靶向人白细胞源性精氨酸氨肽酶基因shRNA重组腺病毒的构建

Construction of recombinant adenovirus expressing shRNA targeting human leukocyte-derived arginine aminopeptidase gene

目的:构建靶向人白细胞源性精氨酸氨肽酶(LRAP)基因的shRNA重组腺病毒,有效抑制LRAP基因在人视网膜血管内皮细胞中的表达。方法:设计并合成3对靶向人LRAP基因shRNA的单链寡核苷酸,退火后克隆至腺病毒穿梭载体pRNAT-H1.1/Adeno得到3个重组穿梭质粒。将重组穿梭质粒和腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183-AD-1中进行同源重组形成腺病毒表达质粒,并在293a细胞中包装形成重组腺病毒颗粒。重组腺病毒经扩增和滴度测定后感染原代培养的人视网膜血管内皮细胞,应用荧光实时定量PCR测定LRAP mRNA的相对表达量,筛选得到沉默效率最高的LRAP shRNA重组腺病毒,并应用Western blot法验证经RNA干扰后视网膜血管内皮细胞中LRAP的蛋白表达水平。结果:PCR、酶切和DNA测序证实LRAP shR-NA重组穿梭质粒和表达质粒的插入序列正确。收获病毒后的PCR证实重组腺病毒颗粒包装成功。实时荧光定量PCR筛选得到具有最大沉默效率的重组腺病毒,其沉默效率达到约79%。Western blot法证实经RNA干扰后,人视网膜血管内皮细胞中的LRAP蛋白水平显著降低。结论:成功构建了靶向人LRAP基因的shRNA重组腺病毒,可有效抑制人视网膜血管内皮细胞中LRAP基因的表达。

AIM：To construct the recombinant adenovirus expressing small hairpin RNA（shRNA） targeting human leukocyte-derived arginine aminopeptidase（LRAP） gene and silence the expression of LRAP in human retinal microvascular endothelial cells（HRMECs）.METHODS： Three pairs of oligonucleotides coding for shRNAs targeting human LRAP gene were designed and synthesized,and were cloned into the shuttle vector pRNAT-H1.1/Adeno after annealing.The three constructed shuttle plasmids,through homologous recombination with adenoviral backbone vector pAdEasy-1,were transformed into E.coli BJ5183-AD-to produce recombinant adenoviral plasmids.And then the recombinants linearized with Pac I were transfected into 293a cells to package adenoviruses.After amplification and titter determination,the recombinant adenoviruses were used to infect the primary HRMECs.Quantitative real-time PCR was taken to determine the relative amount of LRAP mRNA to screen the adenovirus with the highest silencing efficiency.The level of LRAP protein after RNA interference in HRMECs was further determined by Western blotting.RESULTS： PCR,restriction digestion and DNA sequencing confirmed that the purpose shRNA-coding sequences were correctly inserted into the shuttle vectors and adenoviral plasmids,and the recombinant adenoviruses were packaged successfully in 293a cells.The most effective adenovirus with the silencing efficiency up to 79% was selected by quantitative real-time PCR,which significantly lowered the expression of LRAP in HRMECs.CONCLUSION： The recombinant adenovirus expressing shRNA effectively silencing LRAP gene was constructed successfully,which would facilitate further study of the role that LRAP plays in the development of diabetic retinopathy.