[Construction of herpes simplex virus-1 virion protein 22-mediated microdystrophin gene recombinant adenovirus and study on the property of protein transduction].
OBJECTIVE
To construct the recombinant adenovirus containing herpes simplex virus-1 virion protein (VP) 22 and human microdystrophin gene, then the adenovirus was transfected into C2C12 myoblast and studied on the property of protein transduction with VP22-mediated microdystrophin in C2C12 myoblast.


METHODS
The full-length VP22 cDNA was obtained from recombinant plasmid pSINrep5-VP22 with PCR, and the product was directionally inserted into pShuttle-CMV to acquire the plasmid pCMV-VP22. Microdystrophin cDNA was obtained from recombinant plasmid pBSK-micro digested with restrictive endonuclease NotI, and the product was directionally inserted into pCMV-VP22 to acquire the plasmid pCMV-VP22-MICDYS. The plasmid of pCMV-VP22-MICDYS was lined with Pme I, and the fragment containing VP22-microdystrophin was reclaimed and transfected into E1 coli BJ5183 with plasmid pAdeasy-1. After having been screened by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the cytopathic effect of cells and by PCR method to acquire the recombinant adenovirus Ad-VP22-MICDYS. Finally, the C2C12 myoblast were transfected with the recombinant adenovirus Ad-VP22-MICDYS and Ad-MICDYS, and the expression of microdystrophin was detected by RT-PCR, Western blot and immunocytochemistry.


RESULTS
The recombinant adenovirus including VP22 and microdystrophin gene was successfully constructed. VP22 transferred VP22-microdystrophin fused protein from infected C2C12 myoblast into uninfected cells and enhance the expression of microdystrophin in myoblast.


CONCLUSIONS
Recombinant adenovirus containing VP22 and microdystrophin gene was constructed successfully. VP22 can enhance the expression with microdystrophin in myoblast. It lays the foundation for further studying on VP22-mediated recombinant including microdystrophin gene to cure Duchenne muscular dystrophy.
Xiong
Fu
F
Department of Medical Genetics, Southern Medical University, Guangzhou 510515, China.
Zhang
Cheng
C
Zheng
Hui
H
Xiao
Shao-bo
SB
Pan
Yong-fei
YF
Xu
Yong-feng
YF
Liu
Zheng-shan
ZS
Li
Yong
Y
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D000818:Animals; D002460:Cell Line; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000378:metabolism / Q000821:virology; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic; D015678:Viral Structural Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000378:metabolism
D016189:Dystrophin; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22