| Tissue-type plasminogen activator(t-PA) is a key enzyme in fibrinolysis system, the physiological role of which is to convert zymogen plasminogen into an active form of serine proteinase plasmin, which then initiates or accelerates the process of fibrinolysis and degrades the fibrin network of thrombi and blood clots. The activation of plasminogen by t-PA occurs preferentially on clots, which results in considerable enhancement of plasmin generation while preventing systemic activation of plasminogen. Under pathophysiological conditions such as acute myocardial infarction and other thromboembolic disorders, the lytic potential of the patient might not be sufficient for rapid disruption of a thrombus; intravenous administration of plasminogen activators such as human t-PA, u-PA or the bacterial plasminogen cofactors streptokinase or staphylokinasse has been shown to cause effective lysis of stable coronary thrombi. Human t-PA has attracted particular interest as a therapeutic agent due to its natural role in the lysis process, especially its relatively high affinity to the fibrin. However, t-PA has some shortages all the same, its half life is only 3-5 minutes. So when used as therapeutic agent i.v., it must be given continuously in abundance and the cost will be very high consequently.In order to reduce the clearance rate in the plasma, and to increase its affinity to the fibrin, many variants of t-PA have been developed in recent years. In our experiment, rPA, the deleted mutant of tPA cDNA, was initially obtained by the PCR technology. The rPA was then mutated by substituting the KHRR with AAAA at positions 296-299 (rPA(K)), and A at position 473 with S (rPA(A)) and with both (rPA(KA)) at the same DNA, respectively,utilizing site-directed mutagenesis method. The four above mutants were subcloned into the prokaryotic expression vector pET-28a(+) separately and the expression vectors pErA, pErA(K), pErA(A) and pErA(KA) were identified with restriction enzyme digestion and sequencing. The correct recombinants were transformed into E.coli strain BL21 (DE3) and induced by IPTG, the lysates of the bacteria were separated on SDS-PAGE. The result indicated that the expression of mutants pErA(K) and pErA(KA) were realized, however, pErA and pErA(A) could not be expressed under the same conditions. The molecular weight of expressed proteins of interest was 39.6kD, in accordance with that of the estimated and the maginutide of product makes up 35.97% and 37.71% total proteins of E.Coli, separately. The specific reactivity with anti-tPA antibodies was shown by Western blotting analysis. After being primarily purified and refoldded, the expressed proteins displayed relatively high fibrin lysis activity in vitro on a modified Fibrin Agarose Plate Assay (mFAPA). All above laid a foundation for further purification, activity assay in vivo and large scale production.As for the eukaryotic expression system, it can process the mature mRNA properly, improve the preciseness rate of the products, and besides this, the products of it can be secreted into the medium, which is of great benefits to the production and purification of the officinal protein.The signal peptide(SP) of t-PA was achieved by PCR, and after that, rPA and rPA(K) were added the signal peptide by further PCR with SP as 5'-upperstream primer and formerly 3'- downstream primer, respectively. Then the two eukaryotic expression vectors, pCSRA and pCSRK were constructed and expressed in eukaryotic expression system and were analyzed for a variety of properties. The recombinants were transfected into COS-7 with liposome at first and then the supernant of the cell was collected in different time phase, 24h, 48h and 72h, detected with FAPA(Fibrin Agarose Plate Assay).The experiment indicated that the vectors of pCSRA and pCSRK were expressed successfully and the products had good biological activities.To establish a stable cell line expressing t-PA variants, liposome-mediated cotransfection of pCSRA and pCSRK each with pSV2-dhfr were carried out on CHO-d...
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