中文摘要：

目的获得能够过表达小眼畸形相关转录因子（mitf）的重组腺相关病毒载体,用于mitf基因突变相关疾病的基因治疗。方法利用PCR扩增获得目的基因,构建携带目的基因mitf的重组腺相关病毒载体,利用人工脂质体法将p AAV-RC,p Helper,以及p AAV-GFP系列载体转染到HEK293细胞中,采用细胞内质粒DNA同源重组法进行重组腺相关病毒载体构建,利用柱纯化法纯化病毒载体,q PCR法测定重组腺相关病毒载体滴度,病毒载体感染HEK293细胞进行病毒有效性及安全性鉴定,real-time PCR与western blot法进行mitf表达的鉴定。结果 PCR扩增和测序结果一致证实成功获得目的基因并且成功构建mitf基因腺相关病毒载体,测得高水平病毒滴度1.0^＊10^12vg/ml,病毒载体感染HEK293细胞转染效率为86.3%,并未发现HEK293细胞死亡,Real-time PCR及Western blot检测构建Mitf基因转录m RNA与表达的蛋白与正常MITF一致。结论成功构建的携带mitf基因的重组腺相关病毒载体为未来进行mitf突变导致的遗传性疾病的基因治疗提供新思路。

英文摘要：

Objective To construct a recombinant adeno-associated virus vector for potential gene therapy for mitf mutations. Methods We acquired the target gene via PCR amplification. p AAV-RC, p Helper and p AAV-GFP were transfected into HEK293 cells using the artificial liposome method and the recombinant adeno-associated virus vector product was purified via column purification. Titers of the recombinant adeno-associated virus vector were determined by q PCR. The recombinant adeno-associated virus vector was used to infect 293 cells. Real-time PCR and western blot were used to identify expression of the recombinant adeno-associated virus vector carrying mitf. Results PCR amplification and DNA sequencing illustrated 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 efficiency 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.