疼痛相关融合基因的构建及siRNA对其抑制作用的研究  

Construction of pain-related fusion gene plasmids and inhibitory effect of small interfering RNA

作　　者：陈娜[1] 冯泽国[1] 张砡[1] 李冠华[1] 王维[1] 

机构地区：[1]解放军总医院麻醉科,北京100853

出　　处：《军医进修学院学报》2009年第3期351-353,共3页Academic Journal of Pla Postgraduate Medical School

基　　金：国家自然科学基金资助(30672028)~~

摘　　要：目的:通过构建用于RNAi筛选的模拟靶基因,筛选出起作用的siRNA,为进一步研究以Cav2.2e[37a]为靶标的基因治疗提供依据。方法:构建了EGFP-e37a/e37b融合基因,将其放入载体质粒中进行表达。按照siRNA的设计原则设计了四对siRNA(编码为I号、Ⅱ号、Ⅲ号、IV号),并将其放入质粒表达载体中,用siRNA表达载体与融合基因表达载体共转染BHK细胞进行了RNAi试验。用荧光显微镜、流式细胞仪等方法检测RNAi效果。结果:EGFP-e37a/e37b融合基因通过PCR、测序等方法鉴定为正确;在荧光显微镜下可见siRNAⅡ使细胞的荧光效率明显受到抑制,流式细胞术可见siRNAⅡ的RNAi效率在24h达到了90%左右,其余3条siRNA抑制作用不明显。结论:本研究将目的基因e37a与EGFP基因进行了融合构建,通过EGFP的绿色荧光效应显示目的靶基因在细胞内的表达,并观察到RNAi抑制效果,筛选出了有功能的siRNA。Objective： To study the inhibitory effects of small hairpin RNA （ shRNA ） expression vector on expression of the Cav2.2e [ 37a ] gene. Methods： Expression plasmid EGFP-e37a/e37b was constructed. BHK cell line was co-transfected with the expression plasmid EGFP-e37a and level of EGFP-e37a fusion protein was quantified by fluorescent microscopy and fluorescence activated cell sorting （ FACS ）. Results： The clones were identified by PCR and DNA sequence analysis. The results were shown as expected, siRNAII could inhibit 90% of EGFP-e37a expression. Conclusion： The EGFP-e37a/e37b fusion gene plasmids can be constructed and expressed in BHK cell line, and RNA interference （ RNAi ） can effectively reduce the levels of e37a transcripts and protein expression, thus providing a fresh approach to screening the efficient small interfering RNA （ siRNA ）.

关 键 词：SIRNA RNAI 钙通道 N型 Cav2.2e[37a] 

分 类 号：R394[医药卫生—医学遗传学] R733.7[医药卫生—基础医学]