Method for efficiently preparing recombinant human interluekin-33 protein

Abstract

The invention relates to a recombinant vector, method and recombinant pichia pastoris engineering strain for efficiently preparing a recombinant human interluekin-33 (rhIL33-Fc) protein and a produced recombinant protein. The method comprises the following steps of (1) constructing an expression vector of a human interluekin-33 protein, wherein the expression vector contains coded sequences of an expression regulation element, a secretion signal peptide, human interluekin-33 and an IgG1-Fc fusion protein; (2) constructing and screening an efficient expression strain of pichia pastoris of the human interluekin-33 protein; (3) optimizing the fermentation pH value, temperature and carbon source of the engineering strain; and (4) separating, purifying and physicochemically authenticating the human interluekin-33 protein. By using the method provided by the invention, the recombinant human interluekin-33 protein with an industrial value can be efficiently obtained.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for producing recombinant human interleukin-33 protein (rhIL33-Fc) by Pichia pastoris, a Pichia pastoris engineering bacterium that expresses rhIL33-Fc efficiently, and the expressed recombinant protein. Background technique [0002] Interleukin-33 (IL-33) is a member of the IL-1 family, similar to IL-1β and IL-18, and plays an important role in the body's immune response. IL-33 is a 30kDa protein, which is relatively conservative in evolution, and the human-derived sequence has 54% homology with the mouse-derived sequence. Normal IL-33 is mainly expressed in endothelial cells and epidermal cells, and its release is often accompanied by body damage or cell necrosis, so it is also considered as an alarm protein (Alarmin). IL-33 recruits MyD88, IRAK1 / 4 and TRAF6 by binding to the receptor ST2 on the cell surface, which in turn leads to the activation of ...

AI Technical Summary

Problems solved by technology

It is well known that recombinantly expressed proteins in Escherichia coli tend to form inclusion bodies, which in turn involves a complex denaturation process, and the entire system will produce a large amount of endotoxin and many other unfavorable factors

Therefore, preparing interleukin-33 (rhIL-33) by traditional methods greatly increases the production cost, and the production technology is complicated and the production process is unstable.

At the same time, the molecular weight of recombinant human interleukin-33 (rhIL-33) prepared by Escherichia coli is about 18kDa, which is easily removed by glomerular filtration when it is used in animals or humans, seriously affecting its Potential physiological functions and application prospects as a drug candidate

In particular, there is no report on the successful expression and preparation of the fusion protein (rhIL33-Fc) of recombinant human interleukin-33 (rhIL-33) and Fc, and there is no successful use of Pichia pastoris to express and prepare recombinant human leukocytes. Interleukin-33 (rhIL-33) report

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