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

张守梅 黄婷婷 王栋 刘芳 许家军*

doi:10.16098/j.issn.0529-1356.2017.02.001

PDF(768 KB)

解剖学报 ›› 2017, Vol. 48 ›› Issue (2) : 121-127. DOI: 10.16098/j.issn.0529-1356.2017.02.001

神经生物学

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

张守梅黄婷婷王栋刘芳许家军*

作者信息 +

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

ZHANG Shou-mei HUANG Ting-ting WANG Dong LIU Fang XU Jia-jun*

Author information +

文章历史 +

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.

关键词

Key words

Pim-1 / Adeno-associated virus 2 vector / Infection / Retina / Western blotting / Rat

引用本文

EndNote

Ris (Procite)

Bibtex

导出引用

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 https://doi.org/10.16098/j.issn.0529-1356.2017.02.001

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 https://doi.org/10.16098/j.issn.0529-1356.2017.02.001

参考文献

［1］Kumar A, Mandiyan V, Suzuki Y, et al. Crystal structures of proto-oncogene kinase Pim1: a targetof aberrant somatic hypermutations in diffuse large cell lymphoma［J］. J Mol Biol, 2005, 348(1):183-193.

［2］Eichmann A, Yuan L, Breant C, et al. Developmental expression of pim kinases suggests functions also outside of the hematopoietic system［J］. Oncogene, 2000, 19(9):1215-1224.

［3］Mondello P, Cuzzocrea S, Mian M. Pim kinases in hematological malignancies: where are we now and where are we going ［J］? J Hematol Oncol, 2014, 7(1):95.

［4］Yin J, Shine L, Raycroft F, et al. Inhibition of the Pim1 oncogene results in diminished visual function［J］. PLoS One, 2012, 7(12):e52177.

［5］Harvey AR, Hellstrm M, Rodger J. Gene therapy and transplantation in the retinofugal pathway［J］. Prog Brain Res, 2009, 175:151-161.

［6］Harvey AR, Hu Y, Leaver SG, et al. Gene therapy and transplantation in CNS repair: the visual system［J］. Prog Retin Eye Res, 2006, 25(5):449-489.

［7］Dinculescu A, Glushakova L, Min SH, et al. Adenoassociated virus-vectored gene therapy for retinal disease［J］. Hum Gene Ther, 2005, 16(6):649-663.

［8］Huang TT, Xu JJ, Cao WL, et al. Comparison of adeno-associated viral 2 vector and lentiviral vector transfection in rat retina after intravitreal injection［J］. Acta Anatomica Sinica, 2016, 47(2):191-196. (in Chinese)

黄婷婷，许家军，曹文珞，等. 玻璃体注射腺相关病毒2和慢病毒转染大鼠视网膜的比较［J］. 解剖学报，2016，47(2):191-196.

［9］Harvey AR, Kamphuis W, Eggers R, et al. Intravitreal injection of adenoassociated viral vectors results in the transduction of different types of retinal neurons in neonatal and adult rats：a comparision with lentiviral vectors［J］. Mol Cell Neurosci, 2002, 21(1):141-157.

［10］Bennett J, Duan D, Engelhardt JF, et al. Real-time, noninvasive in vivo assessment of adeno-associated virus-mediated retinal transduction［J］. Invest Ophthalmol Vis Sci, 1997, 38(13):2857-2863.

［11］Sarra GM, Stephens C, Schichtenbrede FC, et al. Kinetics of transgene expression in mouse retina following sub-retinal injection of recombinant adeno-associated virus［J］. Vision Res, 2002, 42(4):541-549.

［12］Bemelmans AP, Duque S, Riviere C, et al. A single intravenous AAV9 injection mediates bilateral gene transfer to the adult mouse retina［J］. PLoS One, 2013, 8(4): e61618.

［13］Xiong W, Cepko C. Distinct expression patterns of AAV8 vectors with broadly active promoters from subretinal injections of neonatal mouse eyes at two different ages［J］. Adv Exp Med Biol, 2016, 854:501-507.

［14］Hellstrom M, Harvey AR. Retinal ganglion cell gene therapy and visual system repair［J］. Curr Gene Ther, 2011, 11(2):116-131.

［15］Huang Y, Xu Y, Cheng Q, et al. The expression changes of myelin and lymphocyte protein (MAL) following optic nerve crush in adult rats retinal ganglion cells［J］. J Mol Neurosci, 2014, 54(4):614-621.

［16］Sun F, Park KK, Belin S, et al. Sustained axon regeneration induced by co-deletion of PTEN and SOCS3［J］. Nature , 2011, 480(7377):372-375.

［17］O’Donovan KJ, Ma K, Guo H, et al. B-RAF kinase drives developmental axon growth and promotes axon regeneration in the injured mature CNS［J］. J Exp Med, 2014, 211(5):801-814.

［18］Zippo A, De RA, Bardelli M, et al. Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro［J］. Blood, 2004, 103(12):4536-4544.

［19］Willert M, Augstein A, Poitz DM, et al. Transcriptional regulation of Pim-1 kinase in vascular smooth muscle cells and its role for proliferation［J］. Basic Res Cardiol, 2010, 105(2):267-277.

［20］Hu S, Yan G, Xu H, et al. Hypoxic preconditioning increases survival of cardiac progenitor cells via the pim-1 kinase-mediated anti-apoptotic effect［J］. Circ J, 2014, 78(3):724-731.

［21］Stumpner J, Smul TM, Redel A, et al. Desflurane-induced and ischaemic postconditioning against myocardial infarction are mediated by Pim-1 kinase［J］. Acta Anaesthesiol Scand, 2012, 56(7):904-913.

［22］Feldman JD, Vician L, Crispino M, et al. KID-1, a protein kinase induced by depolarization in brain［J］. J Biol Chem, 1998, 273(26):16535-16543.

［23］Konietzko U, Kauselmann G, Scafidi J, et al. Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP［J］. Embo J, 1999, 18(12):3359-3369.

［24］Zhang Y, Parsanejad M, Huang E, et al. Pim-1 kinase as activator of the cell cycle pathway in neuronal death induced by DNA damage［J］. J Neurochem, 2010, 112(2):497-510. ［25］Yata K, Matchett GA, Tsubokawa T, et al. Granulocyte-colony stimulating factor inhibits apoptotic neuron loss after neonatal hypoxia-ischemia in rats［J］. Brain Res, 2007, 1145:227-238.