刘姿麟,林慕之,况春燕,刘兴德
LIU Zilin,LIN Muzhi,KUANG Chunyan,LIU Xingde

• 1:贵州医科大学附属人民医院
• 2:贵州医科大学
• 3:贵州省人民医院心内科

摘要(Abstract)：

目的:构建大鼠睡眠因子1(Slfn1)基因的发夹状RNA质粒,并进行腺病毒载体包装。方法:根据Rat slfn1基因的转录本设计并合成3个干扰RNA靶点ShRNA-Slfn1(1)、ShRNA-Slfn1(2)及ShRNA-Slfn1(3),并将单链的引物退火成双链oligo序列,连接入双酶切线性化的RNA干扰载体(p DKD-CMV-U6-shRNA)的U6启动子下游,替换掉原来的ccd B基因;利用Admax系统,将3种目的穿梭质粒分别与腺病毒骨架质粒共转染到HEK293细胞中重组获得病毒后进行小量扩增及病毒滴度测定;利用Slfn1过表达腺病毒质粒中目的基因携带Flag标签,通过Western Blot检测Slfn1过表达腺病毒质粒与靶点质粒共转染293T细胞中Flag表达,观察靶点质粒对Slfn1表达的影响。结果:经菌落PCR鉴定获得阳性克隆并测序验证正确,表明构建质粒成功;腺病毒包装ShRNA-Slfn1(1)、ShRNA-Slfn1(2)及ShRNA-Slfn1(3)的病毒滴度分别为1.0×1014ifu/L,2.5×1014ifu/L,6.3×1013ifu/L,Western Blot证实ShRNA-Slfn1(1)及ShRNA-Slfn1(3)能显著降低转染293T细胞中Flag的表达,确定shRNA-Slfn1(1)及shRNA-Slfn1(3)为目的质粒。结论:成功构建了大鼠shRNA-slfn1的重组腺病毒载体。
Objective:To construct hairpin shaped RNA plasmid(Ad-shRNA-Slfn1) of rat sleep factor 1(Slfn1) gene,and to package it in adenovirus vector.Methods:Three pairs of interference sequence,ShRNA-Slfn1(1),ShRNA-Slfn1(2),and ShRNA-Slfn1(3),were designed and synthesized according to the sequence of Slfn1 gene.The single primer was annealed into double-stranded oligo sequences and connected into the U6 promoter downstream of linearized RNA interference carrier of p DKD-CMV-shRNA,which replaced the original ccd B gene.Three kinds of shuttle vectors were respectively transfected into HEK293 cells with backbone plasmid using Admax system.The obtained recombinant adenovirus was amplified in a little quantity and determined by titer.Plasmid was identified by Western Blot using flag antibody.Results:Digestion PCR analysis and sequencing showed that shRNA-Slfn1 was successfully constructed and the titer results of virus were 1.0 × 1014(ifu/L),2.5 ×1014(ifu/L),and 6.3 × 1013(ifu/L) respectively.ShRNA-Slfn1(1) and ShRNA-Slfn1(3) can significantly reduce the Slfn1 protein expression proved by western blot.Conclusion:The recombinant adenovirus vector of rat Ad-shRNA-Slfn1 was successfully constructed.

关键词(KeyWords)： 睡眠因子1;RNA干扰;腺病毒载体;短发夹RNA;构建;大鼠
Schlafen1;RNA interference;adenoviral vector;short hairpin RNA;construction;rats

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81360034,81560056)

作者(Author): 刘姿麟,林慕之,况春燕,刘兴德
LIU Zilin,LIN Muzhi,KUANG Chunyan,LIU Xingde

DOI: 10.19367/j.cnki.1000-2707.2017.02.003

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