组织蛋白酶B-RNAi-LV载体构建及其对小鼠脑微血管内皮细胞系bEND.3的影响

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。

Construction of CTSB-RNAi-LV vector and its effect on mouse brain micvascular endothelial cell line bEND.3

Objective To construct an efficient vector of CTSB-RNAi-LV，and to investigate CTSB-RNAi-LV effect on mouse brain micvascular endothelial cells in vitro. Methods To devise and synthesize including 19 nt DNA oligo of interfering Cathepsin B gene，the DNA oligo was cloned into the expression plasmid of IentiviraI vector［pGCSIL-GFP,which carried green fluorescent protein(GFP)］．The correct Cathepsin B gene was confirmed by endoenzyme digestion and sequencing．Then it was named pGCSIL-GFP-CTSB. pGCSIL-GFP-CTSB was designed and constructed, CTSB-RNAi-LV was produced by 293T cells foIlowing the co-transfection of pGCSIL-GFP-CTSB and packaging plasmids-pHelper1.0 and pHelper2.0. The resulting CTSB-RNAi-LV was used to infect the mice brain micvascular endothelial cell line (bEND.3), Cathepsin B mRNA and Cathepsin B in bEND.3 were dectected by RT-PCR and Western blotting. Results 1. pGCSIL-GFP-CTSB was successfully constructed;2.CTSB-RNAi-LV could be expressed in bEND3;3.CTSB-RNAi significantly inhibited the expression of CTSB in bEND.3.ConclusionpGCSIL-GFP-CTSB was constructed and CTSB-RNAi-LV was produced succes