靶向Slug基因的腺相关腺病毒载体的构建与鉴定  

Construction and identification of a recombinant adeno-associated virus-2 vector carrying an siRNA targeting the slug gene

作　　者：严明总[1] 张克君[2] 王齐全[3] 欧树安[2] 

机构地区：[1]海南省人民医院检验科,海南省海口市527102 [2]海南医学院附属医院普外科,海南省海口市527000 [3]海口市人民医院消化内科,海南省海口市527102

出　　处：《世界华人消化杂志》2011年第11期1179-1183,共5页World Chinese Journal of Digestology

摘　　要：目的:构建并制备携带目的基因的rAAV-EGFP-Slug-siRNA病毒载体.方法:首先构建pDC316-EGFP-Slug-siRNA质粒,以PCR鉴定正确的pDC316-EGFP-Slug-siRNA克隆为模板扩增EGFP-Slug-siRNA片段,EcoRⅠ和SalⅠ双酶切回收的PCR产物;酶切pSNAV2.0-lacz-α载体质粒,并与上述产物进行连接;转化感受态大肠杆菌DH5α,得到重组质粒pSNAV2.0-EGFP-Slug-siRNA,酶切和测序对其进行鉴定.建立载体细胞株BHK/Slug-siRNA,大规模制备rAAV2-EGFP-Slug-siRAN并对其纯化、鉴定和滴度测定.结果:携带编码靶向Slug的小发夹状干扰RNA序列的腺相关病毒载体质粒pSNAV2.0-EGFP-Slug-siRNA,经PCR、酶切和测序鉴定,质粒构建正确.将构建成功的载体质粒与重组Ⅰ型单纯疱疹病毒HSV1-rc/?UL2共转染包装细胞BHK-2,成功复制和包装出重组腺相关病毒rAAV2-EGFP-Slug-siRNA,经检测目的片段插入成功,滴度为9.23×1010puf.结论:成功制备出高滴度的携带目的基因的rAAV-EGFP-Slug-siRNA病毒载体.AIM：To construct a recombinant adeno-associated virus-2 vector carrying a small interfering RNA（siRNA）targeting the slug gene（rAAV2-slug-siRNA）.METHODS：A double-stranded siRNA targeting the slug gene was designed,synthesized and cloned into the pDC316-EGFP vector.The result-ing vector containing slug siRNA was confirmed by RT-PCR and direct sequencing.EGFP-slug-siRNA sequence was amplified,double digested with EcoR I and Sal I,and ligated to pSNAV2.0-lacz-αplasmid digested with the same enzyme pair.After the resulting recombinant plasmidwas transformed into DH5α,bacterial colonies containing the recombinant plasmid was screened on LB agar plates.pSNAV2.0-EGFP-Slug-siRNA plasmid DNA was prepared,purified,identified,and transfected into BHK cells by means of Lipofectamine.BHK cells expressing slug-siRNA（BHK-slug-siRNA）were obtained and subsequently infected with recom-binant herpes simplex virus l（HSV1-rc/△UL2） to package the rAAV2-slug-siRNA to obtain a functional and infectious virus（rAAV2-slug-siRNA）.RESULTS：A recombinant adeno-associated virus-2 vector carrying a slug siRNA was constructed successfully.The presence of the target sequence in the vector was confirmed by RT-PCR and enzyme digestion.By transfecting the pSNAV2.0-EGFP-Slug-siRNA plasmid into BHK cells,BHK-slug-siRNA was obtained and infected with HSV1-rc/△UL2 to package the rAAV2-slug-siRNA to obtain a functional and infectious virus.The titer of the recombinant virus was 9.23 ×10^10 PFU.CONCLUSION：A high-titer recombinant adeno-associated virus-2 vector carrying a slug siRNA has been constructed successfully.

关 键 词：SLUG基因 小干扰RNA 重组腺相关腺病毒 

分 类 号：R346[医药卫生—基础医学]