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目的构建携带大鼠原癌基因Pim-1的重组腺相关病毒2载体(rAAV2-Pim-1),检测其体内感染大鼠视网膜的细胞类型及目的基因Pim-1在视网膜中的表达。方法 p AOV-CAGMINI-EGFP-2A-MCS-3FLAG载体及Pim-1基因PCR产物用Nhel酶切,琼脂糖凝胶电泳鉴定后回收载体及目的基因DNA并连接转化,鉴定质粒阳性克隆及测序。rAAV2-Pim-1表达质粒p AOV-CAGMINI-EGFP-2A-Pim-1-3FLAG及包装质粒p AAV-RC和辅助质粒p Helper,通过Lipofectamine 2000共转染293细胞,纯化获得高滴度的rAAV2-Pim-1。大鼠玻璃体注射rAAV2-Pim-1,用免疫荧光组织化学检测其感染视网膜的细胞类型;用Real-time PCR和Western blotting检测Pim-1在视网膜中的表达。结果rAAV2-Pim-1质粒构建成功并且核苷酸序列比对正确;质粒转染293细胞后出现绿色荧光;包装出的病毒浓缩滴度为5.7×1015vg/L。rAAV2-Pim-1组体内感染视网膜神经节细胞(RGCs)达71%,并感染少量无长突细胞,几乎不感染星形胶质细胞;Pim-1 mRNA和蛋白在视网膜中的表达约为rAAV2-EGFP组的6.61倍和2.29倍。结论成功构建rAAV2-Pim-1病毒载体,并在感染后的大鼠视网膜RGCs中过表达Pim-1。
Objective To construct the recombinant adeno-associated virus 2 vector (rAAV2-Pim-1) carrying the rat proto-oncogene Pim-1 and detect the cell type and the expression of target gene Pim-1 in the retina of the infected rat retina in vivo. Methods p AOV-CAGMINI-EGFP-2A-MCS-3FLAG vector and Pim-1 gene PCR products were digested with Nhel and identified by agarose gel electrophoresis. The vector and target gene DNA were recovered and ligated. The positive clones were identified and sequenced. rAAV2-Pim-1 expression plasmid pAOV-CAGMINI-EGFP-2A-Pim-1-3FLAG and packaging plasmid pAAV-RC and helper plasmid p Helper were co-transfected into 293 cells by Lipofectamine 2000 and purified to obtain high titer rAAV2 -Pim-1. Rats were injected intravitreally with rAAV2-Pim-1. The cell types of the infected retina were detected by immunofluorescence histochemistry. The expression of Pim-1 in the retina was detected by Real-time PCR and Western blotting. Results The rAAV2-Pim-1 plasmid was constructed successfully and the nucleotide sequence alignment was correct. The green fluorescence appeared after the plasmid was transfected into 293 cells. The packaged virus titer was 5.7 × 1015vg / L. The number of retinal ganglion cells (RGCs) infected by rAAV2-Pim-1 reached 71% in vivo and infected a small number of amacrine cells with little infection of astrocytes. The expression of Pim-1 mRNA and protein in the retina was about rAAV2 6.61-fold and 2.29-fold for the -EGFP group. Conclusion The rAAV2-Pim-1 virus vector was successfully constructed and Pim-1 was overexpressed in the infected rat retinal RGCs.