Construction of a recombinant plasmid expressing microdystrophin gene fused with HSV-1 VP22 and its biological characterization / 中华神经医学杂志

ABSTRACT

Objective To construct a recombinant plasmid expressing human microdystrophin gene fused with VP22 ofhmnan herpes simplex virus 1(HSV-1),and investigate the expression of microdystrophin in vitro and the characteristics of VP22-mediated protein transduction.Methods Full length HSV-1 VP22 gene was amplified by PCR from the plasmid pSINrep5-VP22 and cloned into the eukatyotic expression vector pAVXl to conslruct recombinant plasmid pAVP22.Microdystrophin cDNA was obtained from the recombinant plasmid pBSK-MICRO digested with Not Ⅰ,and the product Was inserted into plasmid pAVP22 to construct the plasmid pAVP22-MICDYS. 3T3 cells were transfected with pAVP22-MICDYS,and the expression of microdystrophin was detected by RT-PCR,Western blotting and immtmocytochemislry.The supematant of 3T3 cells transfected with pAVP22-MICDYS were collected to infect human mesenchymal cells(MSCs),and the expression of the fusion protein VP22-microdystrophin in the cells was detected using immunohistochemistry to assess VP22-mediated protein transduction. Results The recombinant plasmid expressing microdystrophin-VP22 fusion gene capable of in vitro expression of the fusion protein was constructed successfully.VP22 was shown to enhance the expression efficiency of microdystrophin in 3T3 cells and transduce VP22-microdystrophin fusion protein from 3T3 cells to human MSCs. Conclusion The recombinant plasmid expressing microdystrophin-VP22 fusion gene with protein transduction capacity provides an important basis for further study of the fusion protein in treatment of Duchenne muscular dystrophy.