Construction Of Human Tissue-Type Plasminogen Activator Prokaryotic Expression Vector And Its Preliminary Expression In Escherichia Coli

Objective:The Prokaryotic expression vectors of Human Tissue-type PLasminogen activator(tPA) have been constructed to identify whether it can be expressed primarily in Escherichia Coli or not, explore the optimum expressive condition, with a hope of obtaining active target proteins after purification and renaturation, which can satisfy the need of production and clinical use.Method:Selected the prokaryotic expression vector pET-28a, The EST-tPA plasmid was amplified from the wild-type tPA recombinant mRNA as a template. The forward and reverse primers were designed. The sequence of RGDS gene was added to the C end of tPA. The target gene of GRDS-tPA can be amplified by means of PCR and recovered by Gel Extraction. The target gene of tPA and the pET-28a vector were double-digested by NcoⅠ and XhoⅠ. The fragments could be connected through cohesive terminus of NcoⅠ and XhoⅠ, which were relatively complemented. The constructions then were transferred into the competent cells named XL-1, with selections, double-digested identifications, plasmid recombination and sequencing. The produced competent cell BL21(DE3) were ready for pET28a-tPA transformation. The SDS-PAGE could show the rtPA expressed in the Escherichia Coli.Result:The target gene rtPA showed a brand of1700bp by Agarose gel electrophoresis, the identification was positive. The correct pET28a-tPA sequence could be identified through sequencing. the target rtPA protein showed a brand of53KD by SDS-PAGE, it could be determined as non-activity inclusion body. After the optimization of the expression condition, rtPA could get a good harvest at37℃for4hours. The expression could be the best at the25℃for8hours, and the expression at17℃for12hours could be lower than the Formers.Conclusion:(1)The Prokaryotic expression vectors of Human Tissue-type PLasminogen activator(tPA) have been constructed successfully, and can be expressed in the Escherichia Coli effectively.(2)The expression of rtPA in Escherichia Coli has a good relationship with induced time, temperature, the concentration of IPTG. Long-time induction and low temperature may lead to low expression of rtPA.