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

doi:10.16098/j.issn.0529-1356.2017.02.001

摘要/Abstract

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 pAOV-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pAOV-CAGMINI-EGFP-2A-Pim-1-3FLAG及包装质粒pAAV-RC和辅助质粒pHelper，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。

Abstract:

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 pAOV-CAGMINI-EGFP-2A-MCS-3FLAG vector and Pim-1 PCR product were cleaved by Nhel enzyme. The recovered vector and target gene DNA were identified after agarose 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 pAAV-RC and helper plasmid pHelper were co-transfected into 293 cells through using the Lipofectamine 2000, then high titer of rAAV2-Pim-1 was purified. After rAAV2-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 after plasmid transfection into 293 cells; The virus titer was 5.7×10¹⁵vg/L. After rAAV2-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 vector is successfully constructed and Pim-1 is overexpressed in the RGCs of infected rat retina.

Key words: Pim-1, Adeno-associated virus 2 vector, Infection, Retina, Western blotting, Rat

张守梅黄婷婷王栋刘芳许家军. 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[J]. 解剖学报, 2017, 48(2): 121-127.

ZHANG Shou-mei HUANG Ting-ting WANG Dong LIU Fang XU Jia-jun. Construction of Pim-1 recombinant adeno-associated virus 2 vector and its infection in rat retina[J]. Acta Anatomica Sinica, 2017, 48(2): 121-127.

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