关键词: 慢病毒, 组织蛋白酶B, 293T细胞, 内皮细胞, 绿色荧光蛋白, 免疫印迹法, 小鼠

Abstract: 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

Key words: Lentivirus, Cathepsin B, 293T cells, Endothelial cells（bEND.3）, Green fluorescent protein, Western blotting, Mouse