Expression box capable of efficiently achieving secretory expression of human FGF21 protein and application of expression box

Abstract

The invention belongs to the technical field of molecular biology and particularly relates to a method for efficiently expressing a recombinant human fibroblast growth factor 21 (rhFGF21) gene by using bacillus subtilis. Firstly, the method adjusts the transcription and translation efficiency of a target protein gene by adding different types of cistron sequences to the 5' end of the target protein gene so as to optimize the soluble expression level of the protein gene, and the method can be applied to the optimal expression of different types of heterologous protein; secondly, the method further optimizes disulfide bond related genes to construct a vector to promote the disulfide bond folding efficiency of rhFGF21, overexpresses a molecular chaperone system to construct a vector suitablefor the folding efficiency increasing of rhFGF21 protein, and knocks out various extracellular protease genes to construct a bacillus subtilis gene engineering strain for improving the extracellular stability of rhFGF21; and finally, the method provides a bacillus subtilis gene engineering strain with good application prospect and capable of achieving efficient secretory expression of the rhFGF21protein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for highly expressing recombinant human fibroblast growth factor 21 (rhFGF21) protein using bacillus. Background technique [0002] Protein expression technology is one of the core technologies of modern biology. Protein expression can not only be used in biological research, but also provide commercial protein products, such as recombinant vaccines, recombinant insulin, cytokines and other products. Currently commonly used expression systems include Escherichia coli, yeast, insect cells and mammalian cells, etc., but each of them has obvious advantages and disadvantages. Escherichia coli expression system is the most well-studied and has a variety of options. The most commonly used is Novagen's pET expression system, which uses phage T7 RNA polymerase to specifically transcribe the target gene behind the T7 promoter. Although it has the advantages of high expression effici...

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Problems solved by technology

[0004] At present, the expression of FGF21 is mainly carried out in Escherichia coli and yeast. As a host cell for protein expression, Escherichia coli has low secretion efficiency, endotoxin, and has caused immune reactions and other unfavorable factors, which will increase the cost of subsequent protein purification processes; Yeast Bacteria, as a fungus, has good protein secretion ability, but the optimum growth temperature is generally around 30°C, the growth is slow and a large amount of cooling water is required in the fermentation process, which is not conducive to energy saving and emission reduction

In addition, the currently commonly used yeast expression system generally uses methanol as an inducer and carbon source, which has certain hidden dangers in production safety, thus further increasing production costs.

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