Cloning T-PA Gene And Constructing PcDNA3.1(+)/t-PA Expression Vector

Objective, Clone tissue-type plasminogen activator (t-PA) gene and construct a new kind of recombinant vector containing human tissue-type plasminogon activator (t-PA) cNDA neither cytotoxiaty nor actovating prot-oncogenes.Methods, The RNA was extracted from fetal lung with efficient Trizol reagent. The t-PA cDNA was obtained from it by reverse transcription polymerase chain reaction (RT-PCR) and was amplified, purified and retrieved. To digest pcDNAS.l (+)/t-PA plasmid and t-PA gene with dual-endonuclease, we retrieved a big fragment from pcDNA3.1 (+) and a 1.9 kb fragment from t-PA gene. Recombined the big fragment and 1.9 kb fragment. The recombination pcDNA3.1 (+)/t-PA plasmid and t-PA gene was identified and sequenced by mono-endonuclease and dual-endonuclease.Result, we succeed in doing t-PA gene from fetal lung and construct a sort of eukaryon expression plasmid vector with pcDNA3.1 (+). A 6.9 kb fragment was found in the recombination pcDNA3.1 (+)/t-PA after digesting with mono-endonuclease. Digesting the 6.9 kb fragment with dual-endonuclease, we obtained two fragments .One is 5.0 kb, another is 1.9 kb. It was identified by sequencing that the length of the 6.9 kb fragment was same as that human being's.Conclusion, A new eukaryon expression plasmid with t-PA gene has been constructed. It established the basics for gene treating localthrombosis after angioplasty.