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Mutant protein of osteoprotegerin, as well as relevant product and application thereof

A technology of osteoprotegerin and related products, applied in the fields of application, peptide/protein composition, animal/human protein, etc., can solve problems that affect clinical application and increase tumor risk

Active Publication Date: 2019-02-05
北京济全生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the reasons is that OPG was found to be defective, which affected its clinical application: the structure of RANKL is similar to tumor necrosis factor related apoptosis-inducing ligand (TRAIL), and the combination of OPG and RANKL At the same time, it can also combine with TRAIL, inhibit the anti-cancer effect of TRAIL, and increase the risk of tumor occurrence

Method used

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  • Mutant protein of osteoprotegerin, as well as relevant product and application thereof
  • Mutant protein of osteoprotegerin, as well as relevant product and application thereof
  • Mutant protein of osteoprotegerin, as well as relevant product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Preparation of wild-type human osteoprotegerin OPGwt and wild-type human osteoprotegerin mutant proteins OPGS73A, OPGS73V, OPGS73G, OPGS73L, OPGK94A and OPGR122G

[0108] 1. Preparation of wild-type human osteoprotegerin OPGwt

[0109] The amino acid sequence of wild-type human osteoprotegerin OPGwt is the 154th to 345th amino acid residues of SEQ ID No.2. Wild-type human osteoprotegerin OPGwt contains amino acid residues 22-201 of human full-length osteoprotegerin (amino acid sequence is GenBank AccessionNumber NP_002537.3 (26-JUN-2017) or SEQ ID No.9).

[0110] Wild-type human osteoprotegerin OPGwt was prepared according to the method of the following literature: Mengmeng Jin, et al. Sortase A-aided Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain. Appl Microbiol Biotechnol (2017) 101:4923–4933. DOI10.1007 / s00253 -017-8188-6. The specific method is as follows:

[0111] 1.1 Preparation of DNA molecules

[0112] The D...

Embodiment 2

[0169] Example 2, Affinity Determination of Wild-type Human Osteoprotegerin OPGwt and Human Osteoprotegerin Mutant Proteins OPGS73A, OPGS73V, OPGS73G, OPGS73L, OPGK94A and OPGR122G with RANKL and TRAIL

[0170] The surface plasmon resonance (Surface Plasmon Resonance, SPR) technology was used, and the equipment used was BIAcore 3000 (GE Healthcare), and RANKL or TRAIL was used as the stationary phase to immobilize on a certain channel on the surface of the CM5 chip (diluted into 10mM sodium acetate pH5.5 ), the response value increased by about 2000RU is better; TNFRSF9 was fixed in another channel as an internal reference; the OPGwt prepared in Example 1 and the mutant proteins OPGS73A, OPGS73V, OPGS73G, OPGS73L, OPGK94A and OPGR122G of human osteoprotegerin were used as mobile phases, and BIAcore buffer (10mM HEPES, 150mM NaCl, 0.005% Tween 20, pH 7.4) prepared OPGwt and human osteoprotegerin mutant proteins OPGS73A, OPGS73V, OPGS73G, OPGS73L, OPGK94A and OPGR122G at differen...

Embodiment 3

[0182] Example 3, Biological activity of wild-type human osteoprotegerin OPGwt and mutant proteins OPGS73A, OPGS73V, OPGS73G, OPGS73L, OPGK94A and OPGR122G of human osteoprotegerin

[0183] 1. Inhibition of RANKL-mediated osteoclast activation experiment

[0184] The differentiation and maturation of RANKL-induced osteoclasts are marked by the formation of multinucleated cells and the expression of tartrate-resistant acid phosphatase (TRAP) in multinucleated cells.

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Abstract

The invention discloses a mutant protein of osteoprotegerin, as well as a relevant product and application thereof. When the mutant protein of the osteoprotegerin is compared with wild osteoprotegerin, a 73rd amino acid residue of a full-length sequence of the wild osteoprotegerin is substituted; the mutant protein has affinity activity for RANKL (Receptor Activator of NF-kappaB ligand), but the affinity activity of the mutant protein for TRAIL is lower than that of the wild osteoprotegerin. As proved by experiments, compared with the corresponding wild osteoprotegerin, the mutant protein of the osteoprotegerin provided by the invention has the advantages that no significant difference exists in the capability of inhibiting RANKL induced osteoclast cell differentiation, the defect that theanti-tumor effect of TRAIL is inhibited by the wild osteoprotegerin can be overcome, and the effect of preventing and treating bone resorption related diseases is achieved more efficiently and safely.

Description

technical field [0001] The invention relates to a mutant protein of osteoprotegerin and related products and applications in the field of biotechnology. Background technique [0002] Bone resorption-related diseases represented by osteoporosis and arthritis are common and frequently-occurring diseases in the world, which have seriously affected human health. The pathological feature of bone resorption-related diseases is bone resorption-formation imbalance, and the most important signal transduction pathway regulating bone resorption and formation balance has been proved to be the OPG / RANKL / RANK system. In this system, osteoprotegerin (Osteoprotegerin, OPG) plays a vital role as the central link. [0003] In 1997, two research groups in the United States and Japan discovered the OPG protein at the same time. It belongs to the TNF receptor superfamily member. It is a soluble and secreted glycoprotein consisting of 401 amino acids. It is expressed in osteoblasts / bone mesench...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K19/00C12N15/12C12N5/10A61K38/17A61P19/08
CPCA61K38/00C07K14/70578C07K2319/30C07K2319/31
Inventor 王玮尚腾飞
Owner 北京济全生物科技有限公司
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