论文部分内容阅读
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 an RNA interference vector targeting mouse h1-calponin using pSOS-HUS system and validate its interference efficiency. Methods Three segments of oligonucleotide (A, B, C) fragments targeting mouse h1-calponin cDNA were designed by on-line software and annealed to double strands. The oligonucleotides were inserted into pSOS- A / B / C. The h1-calponin cDNA was inserted into the vector and fused with GFP to obtain RNAi-A / B / C3 vectors. After vector identification and sequencing, the vector was transfected into HEK293T Cell lines, observed by inverted fluorescence microscopy GFP fluorescence intensity to determine the carrier interference effect. Results Three RNA interference vectors targeting h1-calponin were successfully constructed. The results of fluorescence microscopy showed that the fluorescence intensity of group A was weaker than that of empty vector pSOS-HUS (without RNA interference fragment) Most notably, B remained essentially unchanged and C had a slightly weaker fluorescence intensity. Conclusion The RNAi vector targeting h1-calponin was successfully constructed.