Construction and qualification of recombinant mitf adeno-associated virus gene vector
SHI Xunbei
LIN Chang
YANG Shiming
WU Nan
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.
Keywords：MITFadeno-associated virus vectorgene therapy
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 841-845 )