A preparation process for separating and purifying recombinant human pro-urokinase from recombinant Escherichia coli fermentation broth

Abstract

A preparation process for separating and purifying recombinant human pro-urokinase from recombinant Escherichia coli fermentation broth applied in the technical field of biopharmaceuticals, the preparation process comprises the following steps, using the artificially synthesized human pro-urokinase whole gene sequence as a template, by Obtain the target gene fragment of human prourokinase by PCR, construct a prokaryotic expression vector containing human prourokinase, transform the expression vector into Escherichia coli, screen and pick positive clones, which are genetically engineered strains of human prourokinase; The engineered strain of prourokinase was inoculated into solid LB medium, picked a single colony and inoculated into liquid LB medium, cultured on a shaker at 32°C overnight, then inoculated into the fermentation medium in proportion, and cultivated on a shaker at 32°C overnight, Then inoculate into the fermenter according to the proportion, raise the temperature to 42° C. to induce culture for 4 hours, collect the bacteria by centrifugation, disrupt the bacteria by ultrasonic, and collect the precipitate by centrifugation, which is the inclusion body of human prourokinase. The invention is not limited by natural resources, has the advantages of less investment, high output, large profit margin, unique thrombolytic mechanism, obvious effect, and no or low toxicity and side effects.

Description

technical field [0001] The invention relates to a preparation process for separating and purifying recombinant human pro-urokinase from recombinant Escherichia coli fermentation broth in the technical field of biopharmaceuticals, in particular to a preparation process for recombinant human pro-urokinase for treating thrombus. Background technique [0002] Thrombotic disease has become one of the main diseases that endanger human health. As my country enters an aging society, there are more than 10 million new cardiovascular and cerebrovascular cases every year, and the drug cost for thrombosis treatment is as high as tens of billions. Thrombotic disease not only has a high incidence rate, but also has a high mortality and disability rate. In the past 20 years, although the treatment of thrombotic diseases has made significant progress, its mortality rate is still the highest among all diseases. About 10 million people in the country develop the disease every year, and more t...

AI Technical Summary

Problems solved by technology

In 1991, Gaetano ORSINI et al. [Eur. J. Biochem. 195, 691-697 (1991)] reported the method of expressing and purifying human prourokinase with recombinant Escherichia coli. They used Sepharose-S, hydroxyapatite and Sephacryl S-200 chromatography successively Column purification of human recombinant prourokinase, although this method can obtain active human recombinant prourokinase, but the protein recovery efficiency and purity are low

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