To Study The Effects Of Transfecting T-PA Gene And Pcnaantisense Oligodexoxynucleotide Together On Angio-Restenosis Resulted From Angioreconstruction

Objective To clone human tissue-type plasminogen activator (t-PA) full-length genome and construct an recombinant eukaryotic expression plasmid containing tandem copies of t-PA genome.Methods The total RNA was obtained from fetal heart with efficient Trizol reagent. The t-PA complementary deoxyribonucleic acid (cDNA) fragment was extracted by reverse transcription polymerase chain reaction (RT-PCR) and was amplified, purified and retrieved. To digest pcDNA3.1(+) eukargotic plasmid and t-PA cDNA gene fragment with dual-endonuclease and retrieved a big fragment from dual-endonuclease digesting pcDNA3.1(+) and a 1.9kb fragment from that t-PA cDNA gene. The t-PA cDNA (1.9kb) fragment was inserted into eukargotic expression plasmid pcDNA3.1(+). The recombinant pcDNA3.1(+)/t-PA was identified by restriction enzyme digestion and polymarase chain(PCR), and assayed sequence of t-PA cDNA.Results The full-length t-PA genome was colned successfully from fetal heart. Restrictive enzyme (HindIII / BamHI) digestion analysis Showed that recombinant eukaryotic expression plasmid containing t-PA genome pcDNA3.1(+)/t-PA been constructed successfully. Sequencing result revealed the sequence of amplified t-PA genome was identical with that in gene bank.Conclusion The recombinant eukargotic expression plasmid pcDNA3.1(+)/t-PA is constructed successfully. It establishs the foundation for gene prevention local thrombosis after angio-reconstruction.Key words　tissue-type;plasminogen activator;gene cloning;eukargon expression plasmid...