表达胃癌相关基因GCRG213小干扰RNA腺相关病毒载体的构建和高滴度病毒制备
胃癌;GCRG213基因;腺相关病毒;siRNA;基因转染,徐世平,吴本俨,王孟薇,高利利,伍银桥,王卫华,尤纬缔,蒋立新,通讯作者:,Constructionofrecombinedadeno-associat
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徐世平, 吴本俨, 王孟薇, 高利利, 伍银桥, 王卫华, 尤纬缔, 解放军总医院南楼消化科 北京市 100853
蒋立新, 北京本元正阳基因技术有限公司 北京市 100176
国家自然科学基金资助项目, No. 30370635
通讯作者: 吴本俨, 100853, 北京市复兴路28号, 解放军总医院南楼消化科. benyanwu@vip.sina.com
电话: 010-66939443
收稿日期: 2007-03-06 接受日期: 2007-03-19
Construction of recombined adeno-associated virus vector expressing GCRG213 short interference RNA and preparation of high-titer virus
Shi-Ping Xu, Ben-Yan Wu, Meng-Wei Wang, Li-Li Gao, Yin-Qiao Wu, Li-Xin Jiang, Wei-Hua Wang, Wei-Di You
Shi-Ping Xu, Ben-Yan Wu, Meng-Wei Wang, Li-Li Gao, Yin-Qiao Wu, Wei-Hua Wang, Wei-Di You,Department of Gastroenterology, South Building, General Hospital of Chinese PLA, Beijing 100853, China
Li-Xin Jiang,Beijing AGTC Gene Technology Company LTD, Beijing 100176, China
Supported byNational Natural Science Foundation of China, No. 30370635
Correspondence to:Dr. Ben-Yan Wu, Department of Gastroenterology, South Building, General Hospital of Chinese PLA, Beijing 100853, China. benyanwu@vip-sina.com
Received: 2007-03-06 Accepted:2007-03-19
Abstract
AIM: To construct the recombinant adeno-associated virus vector expressing gastric cancer related gene GCRG213 short interference RNA (rAAV-GCRG213-siRNA) and use it for the preparation of high-titer virus.
METHODS: IMG800-GCRG213-siRNA plasmid, which was constructed with GCRG213 siRNA, was digested with BamHⅠ and BglⅡ. A 410-bp fragment, which contained U6 promoter and GCRG213 siRNA, was obtained and inserted into the adeno-associated virus vector plasmid pSNAV2.0 that was digested with BamHⅠ. The positive vectors were analyzed through enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into BHK cells using LipofecLamineTM2000. The G418-resistant cells were obtained and infected with HSVl-rc/ΔUL2, which had the function of packaging rAAV. After purification, the target vector and virus were collected ......
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