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
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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Cav2.2e37a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。Abstract: Objective: To study the inhibitory effects of small hairpin RNA（shRNA） expression vector on expression of the Cav2.2e37a 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）.
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