构建大鼠白细胞介素1β基因shRNA腺病毒载体  

Construction of a recombinant adenovirus vector expressing sh RNA targeting interleukin-1beta gene in rats

作　　者：赵晓龙[1] 陈建平[2] 张宇[3] 李航[1] 刘艳芳[1] 高文燕[1] 韩磊[1] 邓亚南 

机构地区：[1]山西医科大学,山西省太原市030001 [2]山西医学科学院山西大医院麻醉科,山西省太原市030032 [3]山西医科大学生理学教研室,山西省太原市030001

出　　处：《中国组织工程研究》2015年第18期2923-2927,共5页Chinese Journal of Tissue Engineering Research

基　　金：山西省自然科学基金(2010011049-1)~~

摘　　要：背景:特异性下调白细胞介素1β蛋白的表达可以有效缓解外周神经损伤后病理性疼痛。与si RNA相比,shR NA可以更加稳定、高效地抑制目的基因的表达,但是单纯的shR NA无法高效地进入靶细胞中发挥对目的基因的下调作用,而腺病毒载体具有宿主范围广、感染效率高以及能够在宿主细胞中稳定表达等特点。目的:构建大鼠白细胞介素1β基因的shR NA腺病毒载体并检测其对目的基因表达的影响。方法:根据NCBI查询获得的大鼠白细胞介素1β基因序列设计3条siR NA并合成相应的shR NA,分别为shR NA1、shR NA2、shR NA3。采用3’和5’单链退火得到的sh RNA片段与经过BamHⅠ和EcoRⅠ双酶切的pH BAd/U6/GFP干扰载体连接,构建白细胞介素1βshR NA腺病毒载体穿梭质粒。测序后将穿梭质粒与骨架质粒共转染HEK293细胞,进行白细胞介素1β基因的shR NA腺病毒载体(rA d/shR NAs)的包装与扩增,分别为rA d/shR NA1、rA d/shR NA2、rA d/shR NA3。将r Ad/shR NAs感染大鼠H9C2细胞,使用荧光显微镜观察其感染效率,采用Western Blot检测r Ad/sh RNAs对目的基因表达的影响。结果与结论:测序结果显示白细胞介素1β基因的3条sh RNA腺病毒载体穿梭质粒中的序列分别与所设计的3条sh RNA序列相同;并成功构建了大鼠白细胞介素1β基因的shR NA腺病毒载体(r Ad/sh RNA1、rA d/shR NA2、rA d/sh RNA3)。rA d/shR NA1、rA d/sh RNA2、r Ad/shR NA3均可以下调白细胞介素1β的表达,其中r Ad/shR NA2的下调效果最明显。BACKGROUND：Specific down-regulation of interleukin 1 beta （IL-1β） may al eviate the pain behaviors effectively after peripheral nervous injury. Compared with smal interference RNA （siRNA）, short hairpin RNA （shRNA） could inhibit the expression of target gene more stably and efficiently. However, simple shRNA could not enter target cel s to down-regulate target gene efficiently. Adenovirus vectors have wide host range, high infection efficiency and stable expression in host cel s. OBJECTIVE：To construct recombinant adenovirus vector expressing shRNA targeting IL-1βgene and detect its effect on the expression of target gene. METHODS：Three siRNAs were designed on the basis of the nucleotide sequence of IL-1βobtained from NCBI and then three shRNAs （shRNA1, shRNA2 and shRNA3） were synthesized. The annealed shRNA product and＆nbsp;adenovirus vector pHBAd/U6/GFP digested by BamH I and EcoR I were connected to construct the recombinant adenovirus vector shuttle plasmid expressing shRNA targeting IL-1β. After sequencing, HEK 293 cel s were co-transfected by the shuttle plasmid and skeleton vector, and three recombinant adenovirus vector expressing shRNA targeting IL-1β（rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3） were packaged and amplified. Rats H9C2 cel s were infected by recombinant adenovirus vector expressing shRNA targeting IL-1βand fluorescence microscope was used to observe the infection efficiency. The effect of recombinant adenovirus vector expressing shRNA targeting IL-1βon the expression of target gene was detected by western blot assay. RESULTS AND CONCLUSION：The sequencing results showed that the sequences of three shRNAs adenovirus vector shuttle plasmid were consistent with the sequences of three designed shRNAs. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 were constructed successful y. rAd/shRNA1, rAd/shRNA2 and rAd/shRNA3 could down-regulate the expression of IL-1βin rat H9C2 cel s and the down-regulation effect of rAd/shRNA2 was the most significant.

关 键 词：疼痛 实验动物 基因病毒载体及相关因子模型 神经病理性疼痛 白细胞介素1Β 基因表达 RNA干扰 小干扰RNA 短发卡RNA 腺病毒载体 载体质粒 HEK293细胞 大鼠H9C2细胞 山西省自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]