Construction and Detection of a Novel Type of Recombinant Human rAAV2/2-ND4  

作　　者：Han Pei Xing Wan Weikun Hu Xiaoyan Dong Bin Li 

机构地区：[1]Department of Ophthalmology,Tongji Medical College,Huazhong University of Science & Technology

出　　处：《Eye Science》2013年第2期55-59,共5页眼科学报（英文版）

基　　金：financially supported by National Natural Science Fund.Molecule mechanism of the resistance of HRD1 against diabetic retinopathy (NO.81271015);Molecule mechanism of the resistance of P58IPK against diabetic retinopathy (NO.30872823)

摘　　要：Purpose:To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expression level in mitochondria for potential application in gene therapy for Leber's hereditary optic neuropathy (LHON). Methods:A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting sequence that induces the translocation of this gene into mitochondria.This recombinant adeno-associated virus type 2/serotype 2 (rAAV2/2)-mediated NADH dehydrogenase subunit 4 (ND4) gene was constructed, purified, condensed, and amplified by PCR.The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigeninlabeled H1 probe.Expression of ND4 in mitochondria was evaluated by immunofluorescence. Results: The constructed rAAV2/2-ND4 specifically amplified the target gene band of ND4 and the physical titer of ND4 gene was 1.0×1011 vg/mL,confirming that the recombinant adenovirus vector contained the ND4 target gene. Expression of the ND4 gene was detected in mitochondria by immunofluorescence. Conclusion:A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.Purpose： To construct a novel AAV-mediated gene delivery of the human ND4 complex I subunit and to detect its expres- sion level in mitochondria for potential application in gene therapy for Leber＇s hereditary optic neuropathy （LHON）. Methods： A novel type of normal human ND4 gene was synthesized artificially to contain a mitochondrial targeting se- quence that induces the translocation of this gene into mito- chondria. This recombinant adeno-associated virus type 2/ serotype 2 （rAAV2/2）-mediated NADH dehydrogenase sub- unit 4 （ND4） gene was constructed, purified, condensed, and amplified by PCR. The physical titer of rAAV2/2-ND4 was determined by slot-blot hybridization using a digoxigenin- labeled H1 probe. Expression of ND4 in mitochondria was e- valuated by immunofluorescence. Results： The constructed rAAV2/2-ND4 specifically ampli- fied the target gene band of ND4 and the physical titer of ND4 gene was 1.0x 10~1 vg/mL, confirming that the recombi- nant adenovirus vector contained the ND4 target gene. Ex- pression of the ND4 gene was detected in mitochondria by im- munofluorescence. Conclusion： A new type of rAAV2/2-ND4 was successfully constructed and may have potential in gene therapy for LHON.

关 键 词：重组腺相关病毒 基因检测 ND4基因 重组腺病毒载体 类型 基因治疗 线粒体 PCR扩增 

分 类 号：R774.6[医药卫生—眼科]