Novel production method of thrombolytic drug rPA (recombinant plasminogen activator)

Abstract

The invention provides a method of preparing a thrombolytic drug rPA (recombinant plasminogen activator). The method comprises the steps of: conducting secretion expression on the rPA via saccharomycete for screening to form a high expression strain, conducting the secretion expression on a ligand, erythrina indica trypsin inhibitor (ETI) via the saccharomycete for screening to form a high expression strain, coupling the ETI as the ligand with a stationary phase to prepare an affinity chromatography medium, performing affinity chromatography on the rPA by using the obtained affinity chromatography medium, and separating and purifying the rPA. The method is high in yield and simple in technology, and completely solves the problem that a denaturation and renaturation process of an escherichia coli expressed inclusion body cannot remove endotoxin in prokaryotic expression in the prior art, and the production cost is greatly lowered.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a new production method of thrombolytic drug rPA Background technique [0002] Thrombotic complications caused by cardiovascular diseases seriously threaten human life and are one of the main diseases leading to death and disability. The mortality rate of acute myocardial infarction (AMI) caused by thrombus can be as high as 30%. About 3.5 million people in China die from various cardiovascular diseases every year, accounting for the first of all causes of death, with an average death every ten seconds a patient. More than half of the people have myocardial infarction or sudden death for the first time. About 1.5 million people in the United States have myocardial infarction every year. Intravenous thrombolytic therapy is the most important progress in the treatment of AMI, and it is the main method to treat acute myocardial infarction and reduce mortality. ...

AI Technical Summary

Problems solved by technology

The second-generation thrombolytic drugs are represented by recombinantly expressed tPA (alteplase, rt-PA). This type of drug has certain specificity for thrombolysis, but its half-life is short and the administration method is complicated. , and then two intravenous infusions, and the dose needs to be adjusted according to body weight

[0004] The third-generation thrombolytic drug rPA (Reteplase) is a regional deletion mutant of tissue plasminogen activator (tPA), developed by Boehringer Mannheim in Germany, approved by the FDA in 1996, with a trade name of Retavase, which is a molecular weight of The 39.6KD single-chain non-glycosylated polypeptide is obtained from the inactive inclusion body expressed by E.coli through renaturation in vitro. This mutant contains 9 pairs of disulfide bonds, so it is very difficult to renature, and the renaturation yield is very high. Low (below 5%), the above reasons make rPA expensive, and it is still difficult to popularize in the domestic market

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