Recombinant engineering bacterium capable of efficiently expressing liraglutide precursor and application of recombinant engineering bacterium

Abstract

The invention provides a recombinant engineering bacterium for efficiently expressing a liraglutide precursor and an application of the recombinant engineering bacterium. According to the invention, asignal peptide and an enterokinase restriction enzyme cutting site are designed at the N end of a liraglutide precursor molecule Arg34G LP-1 (7-37), the C end is connected with termination codon, a target gene is formed, then, the target gene is inserted between two restriction enzyme cutting sites of an expression vector; the recombinant engineering bacterium for expressing the liraglutide precursor is constructed, the engineering bacterium is subjected to high-density fermentation culture and is expressed in the form of an inclusion body of a fusion protein, the expression quantity of the recombinant fusion protein is high, the recombinant fusion protein accounts for about 25%-35% of the total protein of the bacterium, and the expression quantity of the inclusion body of a target protein reaches 15-20g / L. The inclusion body is low in impurity protein content, beneficial to separation and purification, and high in purification efficiency and good in stability, the production cost isgreatly reduced, the production efficiency is improved, and the inclusion body has the good application prospect in the field of diabetes treatment medicine preparation.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium for highly expressing liraglutide and its application. Background technique [0002] In recent years, scientists have developed GLP based on the physiological mechanism of glucagon-like peptide 1 (GLP-1) to promote insulin secretion and regulate blood sugar. -1 receptor agonist. GLP-1 has great advantages in the treatment of type 2 diabetes mellitus (T2DM): ①Rely on the increase of human blood sugar concentration to regulate the body's blood sugar; ②Promote the proliferation and differentiation of islet β cells, inhibit their apoptosis, promote the secretion of insulin, and increase the body's insulin Sensitivity; ③Inhibit gastrointestinal emptying, increase satiety, reduce food intake, and reduce weight. But the only shortcoming is that GLP-1 is easily degraded by dipeptidyl peptidase in the human body, and its half-life ...

AI Technical Summary

Problems solved by technology

In terms of production technology, Liraglutide is currently produced by Novo Nordisk using Saccharomyces cerevisiae to express the liraglutide precursor molecule Arg34GLP-1(7-37) in an extracellular way, and after its Lys26 is linked to the fatty acid side chain Excessive amino acids are removed to obtain liraglutide molecules, which are further processed into injections and sold in China as imported drugs. The price is quite expensive and unaffordable for most patients

[0004] At present, prokaryotic expression and eukaryotic expression systems can be used to express foreign genes, but there are some defects in the eukaryotic expression system when expressing the target protein, for example: (1) When using yeast to express the target protein, the product protein will be inhomogeneous Degradation, incomplete signal peptide processing, multimer formation, etc.

Moreover, problems such as protein cleavage and low protein secretion efficiency may occur when expressing proteins in Saccharomyces cerevisiae

(2) The insect baculovirus expression system also has certain defects

In addition, although the baculovirus expression system can perform post-translational modification on the target protein, this modification has certain limitations; although the glycosylation site is the same as in mammalian cells, the properties of the oligosaccharide chain are different. different, unable to produce complex glycan side chains, which may be due to the inability of insect cells to process mature glycans into analogous forms in mammalian cells

(3) Mammalian expression system has relatively high cost and complex technology when expressing foreign genes, and there is potential contamination of animal viruses in the expression process, etc.

However, the amount of target protein produced by this protein expression method is very small, which is not conducive to the subsequent industrial development needs

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