中华耳科学杂志2016，Vol.14Issue(6)：841-845,5.DOI:10.3969/j.issn.1672-2922.2016.06.027
过表达mitf基因腺相关病毒的构建与鉴定
Construction and qualification of recombinant mitf adeno-associated virus gene vector
摘要
Abstract
Objective To construct a recombinant adeno-associated virus vector for potential gene therapy for mitf mu-tations. Methods We acquired the target gene via PCR amplification. pAAV-RC, pHelper and pAAV-GFP were transfected into HEK293 cells using the artificial liposome method and the recombinant adeno-associated virus vector product was puri-fied via column purification. Titers of the recombinant adeno-associated virus vector were determined by qPCR. The recom-binant adeno-associated virus vector was used to infect 293 cells. Real-time PCR and western blot were used to identify ex-pression of the recombinant adeno-associated virus vector carrying mitf. Results PCR amplification and DNA sequencing il-lustrated that the target gene and recombinant adeno-associated virus vector were constructed successfully with high titers (1.0*10^12 vg/ml). Recombinant adeno-associated virus vectors infected 293 cells successfully with an expression efficien-cy of 86.3%. No cell death was found. The constructed mitf gene was equivalent to the normal mitf gene based on real-time PCR and western blot results. Conclusion The recombinant adeno-associated virus vector carrying mitf provides a new way of gene therapy for hereditary diseases caused by mitf mutations.关键词
MITF/腺相关病毒/基因治疗Key words
MITF/adeno-associated virus vector/gene therapy分类
医药卫生引用本文复制引用
史珣贝,林昶,杨仕明,吴南..过表达mitf基因腺相关病毒的构建与鉴定[J].中华耳科学杂志,2016,14(6):841-845,5.基金项目
国家863青年科学家项目(2014AA020510)；国家自然科学基金（面上项目81271082，青年基金81400472）；北京科技创新基地培育与发展专项（z151100001615050）；福建省科技厅重点项目（2014I0003）；中国科协创新驱动助力工程（2016CXQD01）。@@@@National 863 project young scientists(2014AA020510) （2014AA020510）
the National Natural Science Foundation of China (NSFC #81271082,81400472). Special Cultivating and Developing Program of Beijing Science and Technology Innovation Base （z151100001615050)；Key Projects of Fujian Provincial Science and Technology Department (2014I0003) （NSFC #81271082,81400472）
Innovation and drive power en-gineering project of China association for science and technology （）
