Construction of recombinant adenoviral vector carrying human tissue inhibitor of metalloproteinase-1 gene and its expression in vitro.
BACKGROUND
Overexpression of human tissue inhibitors of metalloproteinase-1 (TIMP-1) may play an antitumor role in inhibiting hepatocellular carcinoma (HCC) growth and progression. The aim of the study was to construct a recombinant adenovirus vector carrying hTIMP-1 cDNA for liver gene therapy and observe its expression in vitro.


METHODS
The full-length cDNA of hTIMP-1 was positively cloned into the adenoviral shuttle vector pAdTrack-CMV, and then cotransformed into competent BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Thus, a recombinant adenovirus AdhTIMP-1 containing full-length cDNA of hTIMP-1 was generated by homologous recombination in E. coli. AdhTIMP-1 was then packaged and amplified in adenoviral packaging cells, or human embryonic kidney 293 cells. The viral titer was checked by green fluorescent protein (GFP), and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and RT-PCR.


RESULTS
The recombinant adenovirus vector carrying hTIMP-1 was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. The transcription of TIMP-1 mRNA in 293 cells was checked by RT-PCR and TIMP-1 protein could be detected in the culture by Western blot analysis.


CONCLUSIONS
The successful construction of recombinant adenoviral vector carrying human TIMP-1 and the effective expression in vitro has laid a foundation for further study of its antitumor function and may pave the way for future application in liver gene therapy.
Xia
Dong
D
Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
Yan
Lu-Nan
LN
Tong
Yu
Y
Wang
Xin-Ping
XP
Zhang
Ming-Man
MM
Zhao
Lan-Ying
LY
D000256:Adenoviridae; D001483:Base Sequence; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000628:therapy; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000473:pathology / Q000628:therapy; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D012680:Sensitivity and Specificity; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism*
D012333:RNA, Messenger; D019717:Tissue Inhibitor of Metalloproteinase-3