A kind of peg modification method of recombinant human il-24 protein

An IL-24 and modification method technology, applied in the field of PEG modification of recombinant human IL-24 protein, can solve problems such as low activity, and achieve the effects of high expression level, improved stability, improved stability and biological activity

Active Publication Date: 2020-12-01
BEIJING JIAOTONG UNIV
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Problems solved by technology

[0005] The purpose of the present invention is to overcome the defect of low activity of recombinant human IL-24 protein expressed by Escherichia coli due to lack of glycosylation modification and other reasons

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  • A kind of peg modification method of recombinant human il-24 protein
  • A kind of peg modification method of recombinant human il-24 protein
  • A kind of peg modification method of recombinant human il-24 protein

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Embodiment Construction

[0067] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0068] The PEG modification method of the recombinant human IL-24 protein of the present invention uses the prokaryotic expression vector pET-28a to express the recombinant human IL-24 protein in bacteria through IPTG induction, wherein the initial concentration OD600 of the host bacterium BL21 and the inducing bacterium is 0.6, the final concentration of the inducer IPTG is 0.5mM, induced at 37°C for 5h, and exists in the inclusion body after induction; the recombinant human IL-24 protein is denatured by the denaturant 6M guanidine hydrochloride, the refolding agent is 20mM acetic acid-sodium acetate, 2M urea, 1mM GSSG, 1mM GSH, pH 5.5 dialyzed overnight, in the presence of 1% SDS, purified by gel filtration to obtain high-purity monomeric recombinant human IL-24 protein; fluorescence spectrum showed correct spatial s...

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Abstract

The invention discloses a PEG (Polyethylene Glycol) modification method of recombinant human IL-24 (rhIL024) protein. The PEG modification method comprises the following steps: a, constructing a recombinant prokaryotic expression vector of human IL-24; carrying out induced expression to obtain an inclusion body; b, selecting a denaturing agent for modifying the inclusion body, so as to obtain denatured protein; c, determining a renaturation condition and renaturing the denatured protein to obtain renatured protein; d, filtering and purifying gel under the condition of adding SDS (Sodium Dodecyl Sulfonate) into the renatured protein to obtain purified target protein; e, carrying out site-specific modification on the purified target protein by adopting a PEG modifier, so as to obtain a final product. According to the method disclosed by the invention, the polyethylene glycol is used for carrying out the site-specific modification on the basis of preparing the high-purity recombinant human IL-24 protein; a space barrier is generated at the periphery of modified protein so that the enzymolysis of the protein is reduced and a half-life period is prolonged; the in-vitro tumor-inhibition activity is not influenced, the utilization rate is increased and the stability and biological activity are improved.

Description

technical field [0001] The invention relates to the technical field of tumor biotherapy, in particular to a method for PEG modification of recombinant human IL-24 protein. Background technique [0002] Human interleukin 24 (Interleukin 24, IL-24) is a cytokine discovered in human melanoma cell HO-1 by subtractive hybridization in 1995 by Professor Paul Fisher of Columbia University, and belongs to the IL-10 superfamily member. Nearly two decades of research have shown that IL-24 is an anti-tumor "star" molecule. First, the endogenous expression of IL-24 in normal tissues is highly tissue-specific, and it is only expressed in immune system-related tissues (such as spleen, thymus, and peripheral blood leukocytes) and specific types of cells; secondly, IL-24 has Broad-spectrum and specific anti-tumor activity, can inhibit the growth of various tumors (such as lung cancer, breast cancer, cervical cancer, etc.) It has the ability to stimulate the body's anti-tumor immune respon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C07K1/107A61K38/20A61K47/60A61P35/00
CPCA61K38/00C07K14/54
Inventor 江红马群风郑晓飞张耀王俊峰张驰史亦南葛建林化计磊
Owner BEIJING JIAOTONG UNIV
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