Construction of recombinant eukaryotic hansenula engineering bacterial strains containing medical hirudin genes and production process of recombinant hirudin

Abstract

The invention discloses construction of recombinant eukaryotic hansenula engineering bacterial strains containing medical hirudin genes and a production process of recombinant hirudin, wherein hansenula polytype host cell strains RB-11 are adopted as initial bacterial strains for constructing hansenula high-expression engineering bacterial strains RBHV 145-6 containing hirudin, and the hirudin with high purity and high content is obtained through culture and fermentation. In the invention, the hirudin genes are copied onto host cell chromosomes in a multi-copy mode, in addition, the copying number is kept stable, exogenous gene expression boxes containing the hirudin genes are completely integrated onto the host cell chromosomes, the existence of free plasmids in seed bank cells and passage 40 and 80 generation cells is avoided, the expression quantity and the secretion efficiency of foreign protein are 10 times higher than those of saccharomyces cerevisiae, particularly through beingcompared with that of the polytypic hansenula in China, the expression quantity of the foreign protein is high and is 35.8 percent, and in addition, the excessive glycosylation phenomenon does not exist, so the production process is suitable for target protein through large-scale fermentation production.

Description

technical field [0001] The invention relates to the construction of an engineering strain of eukaryotic Hansenula with extracellular protein expression gene, more specifically, relates to an engineering strain containing a hirudin gene and a method for constructing and producing hirudin. Background technique [0002] Leeches have been used in traditional Chinese medicine for thousands of years and are an indispensable traditional Chinese medicine for promoting blood circulation. Hirudin is a small molecular protein with strong anticoagulant activity secreted by leech salivary glands. It is mainly a polypeptide containing 65 amino acids, with a molecular weight of about 7000 and three disulfide bonds. Seven isomers have been separated and identified. Because hirudin has a strong affinity with thrombin, it can neutralize thrombin at a very low concentration and play an anticoagulant role. It can be widely used in operations such as organ transplantation. The fibrinolytic effe...

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Problems solved by technology

Chinese patent application "A Recombinant Hirudin Encoding Gene and Its Application" (application number 200810103154.8, application date March 31, 2008, publication date August 27, 2008) discloses a method of utilizing Hansenula polymorpha to produce hirudin production, but its exogenous protein expression and secretion efficiency are low, and it will produce excessive glycosylation, so there are serious deficiencies in adapting to large-scale fermentation production

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