[1]赵银娟,来珊珊,李朝军,等.GGPPS基因干扰腺病毒载体的构建及鉴定[J].南京师大学报(自然科学版),2013,36(02):104-107.
　Zhao Yinjuan,Lai Shanshan,Li Chaojun,et al.Construction and Identification of GGPPS Gene Interference Adenovirus[J].Journal of Nanjing Normal University(Natural Science Edition),2013,36(02):104-107.
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GGPPS基因干扰腺病毒载体的构建及鉴定()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第36卷
期数:
2013年02期
页码:
104-107
栏目:
生命科学
出版日期:
2013-06-30

文章信息/Info

Title:
Construction and Identification of GGPPS Gene Interference Adenovirus
文章编号:
1001-4616(2013)02-0104-04
作者:
赵银娟1; 来珊珊2; 李朝军2; 薛 斌2
1.南京林业大学森林资源与环境学院,江苏省有害生物入侵与控制重点实验室,江苏 南京 210037
2.南京大学医学院,江苏省医学分子技术重点实验室,江苏 南京 210093
Author(s):
Zhao Yinjuan1; Lai Shanshan2; Li Chaojun2; Xue Bin2
1.College of Forestry Resources and Environment,Nanjing Forestry University,Jiangsu Key Laboatory for Prevention and Management of Invation Species,Nanjing 210037,China
2.Medical School of Nanjing University,Jiangsu Key Laboratory of Molecular Medicine,Nanjing 210093,China
关键词:
GGPPS基因; 干扰; 腺病毒
Keywords:
GGPPS; interference; adenovirus
分类号:
Q29
文献标志码:
A
摘要:
构建GGPPS基因干扰腺病毒质粒载体并加以鉴定.根据GGPPS基因序列设计GGPPS干扰序列引物,定向克隆至穿梭载体pshuttle-H1的Bgl Ⅱ和Hind Ⅲ位点,Pme Ⅰ酶切线性化的pShuttle-H1-SiGGPPS干扰质粒,并与腺病毒载体(pAdEasy-1质粒)共同转化E.coli BJ5183感受态细菌,产生重组腺病毒载体.用Pac Ⅰ酶切线性化的回收质粒,转染293A细胞包装腺病毒颗粒,在倒置显微镜下观察细胞CPE,用TCID50法测定病毒颗粒的浓度,并初步观察病毒感染PC12细胞对目的基因的干扰效率.经酶切鉴定、测序证实成功构建GGPPS基因干扰腺病毒载体.包装的腺病毒浓缩悬液滴度为1.995×107 PFU/mL.GGPPS在人中具有保守性,该病毒能在人源的WRL-68细胞中成功表达,并且对GGPPS基因干扰效率达70%以上.
Abstract:
To construct the recombinant adenovirus vector expressing small RNA against human GGPPS gene,the interferce primer designed according to GGPPS gene sequence was cloned into pshuttle-H1 vector with Bgl Ⅱ and Hind Ⅲ restriction site.E.coli BJ5183 sensitive bacteria were cotransfected with vector lenealized by Pme Ⅰ enzyme and adenovirus vector pAdEasy-1.The adenovirus was obtained in 293A cells transfected with linearized recombinant adenovirus plasmids.The titer of virus was measured based on the appearing of CPE and was determined by TCID50 assay.The interference efficiency was caculated.The results obtained from DNA sequencing and digestion identification demonstrated that the adenovirus vectors tagerting to human GGPPS gene was constructed.The titer of concentrated virus was 1.995×107 PFU/mL.The interference efficiency of endogenous GGPPS gene in WRL68 cells was above 70% as evidenced by western blot analysis.The interference adenovirus vector of human GGPPS gene is successfully constructed.

参考文献/References:

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相似文献/References:

[1]梁 金,沈 宁,徐 康,等.cnksr2基因干扰腺病毒的构建及鉴定[J].南京师大学报(自然科学版),2013,36(03):103.
　Liang Jin,Shen Ning,Xu Kang,et al.Construction and Identification of cnksr2 Gene Interference Adenovirus[J].Journal of Nanjing Normal University(Natural Science Edition),2013,36(02):103.

备注/Memo

备注/Memo:
收稿日期:2012-10-28.
基金项目:国家自然科学青年基金(31100448)、博士点基金(2113204120004).
通讯联系人:薛斌,副教授,博士,研究方向:细胞生物学.E-mail:xuebin@nju.edu.cn
更新日期/Last Update: 2013-06-30