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  

Construction of recombined adeno-associated virus vector expressing GCRG213 short interference RNA and preparation of high-titer virus

作　　者：徐世平[1] 吴本俨[1] 王孟薇[1] 高利利[1] 伍银桥[1] 蒋立新 王卫华[1] 尤纬缔[1] 

机构地区：[1]解放军总医院南楼消化科,北京市100853 [2]北京本元正阳基因技术有限公司,北京市100176

出　　处：《世界华人消化杂志》2007年第12期1417-1420,共4页World Chinese Journal of Digestology

基　　金：国家自然科学基金;No.30370635~~

摘　　要：目的:构建一个表达胃癌相关基因GCRG213小干扰RNA的重组腺相关病毒载体,制备能靶向性抑制GCRG213表达的重组腺相关病毒.方法:将构建好并经验证的包含胃癌相关基因GCRG213特异性小干扰RNA片段GCRG213-RNAi-2的IMG800-GCRG213i-2质粒用Bam H I+Bg/Ⅱ双酶切,回收410 bp左右目的片段,将pSNAV2.0质粒用BamH I单酶切后同目的片段连接,经酶切和测序鉴定后,用新建质粒脂质体转染BHK-21细胞,G418筛选后大规模培养,再用辅助病毒HSV1-rc/△UL2感染,获取病毒.结果:成功构建包含U6启动子和胃癌相关基因GCRG213特异性RNA干扰片段的重组腺相关病毒载体,并制备出滴度为5×10^(12)vector genome/L高滴度腺相关病毒.结论:载体的构建为进一步研究GCRG213的体内功能打下了基础,也将为更多的基因干预和基因的功能研究提供有效的工具.AIM： To construct the recombinant adenoassociated 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 BamH Ⅰ and Bgl Ⅱ. 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 BamH Ⅰ. The positive vectors were analyzed through enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into BHK cells using LipofecLamine^TM2000. The G418-resistant cells were obtained and infected with HSV1- rc/AUL2, which had the function of packaging rAAV. After purification, the target vector and virus were collected. RESULTS： The target vector, rAAV-GCRG213- siRNA, was successfully constructed, and the rAAV with a titer of 5 × 10^12 vg/L （vg： vector genome） was obtained. CONCLUSION： Recombitant rAAV vector may be used for further investigation of GCRG213 function in vivo and gene therapy.

关 键 词：胃癌 GCRG213基因 腺相关病毒 SIRNA 基因转染 

分 类 号：R735.2[医药卫生—肿瘤]