利用pSOS-HUS系统构建针对小鼠h1-calponin的RNA干扰载体

Construction of pSOS-HUS System Against Mouse h1-Calponin mRNA Based on siRNA Expression Vectors

目的利用pSOS-HUS系统构建针对小鼠碱性调宁蛋白(h1-calponin)的RNA干扰载体并对其干扰效率进行验证。方法利用在线软件设计三段针对小鼠h1-calponin cDNA的寡核苷酸(A、B、C)片段,经退火成为双链后分别装入pSOS-HUS载体,得到载体调宁蛋白-PS-A/B/C;再将h1-calponin cDNA插入该载体和GFP一起融合表达,得到调宁蛋白RNAi-A/B/C3个载体;对上述载体经酶切和测序鉴定后,转染至HEK293T细胞株,通过倒置荧光显微镜观察GFP荧光强度来判断上述载体的干扰效果。结果经酶切和测序证明针对小鼠h1-calponin的3个RNA干扰载体构建成功,荧光显微镜下观察显示,和空载体pSOS-HUS(未插入RNA干扰片段)相比,A组的荧光强度减弱最明显,B的基本不变,C的荧光强度稍有减弱。结论成功构建了针对h1-calponin的RNA干扰载体。

OBJECTIVE To construct pSOS-HUS system based on siRNA expression vectors against mouse hl-calponin mRNA. METHODS Three pairs of primers （A, B,C） with predicted specific RNAi effect on mouse hl-calponin mRNA were chosen using online designing software. Each pair of these primers and the hl-calponin cDNA were cloned into pSOS-HUS to get calponin RNAi-A, calponin RNAi-B, and caiponin RNAi-C. After being confirmed by restriction enzyme digestion and sequencing analysis, they were transfected into HEK293T cells to examine their interference effects on hl-calponin expression using fluorescence microscopy. RESULTS Three pSOS-HUS based RNAi vectors against mouse hl-calponin were generated. Compared with cells transfected with pSOS-HUS, the fluorescence intensity of calponin RNAi-A tranfected ceils was decreased significantly, while there was no obvious reduction of fluorescence intensity in ceils transfected with either caiponin RNAi-B or calponin RNAi-C. CONCLUSIONS A pSOS-HUS based RNAi expression vector with strong interfering effect on mouse hl-calponin is constructed successfully.