Construction and biological activities of human tPA eukaryotic expression plasmid.
BACKGROUND
Tissue-type plasminogen activator(tPA), which is highly efficient and specific in resolution of thrombus, could significantly improve the survival rate and life-quality of the patients with thrombosis. This study was designed to clone human tPA gene, construct eukaryotic expression plasmid pcDNA3.1(+)/tPA, and evaluate their biological activities.


METHODS
The tPA gene was obtained from dead human heart tissue with reverse transcriptase-polymerase chain reaction (RT-PCR). It was cloned to eukaryotic expression plasmid pcDNA3.1(+) by recombination strategy. The eukaryotic expression plasmid pcDNA3.1(+)/tPA was identified by restriction enzyme digestion and sequenced. The pcDNA3.1(+)/tPA was transfected into vascular smooth muscle cell (VSMC) by using lipofection. The tPA expression level was detected by Northern blot and dot blot, and the protein biological activities of tPA were detected by the fibrin plate technique.


RESULTS
The tPA gene was cloned and pcDNA3.1(+)/tPA was constructed. The tPA expression levels of mRNA and protein were highly increased after pcDNA3.1(+)/tPA transfected into VSMC, and the expression of tPA protein showed evident biological activities.


CONCLUSIONS
The present study has laid a foundation for further animal experiment in treating thrombus in transplanted organ by tPA gene transfection.
Wu
Zhong-Jun
ZJ
Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. wzjxdfpxy@zju.edu.cn
Yang
Su-Fen
SF
Zheng
Shu-Sen
SS
Shi
De
D
Wu
Wei-Wei
WW
Li
De-Wei
DW
D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005784:Gene Amplification; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D010959:Tissue Plasminogen Activator