conclusion      The recombinant adenovirus vector of EPO is successfully constructed and significantly enhances the EPO levels after its transfection into SGNs.

参考文献/References:

钟诚, 姜振东, 张学渊. 大鼠促红细胞生成素重组腺病毒载体的构建及其在大鼠SGNs中的表达[J].第三军医大学学报,2012,34(18):1818-1821.

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更新日期/Last Update: 2012-09-18