| Thrombus is the major leading life-threatening disease human faced, including acute myocardial infarction, pulmonic infarction, artery infarction, shock lacked of blood and so on. The mortality is more than 30% caused by acute myocardial infarction.Thrombolytic treatment is considered as the best way to cure thrombus, it is always a hot point to study better thrombolytic drugs with fewer side-effect in the world. Plasminogen activator is the most important one among them. Because they could convert circulating plasminogen to plasmin and dissolve the fibrin made of thrombus framework and eliminate thrombus.The ability of thrombolytic is the rate of dissolving thrombus, thorough and thrombus formed again. A good thrombolytic drug could dissolve not only old thrombus but also new thrombus, and lower rate of thrombus formed again.The thrombolytic drugs could be divided into three-generation on the clinic and study as a rule:Although these first-generation fibrinolytic agents(Streptokinase, Urokinase, and so on) are effective at thrombolysis , they are not fibrin specific; they also convert circulating plasminogen to plasmin. Because plasminogen in the thrombus and in the plasma are in equilibrium, the plasminogen within the thrombus is also gradually depleted. This "plasminogen steal" reduces clot lysis and side-effect at every pore.The second-generation fibrinolytic agents.(tissue plasminogen activator, t-PA;single-chain urokinase-type plasminogen activator, or prourokinase) are fibrin selective in vivo or vitro at animal experiments, with lower side-effect haemorrhage at every pore.When injected into blood, they can be combined to fibrin and eliminate the acyl, converted into SK-fibrinogen. The fibrinogen can be activable converted into fibrin by SK-fibrinogen, While the second-generation fibrinolytic agents have the limitions of a shorter half-life, longer injection time,and the higher rate of thrombus formation once more.The third-generation thrombolytic agents are several types new thrombolytic agents being developed, including TNK-PA, STAR, rPA ,and so on . They have the advantages of lower side-effect, longer half-life. Moreover, the usage of dose is less and the cost of expense is rather lower. The first-generation fibrinolytic agents have the fatal limition such as haemorrhage, side-effect, which have been disused. The new second-generationfibrinolytic agents have been improved, but still not ideal. The scientists are engaged in studing the third-generation fibrinolytic agents with the way of protein engineering in the world. The way is to enhance or keep the activity of catalysis in advance, the means focused on making some amino acid mutate, deleting ,adding or fusing some amino acid domains in order to get the next objects: 1. Improving the fibrin selectivity. 2. Increasing resistance to plasminogen-activator inhibitor. 3. Prolonging the half-life in the serum. 4. Improving thrombolytic efficacy. Reteplase is a single chain nonglycosylated deleted mutant of alteplase, consisted of just the kringle-2 and protease domains. These structural differences result in a fivefold decrease in fibrin binding and an extended half-life and side-effect of hemorrhage. Reteplase is consided as a safe and effective thrombolytic drug.At present reteplase is not on sale in China. Reteplase on sale outside is expressed as inclusion body in E.coli, including 355 amino acids, 9 disulfide bonds, a nonglycosylated, single-chain molecular. The molecular weight is 39kD. It is a complicated denaturing and restoring process to distill and pure reteplase, many disulfide bonds is easy to link wrongly and unstable. The rate of callback is often under the 10% of expressed reteplase.The amount of single injection is more than 30 times than the other recombinant protein such as rhG-CSF, IFNα-2b, so the final products need high purity and low endotoxin. In order to solve the problems, it is one of the instancy tasks to search a new expressed system. Pichia pastoris is a species of methanol yeast, which has a needed peroxide enzyme(AOX1, AOX2)... |
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