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目的构建组织蛋白酶B(Cathepsin B,CTSB)小RNA(siRNA)慢病毒载体,并探讨其对小鼠脑微血管内皮细胞(bEND.3)的影响。方法设计并合成含有干扰Cathesin B基因的19 nt的双链寡DNA片段,将此片段克隆到携有绿色荧光蛋白(GFP)的慢病毒表达载体质粒pGCSIL-GFP上,经测序正确后,命名为pGCSIL-GFP-CTSB,将慢病毒表达载体pGCSIL-GFP-CTSB、慢病毒包装载体pHelper1.0和pHelper2.0 3质粒共同转染于293T细胞,获得携带Cathepsin B基因的RNAi慢病毒(Cathesin B-RNAi-Lentivirus,即CTSB-RNAi-LV),通过Real time-PCR和Western blotting方法观察CTSB-RNAi-LV对bEND.3的影响。结果 1.pGCSIL-GFP-CTSB中携带有正确的Cathesin B siRNA基因;2.目的基因Cathepsin B siRNA被RNAi慢病毒高效地转导入靶细胞bEND.3内,并达到稳定的表达;3.CTSB-RNAi-LV能有效降低bEND.3中Cathepsin B mRNA和蛋白表达。结论成功构建了RNAi慢病毒载体pGCSIL-GFP-CTSB;并成功包装了RNAi慢病毒CTSB-RNAi-LV;CTSB-RNAi-LV能有效地抑制bEND.3中Cathepsin B mRNA和蛋白表达水平下调。
Objective To construct a lentiviral vector containing small interfering RNA (siRNA) targeting Cathepsin B (CTSB) and investigate its effect on mouse brain microvascular endothelial cells (bEND.3). Methods A 19-nt double-stranded oligo DNA fragment containing the Cathesin B gene was designed and synthesized. The fragment was cloned into the lentiviral vector pGCSIL-GFP carrying green fluorescent protein (GFP). After sequencing, the fragment was named pGCSIL-GFP-CTSB. The lentiviral vector pGCSIL-GFP-CTSB and lectin vector pHelper1.0 and pHelper2.0 3 were co-transfected into 293T cells to obtain the RNAi lentivirus carrying Cathepsin B- RNAi-Lentivirus (CTSB-RNAi-LV). The effect of CTSB-RNAi-LV on bEND.3 was observed by Real time-PCR and Western blotting. Cathelin B siRNA gene was carried in pGCSIL-GFP-CTSB; Cathepsin B siRNA of target gene was efficiently transduced into bEND.3 target RNAi by RNAi lentivirus and stably expressed; 3.CTSB- RNAi-LV effectively reduced Cathepsin B mRNA and protein expression in bEND.3. Conclusion RNAi lentiviral vector pGCSIL-GFP-CTSB was successfully constructed and RNAi lentivirus CTSB-RNAi-LV was successfully packaged. CTSB-RNAi-LV could effectively inhibit the down-regulation of Cathepsin B mRNA and protein expression in bEND.3.