Construction and biological activities of human tPA eukaryotic expression plasmid
Zhong-Jun Wu, Su-Fen Yang, Shu-Sen Zheng, De Shi, Wei-Wei Wu and De-Wei Li
Hangzhou, China
Author Affiliations: Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Wu ZJ and Zheng SS), Department of Vascular Surgery, Chongqing Medical University, Chongqing 400016, China (Yang SF, Shi D, Wu WW and Li DW) Corresponding Author: Zhong-Jun Wu, MD, Organ Transplant Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-88215788; Fax: 86-571-87236570; Email: wzjxdfpxy@zju.edu.cn)
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.
2004, Vol. 3 
