Pim-1基因重组腺相关病毒2载体的构建及感染大鼠视网膜  

Construction of Pim-1 recombinant adeno-associated virus 2 vector and its infection in rat retina

作　　者：张守梅[1] 黄婷婷[1] 王栋[1] 刘芳[1] 许家军[1] 

机构地区：[1]第二军医大学基础部人体解剖学教研室,上海200433

出　　处：《解剖学报》2017年第2期121-127,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金(31271282;31571239)

摘　　要：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 a recombinant adeno-associated virus 2 vector（ rAAV2-Pim-1） carrying rat proto-oncogene Pim-1,and to detect infected cell types and the expression of Pim-1 in rat retina infected by rAAV2-Pim-1.Methods The p AOV-CAGMINI-EGFP-2A-MCS-3FLAG vectorand Pim-1 PCR product were cleaved by Nhel enzyme.The recovered vectorand target gene DNA were identified afteragarose gel electrophores and linked to transformation,positive plasmid cloning and sequencing analysis. rAAV2-Pim-1 expression plasmid pAOV-CAGMINI-EGFP-2A-Pim-1-3FLAG,packaging plasmid p AAV-RC and helperplasmid p Helperwere co-transfected into 293 cells through using the Lipofectamine 2000,then high titerof rAAV2-Pim-1 was purified. AfterrAAV2-Pim-1 injection into rat intravitreal body,the infected retinal cell types were detected by immunofluorescence histochemistry staining; the expression of Pim-1 in the retina was quantified by Real-time PCR and Western blotting. Results The construction of rAAV2-Pim-1 plasmid was successful and the nucleotide sequence was right; Green fluorescence in 293 cells was found afterplasmid transfection into293 cells; The virus titerwas 5. 7 × 10^15vg/L. AfterrAAV2-Pim-1 infected rat retina in vivo,the infection percentage of retinal ganglion cells（ RGCs） reached 71%,and a few of amacrine cells and hardly astrocytes were infected; the expression of Pim-1 mRNA and protein in the retina of rAAV2-Pim-1 group was about 6. 61 times and 2. 29 times of the rAAV2-EGFP group respectively. Conclusion rAAV2-Pim-1 virus vectoris successfully constructed and Pim-1 is overexpressed in the RGCs of infected rat retina.

关 键 词：PIM-1 腺相关病毒2载体 感染 视网膜 免疫印迹法 大鼠 

分 类 号：R322.91[医药卫生—人体解剖和组织胚胎学]