Construction of a recombinant plasmid expressing microdystrophin gene fused with HSV-1 VP22 and its biological characterization
10.3760/cma.j.issn.1671-8925.2009.04.001
- VernacularTitle:HSV-1 VP22及microdystrophin基因融合表达重组质粒的构建及其生物学特性的初步研究
- Author:
Fu XIONG
1
;
Cheng ZHANG
;
Song-Lin CHEN
;
Hui ZHENG
;
Shao-Bo XIAO
;
Yong-Fei PAN
;
Mei-Juan YU
;
Shan-Wei FENG
;
Xi-Lin LU
Author Information
1. 南方医科大学
- Keywords:
Microdystrophin gene;
Herpes simplex virus 1 VP22;
Duchenne muscular dystrophy;
Protein transduction
- From:
Chinese Journal of Neuromedicine
2009;8(4):325-330
- CountryChina
- Language:Chinese
-
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.
