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

doi:10.16098/j.issn.0529-1356.2017.02.001

Abstract

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

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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