[1]宋敏,白云,段文元,等.重组VIP腺病毒载体的构建及其在293细胞的表达[J].陆军军医大学学报(原第三军医大学学报),2007,29(09):806-809.
 SONG Min,BAI Yun,DUAN Wen-yuan,et al.Construction, expression and identification of recombinant adenovirus Ad-VIP in 293 cells[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(09):806-809.
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重组VIP腺病毒载体的构建及其在293细胞的表达
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第09期
页码:
806-809
栏目:
论著
出版日期:
2007-05-15

文章信息/Info

Title:
Construction, expression and identification of recombinant adenovirus Ad-VIP in 293 cells
作者:
宋敏白云段文元章波杨晓亚许雪青
第三军医大学基础医学部医学遗传学教研室
Author(s):
SONG Min BAI Yun DUAN Wen-yuan ZHANG Bo YANG Xiao-ya XU Xue-qing
Department of Medical Genetics, College of Medicine, Third Military Medical University, Chongqing 400038, China
关键词:
血管活性肠肽腺病毒基因治疗
Keywords:
vasoactive intestinal peptide adenovirus gene therapy
分类号:
R392.11; R394-33; R394.2
文献标志码:
A
摘要:
目的      构建血管活性肠肽重组腺病毒载体,以期用于体内实验研究。      方法      利用RT-PCR扩增小鼠大脑VIP mRNA,亚克隆到穿梭质粒pAdTrack-CMV,在pAdeasy内同源重组,筛选阳性克隆,酶切、测序鉴定正确,线性化后脂质体法转染293 细胞进行包装、扩增,利用报告基因EGFP对病毒滴度进行监测。PCR、免疫荧光鉴定pAdeasy-VIP感染293 细胞后VIP基因的表达,ELISA检测重组病毒转染后293细胞上清中的表达。      结果      测序、酶切证实VIP基因重组腺病毒载体构建成功。RT-PCR,免疫荧光检测pAdeasy-VIP病毒感染的293细胞,均有VIP的表达。与对照组相比,重组病毒转染后293细胞上清中VIP多肽的表达明显增高。      结论      成功构建了含小鼠VIP基因的重组腺病毒载体,并成功表达VIP多肽。
Abstract:
Objective      To construct a recombinant adenovirus encoding vasoactive intestinal peptide (VIP) for future gene therapy in vivo.        Methods      Full length mouse VIP cDNA linked with an internal ribosome entry site (IRES)-EGFP cassette was subcloned into pAdTrack-CMV shuttle plasmid. The product was linearized to mediate homologous recombination with pAdeasy-1 vector in BJ5183 host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombinant adenovirus DNA was transfected into 293 cells for packaging and amplification of pAdeasy-VIP virus, which was purified by SARTOBIND Membrane Adsorbers. The expression of VIP was monitored by EGFP fluorescence in infected cells. VIP peptide expression on 293 cells was detected by PCR, immunofluorescent method and ELISA.       Results      After transfected with adenovirus DNA, infectious virus was only produced to cause cytopathic effect in the permissive cell line 293 but not in the non-permissive cell line HeLa, confirming only replication defective but not wild type virus was generated. The specific expression of mouse pAdeasy-VIP was verified by RT-PCR and immunofluorescent method in 293 cells after infection with pAdeasy-VIP, but not Ad-EGFP, a similarly constructed control virus. pAdeasy-VIP, but not pAd-EGFP, significantly secretes VIP peptide in supernatant.       Conclusion      We have successfully constructed a recombinant adenovirus pAdeasy-VIP that expresses VIP peptide in vitro.

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更新日期/Last Update: 2008-10-24