CN109306002B

CN109306002B - N139 mutant protein of osteoprotegerin, and related product and application thereof

Info

Publication number: CN109306002B
Application number: CN201710630102.5A
Authority: CN
Inventors: 王玮
Original assignee: Beijing Jiquan Biological Technology Co ltd
Current assignee: Beijing Jiquan Biological Technology Co ltd
Priority date: 2017-07-28
Filing date: 2017-07-28
Publication date: 2021-02-12
Anticipated expiration: 2037-07-28
Also published as: CN109306002A

Abstract

The invention discloses a mutant protein of osteoprotegerin, and a related product and application thereof. Compared with wild type osteoprotegerin, the mutant protein of the osteoprotegerin is substituted at the 139 th amino acid residue of the full-length sequence of the wild type osteoprotegerin; the mutant protein has affinity activity for RANKL but the mutant protein has lower affinity activity for TRAIL than the wild-type osteoprotegerin. Experiments prove that compared with corresponding wild type, the mutant protein of the osteoprotegerin has no obvious difference in the capability of inhibiting RANKL to induce osteoclast differentiation, can overcome the defect that the wild type inhibits TRAIL in an anti-tumor effect, and can more efficiently and safely play a role in preventing and treating diseases related to bone resorption.

Description

N139 mutant protein of osteoprotegerin, and related product and application thereof

Technical Field

The invention relates to a mutant protein of osteoprotegerin in the field of biotechnology, and related products and applications thereof.

Background

Bone resorption-related diseases, such as osteoporosis and arthritis, are common frequently encountered diseases in the world and have a serious influence on human health. The pathological feature of bone resorption-related diseases is bone resorption-formation imbalance, while the most important signaling pathway regulating bone resorption and formation equilibrium has been shown to be the OPG/RANKL/RANK system. In this system, Osteoprotegerin (OPG) plays a crucial role as a central link.

In 1997, OPG proteins were discovered contemporaneously in both the us and japan research groups. It belongs to a TNF receptor superfamily member, is soluble, secretory glycoprotein, is composed of 401 amino acids, is expressed in osteoblast/bone interstitial cells, and is the only osteoclast negative regulation factor discovered at present. Considering the important regulation effect of OPG on osteoclasts, the two research groups immediately search for proteins closely related to OPG, and find out the ligand of nuclear factor kappa B receptor activating factor (RANKL) and the receptor of osteoclast surface membrane Receptor (RANK). The OPG/RANKL/RANK system is known for 2 years, and answers an accurate mechanism for regulating osteoclast by osteoblast: the OPG corresponds to the brake of the entire OPG/RANKL/RANK system. It is used as a pseudo receptor of RANKL, competitively inhibits the combination of RANKL and RANK, thereby inhibiting the activity and differentiation of osteoclast and resisting osteoporosis, so that the RANKL/OPG ratio becomes a key factor in the pathogenesis of osteoporosis. Many hormones and cytokines (e.g., TGF- β, PTH, 1, 25-VD3, glucocorticoids, and estrogens) act through the OPG pathway. After injection of OPG, the bone density of osteoporosis mice was significantly increased, while OPG knockout mice exhibited severe osteoporosis.

The important function of OPG in preventing and treating osteoporosis is revealed to become a competitive star protein. The OPG-Fc developed by the largest biological company in the world, Amgen, has entered into the phase II clinical study, and preliminary experiments show that OPG can inhibit bone resorption in human body and resist osteoporosis. Amgen even collaborated with the U.S. aeronautics and astronautics office (NASA) to develop space experiments for OPG to prevent disuse osteoporosis. While the development of OPG as an anti-osteoporosis drug is vigorously carried out, Amgen company stops the research and development of the protein in 2005 and abandons further research on OPG. Other laboratories have also been on the hold for OPG. One reason for this is that OPG is found to have defects, which affect its clinical use: the structure of RANKL is similar to that of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and OPG can be combined with both RANKL and TRAIL, thereby inhibiting the anticancer effect of TRAIL and increasing the risk of tumor occurrence. TRAIL is an autocrine protein of human body, which can specifically kill tumor cells without obvious cytotoxic effect on normal cells.

The Australian EvoGenix corporation filed a patent for OPG mutant derivatives in 2006 (US 2006/0189528A 1) which involved gene mutation sites mainly including I115, R122 and F128. Through the amino acid substitution of the sites, the binding capacity of the OPG mutant and TRAIL is reduced, thereby reducing the risk of promoting the tumorigenesis.

Disclosure of Invention

The technical problem to be solved by the invention is how to reduce the inhibition of wild-type osteoprotegerin on the anticancer effect of TRAIL while retaining the ability of wild-type osteoprotegerin to inhibit the activation of RANKL mediated osteoclasts.

In order to solve the above technical problems, the present invention provides a mutant protein of wild-type osteoprotegerin, which is a mutant protein consisting of wild-type osteoprotegerin substituted by 1 amino acid residue, wherein the mutant protein is substituted at amino acid residue 139 of the full-length sequence of the wild-type osteoprotegerin as compared with the wild-type osteoprotegerin; the mutant protein has affinity activity for nuclear factor-kappa B receptor activator ligand (RANKL) but the mutant protein has affinity activity for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that is lower than the affinity activity of the wild-type osteoprotegerin for the TRAIL.

In the above mutant proteins, the wild type osteoprotegerin includes full length fragments of wild type osteoprotegerin and also includes active fragments of wild type osteoprotegerin.

In the above mutant proteins, the wild-type osteoprotegerin may be derived from any species, such as osteoprotegerin derived from a mammal, such as a human.

In the mutant protein, the full-length amino acid sequence of the human wild-type osteoprotegerin is SEQ ID No.9, as shown in GenBank Accession Number NP-002537.3 (26-JUN-2017).

In the above mutant protein, the wild-type osteoprotegerin may be an active fragment of human wild-type osteoprotegerin. The amino acid sequence of the active fragment of human wild-type osteoprotegerin may be amino acid residues 22-201 of the full-length sequence.

In the above mutant proteins, the mutant protein includes any variant protein in which the substitution at amino acid residue 139 of the full length sequence of the wild-type osteoprotegerin has been made, whether truncated or full length, whether in monomeric or dimeric form.

Among the above mutant proteins, the comparison between the affinity activity for RANKL and the affinity activity for TRAIL of the mutant protein and the wild-type osteoprotegerin was performed based on proteins of similar sizes.

In the mutant protein, the mutant protein has affinity activity to RANKL, and the affinity activity of the mutant protein to RANKL is more than 70% of that of the wild-type osteoprotegerin to RANKL.

In the above mutant protein, the affinity activity of the mutant protein for TRAIL is lower than that of the wild-type osteoprotegerin, and the affinity activity of the mutant protein for TRAIL is 25% or less of that of the wild-type osteoprotegerin.

In the above mutant protein, the 70% or more may be any one of a1) -a 10):

a1) greater than or equal to 76% to equal to 100%;

a2) greater than or equal to 80% to equal to 100%;

a3) greater than or equal to 81% to equal to 100%;

a4) greater than or equal to 82% to equal to 100%;

a5) greater than or equal to 83% to equal to 100%;

a6) greater than or equal to 84% to equal to 100%;

a7) greater than or equal to 85% to equal to 100%;

a8) greater than or equal to 90% to equal to 100%;

a9) greater than or equal to 92% to equal to 100%;

a10) 80%, 81%, 82%, 83%, 84%, 85%, 88%, 90%, 92%, 95%, or 100%.

In the above mutant protein, the 25% or less may be any one of b1) -b 9):

b1) less than or equal to 20% to equal to 0 or less than or equal to 20% to less than or equal to 16%;

b2) less than or equal to 16% to equal to 0 or less than or equal to 16% to less than or equal to 13%;

b3) less than or equal to 13% to equal to 0 or less than or equal to 13% to less than or equal to 11%;

b4) less than or equal to 11% to equal to 0 or less than or equal to 11% to less than or equal to 10%;

b5) less than or equal to 10% to 0 or less than or equal to 20% to less than or equal to 5%;

b6) less than or equal to 5% to equal to 0 or less than or equal to 5% to less than or equal to 3%;

b7) less than or equal to 3% to equal to 0 or less than or equal to 3% to less than or equal to 1%;

b8) less than or equal to 1% to equal to 0;

b9) 20%, 16%, 13%, 11%, 8%, 7%, 6% or 1%.

In the above mutant protein, the mutant protein may be a protein obtained by substituting the asparagine amino acid residue at position 139 of the wild-type osteoprotegerin with another amino acid residue.

The other amino acid residues can be determined by the technicians in the field according to the affinity activity of the mutant protein to RANKL and the affinity activity to TRAIL, and other amino acid residues can be selected as long as the affinity activity of the mutant protein to RANKL meets any one of the requirements of a1) -a10) and the affinity activity to TRAIL meets any one of the requirements of b1) -b 9).

The other amino acids may be selected from r1) to r 4):

r1) any one of the following 19 amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), threonine (Thr), cysteine (Cys), tyrosine (Tyr), serine (Ser), glutamine (gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), and glutamic acid (Glu); the conformation of the amino acid is L-type;

r2) any one of the following 19 amino acids: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), threonine (Thr), cysteine (Cys), tyrosine (Tyr), serine (Ser), glutamine (gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), and glutamic acid (Glu); the conformation of the amino acid is D-type; d-form amino acids refer to amino acids corresponding to L-form amino acids constituting proteins;

r3) the artificially modified amino acid obtained by chemically modifying r1) or r 2);

r4) rare amino acids occurring in nature.

The chemical modification in r3) can be methylation or phosphorylation.

The rare amino acids present in nature include unusual amino acids constituting proteins and amino acids not constituting proteins, such as 5-hydroxylysine, methylhistidine, gamma-aminobutyric acid, or the like.

In the above mutant protein, the other amino acid may be C1 or C2:

c1, a non-polar amino acid or a polar amino acid;

c2, the other amino acid being valine, glycine, alanine or leucine.

In the mutant protein, compared with the wild type osteoprotegerin, the mutant protein has the sequence that only the 139 th amino acid residue of the full-length sequence of the wild type osteoprotegerin is replaced, and other amino acid residues are not replaced. Specifically, in the mutant protein, the mutant protein is different from the wild-type osteoprotegerin in sequence only at the 139 th amino acid residue of the full-length sequence of the wild-type osteoprotegerin, and other amino acid residues are the same.

Among the above mutant proteins, the mutant protein may specifically be any one of D1 to D4:

d1, a protein with the amino acid sequence of SEQ ID No.6 (named as OPGN139L), or a protein with the amino acid sequence of amino acid residues 5-184 of SEQ ID No. 6;

d2, a protein with the amino acid sequence of SEQ ID No.5 (named as OPGN139V), or a protein with the amino acid sequence of amino acid residues 5-184 of SEQ ID No. 5;

d3, a protein with the amino acid sequence of SEQ ID No.4 (named as OPGN139A), or a protein with the amino acid sequence of amino acid residues 5-184 of SEQ ID No. 4;

d4, a protein with the amino acid sequence of SEQ ID No.3 (named OPGN139G), or a protein with the amino acid sequence of amino acid residues 5-184 of SEQ ID No. 3.

The following products related to the mutant proteins E1 to E3 also belong to the scope of protection of the present invention:

e1, a related biomaterial of said mutant protein, which related biomaterial may be any one of the following B1) to B16):

B1) a nucleic acid molecule encoding the mutant protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism containing the recombinant vector of B4);

B9) a transgenic animal cell line comprising the nucleic acid molecule of B1);

B10) a transgenic animal cell line comprising the expression cassette of B2);

B11) a transgenic animal cell line containing the recombinant vector of B3);

B12) a transgenic animal cell line containing the recombinant vector of B4);

B13) a transgenic plant cell line comprising the nucleic acid molecule of B1);

B14) a transgenic plant cell line comprising the expression cassette of B2);

B15) a transgenic plant cell line comprising the recombinant vector of B3);

B16) a transgenic plant cell line comprising the recombinant vector of B4);

e2, a derivative of said mutant protein, being the following a11 or a 12:

a11, fusion protein obtained by fusing tag protein at the amino terminal or/and the carboxyl terminal of the mutant protein or fusion protein obtained by connecting protein with targeting function with the mutant protein;

a12, and a modifier obtained by modifying the mutant protein by at least one of the following modifications a21 to a 28:

a21, adding leucine zipper;

a22, adding isoleucine zipper;

a23, adding zinc atoms and disulfur groups;

a24, loaded into human serum albumin nanoparticles;

a25, binding to HSA ligand;

a26, conjugated with PEG;

a27, loading by using a polylactic acid-glycolic acid copolymer microsphere slow-release system;

a28, binding to the Fc part (crystallizable fragment) of the antibody;

e3, a mono-or multimer of said mutant protein.

In the above-mentioned related products, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above-mentioned related product, the recombinant microorganism may be a recombinant microorganism obtained by introducing a gene encoding the mutant protein into a recipient microorganism, and the recipient microorganism may be any one of F11 to F14:

f11, prokaryotic microorganisms;

f12, gram negative bacteria;

f13, Escherichia bacterium;

f14, Escherichia coli BL21(DE 3).

Of the above-mentioned biological materials, B9) to B16) may or may not include propagation material.

In the related products, the protein with the targeting function can be antibody or transferrin. The antibody may be a single chain antibody or an intact antibody. The tag protein (protein-tag) refers to a polypeptide or protein which is expressed by fusion with a target protein by using a DNA in vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag protein can be Flag tag protein, His6 tag protein, MBP tag protein, HA tag protein, myc tag protein, GST tag protein or SUMO tag protein, etc.

The invention also provides a medicament for inhibiting RANKL-mediated osteoclast activation.

The medicine for inhibiting the RANKL-mediated osteoclast activation contains the mutant protein or/and the related product.

The drug for inhibiting the RANKL-mediated osteoclast activation can be specifically a drug for preventing and/or treating diseases related to bone resorption, such as a drug for preventing and/or treating osteoporosis, a drug for preventing and/or treating arthritis and the like. In the above, the active ingredient of the medicament may be the mutant protein or/and the related product, and the active ingredient of the medicament may further contain other ingredients, and the other active ingredients of the medicament may be determined by those skilled in the art according to their effect of inhibiting RANKL-mediated osteoclast activation.

In the above drugs, the drug may further contain a carrier or excipient. The carrier material includes, but is not limited to, water-soluble carrier materials (e.g., polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), sparingly soluble carrier materials (e.g., ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (e.g., cellulose acetate phthalate, carboxymethyl cellulose, etc.).

Experiments prove that compared with corresponding wild OPG (OPGwt), the mutant proteins OPGN139A, OPGN139V, OPGN139G and OPGN139L obtained by replacing the asparagine residue (N139) at the 139 th position of the full-length sequence of the human wild-type osteoprotegerin with alanine residue, valine residue, glycine residue and leucine residue respectively have the affinity of 0.88 times, 0.90 times, 0.80 times and 0.92 times of the wild-type with RANKL and the affinity of 0.11 times, 0.06 times, 0.08 times and 0.07 times of the wild-type with TRAIL. Compared with the corresponding wild OPG (OPGwt), the mutant proteins OPGN139A, OPGN139V, OPGN139G and OPGN139L have no significant difference in the capability of inhibiting the differentiation of the osteoclast induced by RANKL. Thus, the mutation of the N139 site does not reduce the inhibiting effect of OPG on RANKL. The inhibition effect of the mutant proteins OPGN139A, OPGN139V, OPGN139G and OPGN139L on TRAIL is far lower than that of wild OPG (OPGwt), and the OPG mutant proteins can overcome the defect that the wild type inhibits the anti-tumor effect of TRAIL and play the role of preventing and treating diseases related to bone resorption more efficiently and safely.

Drawings

FIG. 1 shows the results of the identification of OPGN139G and OPGE96G obtained after molecular sieve purification.

A is identified by SDS-PAGE electrophoresis. Lanes 1-2 are OPGN139G and OPGE96G, respectively, with the purified protein from each lane indicated by an arrow.

B is a molecular sieve purified image after OPGN139G renaturation. The arrow indicates the elution peak of OPGN 139G.

FIG. 2 shows the experiment of OPG inhibiting RANKL to stimulate RAW264.7 cells to differentiate and mature into osteoclasts (upper panel) and the experiment of OPG inhibiting TRAIL induced apoptosis of colo205 cells (lower panel). In the figure, wOPG is wild type human osteoprotegerin OPGwt, T1 is OPGN139G, T101 is OPGN139V, T102 is OPGN139A, T103 is OPGN139L, T2 is OPGE96G, and T3 is positive control OPGR 122G. In the upper panel, the concentration of RANKL and OPG in Negative wells was 0ng/ml and 0ng/ml, respectively; the concentration of RANKL in the wells of RANKL was 50ng/ml and the concentration of OPG was 0 ng/ml; RANKL: the concentration of RANKL in the wells of OPG1:1 was 50ng/ml, the concentration of OPG was 50ng/ml, the concentration of RANKL: the concentration of RANKL in the wells of OPG1:2 was 50ng/ml, the concentration of OPG was 100ng/ml, the concentration of RANKL: the concentration of RANKL in the wells of OPG1:4 was 50ng/ml, the concentration of OPG was 200ng/ml, the concentration of RANKL: the concentration of RANKL in the wells of OPG1:8 was 50ng/ml and the concentration of OPG was 400 ng/ml.

In the lower panel, the concentration of TRAIL and OPG in Negative wells was 0ng/ml and 0ng/ml respectively; the concentration of TRAIL in the wells of TRAIL is 100 ng/ml; TRAIL: the concentration of TRAIL in the wells of OPG1:1 was 100ng/ml, the concentration of OPG was 100ng/ml, TRAIL: the concentration of TRAIL in the OPG1:5 wells was 100ng/ml, the concentration of OPG was 500ng/ml, TRAIL: the concentration of TRAIL in the wells of OPG1:25 was 100ng/ml, the concentration of OPG was 2500ng/ml, TRAIL: the concentration of TRAIL in the wells of OPG1:125 was 100ng/ml and the concentration of OPG was 12500 ng/ml.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

TRAIL in the following examples is of human origin and has an amino acid Sequence such as NCBI Reference Sequence NP-003801.1 (Update Date: 06-MAY-2017) consisting of 281 amino acid residues, a product of Peprotech corporation.

RANKL in the following examples is derived from human and has an amino acid Sequence such as NCBI Reference Sequence NP-003692.1 (Update Date: 02-JUL-2017) consisting of 317 amino acid residues, a product of Abcam corporation.

The application researches two key sites on the wild type human osteoprotegerin which the affinity activity to the nuclear factor kappa B receptor activator ligand is possibly satisfied but the affinity activity to the tumor necrosis factor related apoptosis inducing ligand is lower than that of the wild type human osteoprotegerin, namely N139 and E96, and designs mutant proteins of the wild type human osteoprotegerin, namely OPGN139G, OPGN139A, OPGN139V, OPGN139L and OPGE 96G. See in particular the examples below.

Example 1 preparation of wild type human osteoprotegerin OPGwt and mutant proteins of wild type human osteoprotegerin OPGN139G, OPGN139A, OPGN139V, OPGN139L, OPGE96G and OPGR122G

1. Preparation of wild-type human osteoprotegerin OPGwt

The amino acid sequence of wild-type human osteoprotegerin OPGwt is amino acid residues 154 to 345 of SEQ ID No. 2. The wild-type human osteoprotegerin OPGwt contains the 22 nd to 201 th amino acid residues of the human full-length osteoprotegerin (the amino acid sequence is GenBank Accession number NP-002537.3 (Update Date: 26-JUN-2017) or SEQ ID No. 9)). Wild-type human osteoprotegerin OPGwt was prepared according to the following literature: a-aid Escherichia coli expression system for functional osteoprotegerin cysteine-rich domain.appl Microbiol Biotechnol (2017)101: 4923-4933. DOI 10.1007/s 00253-017-8188-6. The specific method comprises the following steps:

1.1 preparation of DNA molecules

DNA shown in SEQ ID No.1 was prepared. The DNA shown in SEQ ID No.1 is a fusion protein SrtA-LPETG-OPGwt gene fused with Sortase A (Sortase A, SrtA) and LPXTG motifs. The SrtA-LPETG-OPGwt gene expresses a fusion protein SrtA-LPETG-OPGwt shown in SEQ ID No. 2. The N-terminal of SEQ ID No.2 is sortase A which cleaves between Thr and Gly of LPXTG to release OPGwt. The amino acid sequence of OPGwt is amino acid residues 154 to 345 of SEQ ID No. 2. Amino acid residues 158 to 337 of SEQ ID No.2 correspond to amino acid residues 22 to 201 of human full-length osteoprotegerin. A fragment whose amino acid sequence is amino acid residues 22 to 201 of SEQ ID No.9 was taken as a wild type. The amino acid residues 150-154 of SEQ ID No.2 are the LPXTG motif and the amino acid residues 3-149 are the amino acid sequence of sortase A.

1.2 preparation of expression vectors

The fragment between the NcoI and XhoI recognition sites of pET28a (+) (Novagen) (small fragment including the NcoI recognition site and the XhoI recognition site) was replaced with DNA having a nucleotide sequence of nucleotides 1 to 1019 of SEQ ID No.1, and the other sequence of pET28a (+) was kept unchanged to obtain a SrtA-LPETG-OPGwt gene recombinant expression vector, which was designated as pET28 a-SrtA-LPETG-OPGwt. pET28a-SrtA-LPETG-OPGwt contains SrtA-LPETG-OPGwt gene, pET28a-SrtA-LPETG-OPGwt is introduced into E.coli BL21(DE3) to obtain protein with amino acid sequence from amino acid residues 154 to 345 of SEQ ID No.2, the protein contains amino acid residues 22 to 201 of human full-length osteoprotegerin, and the protein is named OPGwt. Amino acid residues 158 to 337 of SEQ ID No.2 are identical to amino acid residues 22 to 201 of human full-length osteoprotegerin.

1.3 obtaining wild type human osteoprotegerin OPGwt

pET28a-SrtA-LPETG-OPGwt was transformed into E.coli BL21(DE3) competent cells, which were uniformly spread on LB plates containing kanamycin, and cultured at 37 ℃ for 16 hours. The single colony was cultured overnight by shaking, the plasmid was extracted for sequencing, and the recombinant E.coli containing pET28a-SrtA-LPETG-OPGwt was named BL21(DE3)/pET28a-SrtA-LPETG-OPGwt as shown by the sequencing result.

B is to beL21(DE3)/pET28a-SrtA-LPETG-OPGwt was inoculated into LB medium containing 100ug/ml kanamycin and amplified (37 ℃, 200rpm), IPTG was added to induce expression (IPTG final concentration of 1mM) when OD600 reached 0.6, and the cells were harvested by ultracentrifugation after induction at 37 ℃ for 5 hours. The thalli is resuspended in 100ml of washing buffer (50mM Tris, 150mM NaCl, 5mM EDTA, 1% v/v Triton X-100, pH 8.0), after ultrasonication, washed again with the washing buffer and centrifuged to retain the precipitate, the process is repeated for a plurality of times to obtain inclusion bodies, and the inclusion bodies are subpackaged and frozen. Inclusion bodies were solubilized in lysis buffer (6M guanidine hydrochloride, 50mM Tris pH 8.5,1mM EDTA,150mM NaCl, 10mM DTT) at a protein concentration of 30mg/ml at room temperature, and renaturation buffer (20mM Na₂HPO₄1M L-arginine, 20% glycerol, 10mM reduced glutathione and 1mM oxidized glutathione, pH 7.3) to a protein concentration of 10mg/ml, filling the solution into dialysis bags and placing into dialysate (20mM Na₂HPO₄Dialyzing at pH 7.3,0.5M L-arginine and 10% glycerol at 4 deg.C for 12h, and replacing dialysate (20mM Na)₂HPO₄pH 7.3,0.2M L-arginine, 5% glycerol), again at 4 deg.C for 12h, and the dialysate (20mM Na) replaced again₂HPO₄pH 7.3) was left at 4 ℃ for 12 h. Ultracentrifuging the obtained protein (20,000g for 10min), purifying by molecular sieve chromatography (Superdex200, GE Healthcare) to obtain wild type human osteoprotegerin OPGwt, packaging, and freezing at-80 deg.C. The buffer used for molecular sieve chromatographic purification was Tris buffer (0.1M Tris,50mM NaCl pH 7.0).

2. Preparation of human osteoprotegerin mutant protein OPGN139G in which the 139 th asparagine residue of human full-length osteoprotegerin was mutated to a glycine residue

The amino acid sequence of human osteoprotegerin mutant protein OPGN139G is SEQ ID No.3, and is a mutant obtained by mutating the 139 th asparagine residue (corresponding to the 275 th amino acid residue of SEQ ID No. 2) of human full-length osteoprotegerin to the 122 th amino acid residue (corresponding to the 122 th amino acid residue of SEQ ID No. 3). The mutant protein OPGN139G of human osteoprotegerin differs in amino acid sequence from wild-type human osteoprotegerin OPGwt only in amino acid residue 275 of SEQ ID No.2 (corresponding to amino acid residue 122 of SEQ ID No. 3).

2.1 preparation of DNA molecules

The aat (codon for asparagine) at position 825-827 of SEQ ID No.1 was replaced with ggt (codon for glycine), and the other nucleotides were not changed to obtain the SrtA-LPETG-OPGN139G gene as a fusion protein fused with the motifs of sortase A and LPXTG. The gene SrtA-LPETG-OPGN139G expresses the fusion protein SrtA-LPETG-OPGN 139G. The amino acid sequence of SrtA-LPETG-OPGN139G is obtained by mutating the 275 th asparagine residue of SEQ ID No.2 to a glycine residue, and keeping the other amino acid residues of SEQ ID No.2 unchanged.

2.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGN139G gene was used to replace the segment between the NcoI and XhoI recognition sites of pET28a (+) (small segment including the NcoI recognition site and the XhoI recognition site), and the other sequence of pET28a (+) was kept unchanged to obtain a SrtA-LPETG-OPGN139G gene recombinant expression vector, which was named pET28a-SrtA-LPETG-OPGN 139G. pET28a-SrtA-LPETG-OPGN139G contains SrtA-LPETG-OPGN139G gene, pET28a-SrtA-LPETG-OPGN139G is introduced into E.coli BL21(DE3), and mutant protein OPGN139G of human osteoprotegerin with the amino acid sequence SEQ ID No.3 can be obtained.

2.3 obtaining human osteoprotegerin mutant protein OPGN139G with mutation of asparagine residue at position 139 of human full-length osteoprotegerin to glycine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGN139G was named BL21(DE3)/pET28a-SrtA-LPETG-OPGN139G by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGN 139G; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGN139G to obtain human osteoprotegerin mutant protein OPGN 139G.

3. Preparation of human osteoprotegerin mutant protein OPGN139A in which the 139 th asparagine residue of human full-length osteoprotegerin was mutated to alanine residue

The amino acid sequence of human osteoprotegerin mutant protein OPGN139A is SEQ ID No.4, and is a mutant obtained by mutating the 139 th asparagine residue (corresponding to the 275 th amino acid residue of SEQ ID No. 2) of human full-length osteoprotegerin to alanine residue (corresponding to the 122 th amino acid residue of SEQ ID No. 4). The mutant protein OPGN139A of human osteoprotegerin differs in amino acid sequence from wild-type human osteoprotegerin OPGwt only in amino acid residue 275 of SEQ ID No.2 (corresponding to amino acid residue 122 of SEQ ID No. 4).

3.1 preparation of DNA molecules

The aat (codon for asparagine) at position 825-827 of SEQ ID No.1 was replaced with gca (codon for alanine), and the other nucleotides were not changed to obtain the SrtA-LPETG-OPGN139A gene as a fusion protein fused with the motifs of sortase A and LPXTG. The gene SrtA-LPETG-OPGN139A expresses the fusion protein SrtA-LPETG-OPGN 139A. The amino acid sequence of SrtA-LPETG-OPGN139A is obtained by mutating the 275 th asparagine residue of SEQ ID No.2 to alanine residue, and keeping the other amino acid residues of SEQ ID No.2 unchanged.

3.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGN139A gene was used to replace the segment between the NcoI and XhoI recognition sites of pET28a (+) (small segment including the NcoI recognition site and the XhoI recognition site), and the other sequence of pET28a (+) was kept unchanged to obtain a SrtA-LPETG-OPGN139A gene recombinant expression vector, which was named pET28a-SrtA-LPETG-OPGN 139A. pET28a-SrtA-LPETG-OPGN139A contains SrtA-LPETG-OPGN139A gene, pET28a-SrtA-LPETG-OPGN139A is introduced into E.coli BL21(DE3), and mutant protein OPGN139A of human osteoprotegerin with the amino acid sequence SEQ ID No.4 can be obtained.

3.3 obtaining mutant protein OPGN139A of human osteoprotegerin in which the 139 th asparagine residue of human full-length osteoprotegerin is mutated into alanine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGN139A was named BL21(DE3)/pET28a-SrtA-LPETG-OPGN139A by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGN 139A; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGN139A to obtain human osteoprotegerin mutant protein OPGN 139A.

4. Preparation of human osteoprotegerin mutant protein OPGN139V in which the 139 th asparagine residue of human full-length osteoprotegerin was mutated to valine residue

The amino acid sequence of human osteoprotegerin mutant protein OPGN139V is SEQ ID No.5, and the mutant protein is a mutant obtained by mutating the 139 th asparagine residue (corresponding to the 275 th amino acid residue of SEQ ID No. 2) of human full-length osteoprotegerin into a valine residue (corresponding to the 122 th amino acid residue of SEQ ID No. 5). The mutant protein OPGN139V of human osteoprotegerin differs in amino acid sequence from wild-type human osteoprotegerin OPGwt only in amino acid residue 275 of SEQ ID No.2 (corresponding to amino acid residue 122 of SEQ ID No. 5).

4.1 preparation of DNA molecules

The aat (codon for asparagine) at position 825-827 of SEQ ID No.1 was replaced with gtt (codon for valine), and the other nucleotides were not changed to obtain the SrtA-LPETG-OPGN139V gene as a fusion protein fused with the motifs of sortase A and LPXTG. The gene SrtA-LPETG-OPGN139V expresses the fusion protein SrtA-LPETG-OPGN 139V. The amino acid sequence of SrtA-LPETG-OPGN139V is obtained by mutating asparagine residue at position 275 of SEQ ID No.2 to valine residue, and keeping the other amino acid residues of SEQ ID No.2 unchanged.

4.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGN139V gene was used to replace the segment between the NcoI and XhoI recognition sites of pET28a (+) (small segment including the NcoI recognition site and the XhoI recognition site), and the other sequence of pET28a (+) was kept unchanged to obtain a SrtA-LPETG-OPGN139V gene recombinant expression vector, which was named pET28a-SrtA-LPETG-OPGN 139V. pET28a-SrtA-LPETG-OPGN139V contains SrtA-LPETG-OPGN139V gene, pET28a-SrtA-LPETG-OPGN139V is introduced into E.coli BL21(DE3), and mutant protein OPGN139V of human osteoprotegerin with the amino acid sequence SEQ ID No.5 can be obtained.

4.3 obtaining mutant protein OPGN139V of human osteoprotegerin in which the 139 th asparagine residue of human full-length osteoprotegerin is mutated into valine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGN139V was named BL21(DE3)/pET28a-SrtA-LPETG-OPGN139V by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGN 139V; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGN139V to obtain human osteoprotegerin mutant protein OPGN 139V.

5. Preparation of human osteoprotegerin mutant protein OPGN139L in which the 139 th asparagine residue of human full-length osteoprotegerin was mutated to leucine residue

The amino acid sequence of human osteoprotegerin mutant protein OPGN139L is SEQ ID No.6, and the mutant protein is a mutant obtained by mutating the 139 th asparagine residue (corresponding to the 275 th amino acid residue of SEQ ID No. 2) of human full-length osteoprotegerin into the 122 th leucine residue (corresponding to the 122 th amino acid residue of SEQ ID No. 6). The mutant protein OPGN139L of human osteoprotegerin differs in amino acid sequence from wild-type human osteoprotegerin OPGwt only in amino acid residue 275 of SEQ ID No.2 (corresponding to amino acid residue 122 of SEQ ID No. 6).

5.1 preparation of DNA molecules

The aat (codon for asparagine) at position 825-827 of SEQ ID No.1 was replaced with ctg (codon for leucine) and the other nucleotides were not changed to obtain the SrtA-LPETG-OPGN139L gene as a fusion protein fused with the motifs of sortase A and LPXTG. The gene SrtA-LPETG-OPGN139L expresses the fusion protein SrtA-LPETG-OPGN 139L. The amino acid sequence of SrtA-LPETG-OPGN139L is obtained by mutating the 275 th asparagine residue of SEQ ID No.2 to leucine residue, and keeping the other amino acid residues of SEQ ID No.2 unchanged.

5.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGN139L gene was used to replace the segment between the NcoI and XhoI recognition sites of pET28a (+) (small segment including the NcoI recognition site and the XhoI recognition site), and the other sequence of pET28a (+) was kept unchanged to obtain a SrtA-LPETG-OPGN139L gene recombinant expression vector, which was named pET28a-SrtA-LPETG-OPGN 139L. pET28a-SrtA-LPETG-OPGN139L contains SrtA-LPETG-OPGN139L gene, pET28a-SrtA-LPETG-OPGN139L is introduced into E.coli BL21(DE3), and mutant protein OPGN139L of human osteoprotegerin with the amino acid sequence SEQ ID No.6 can be obtained.

5.3 obtaining human osteoprotegerin mutant protein OPGN139L with mutation of asparagine residue at position 139 of human full-length osteoprotegerin to leucine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGN139L was named BL21(DE3)/pET28a-SrtA-LPETG-OPGN139L by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGN 139L; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGN139L to obtain human osteoprotegerin mutant protein OPGN 139L.

6. Preparation of human osteoprotegerin mutant protein OPGE96G having glutamic acid residue at position 96 mutated to glycine residue

The human osteoprotegerin mutant protein OPGE96G is a protein obtained by mutating the glycine residue at position 122 of SEQ ID No.3 (corresponding to position 139 of human full-length osteoprotegerin) to an asparagine residue, and mutating the glutamic acid residue at position 79 of SEQ ID No.3 (corresponding to position 96 of human full-length osteoprotegerin) to a glycine residue, while keeping the other amino acid residues of SEQ ID No.3 unchanged. In this application as a control.

6.1 preparation of DNA molecules

The gag (codon for glutamic acid) at position 696-698 of SEQ ID No.1 was replaced with ggt (codon for glycine), and the other nucleotides were not changed to obtain the SrtA-LPETG-OPGE96G gene as a fusion protein fused with the motifs of sortase A and LPXTG. The gene SrtA-LPETG-OPGE96G expresses the fusion protein SrtA-LPETG-OPGE 96G. The amino acid sequence of SrtA-LPETG-OPGE96G is obtained by mutating the 232 nd glutamic acid residue of SEQ ID No.2 to glycine residue, and keeping the other amino acid residues of SEQ ID No.2 unchanged.

6.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGE96G gene was used to replace the fragment between the NcoI and XhoI recognition sites of pET28a (+) (small fragment including the NcoI recognition site and the XhoI recognition site), and the other sequence of pET28a (+) was kept unchanged to obtain a recombinant expression vector of SrtA-LPETG-OPGE96G gene, which was named pET28a-SrtA-LPETG-OPGE 96G. pET28a-SrtA-LPETG-OPGE96G contains SrtA-LPETG-OPGE96G gene, and pET28a-SrtA-LPETG-OPGE96G is introduced into E.coli BL21(DE3) to obtain human osteoprotegerin mutant protein OPGE 96G.

6.3 obtaining human osteoprotegerin mutant protein OPGE96G in which the 96 th glutamic acid residue of human full-length osteoprotegerin is mutated into glycine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGE96G was named BL21(DE3)/pET28a-SrtA-LPETG-OPGE96G by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGE 96G; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGE96G to obtain human osteoprotegerin mutant protein OPGE 96G.

7. Preparation of human osteoprotegerin mutant protein OPGR122G in which arginine residue at position 122 of human full-length osteoprotegerin was mutated to glycine residue

US 2006/0189528 a1 discloses a mutant protein of human osteoprotegerin R122G. R122G was prepared as a positive control in this application.

The amino acid sequence of the mutant protein R122G of human osteoprotegerin (referred to herein as OPGR122G) is SEQ ID No.9, a mutant comprising amino acid residues 22-194 of human full-length osteoprotegerin, but having the arginine residue at position 122 of human full-length osteoprotegerin mutated to a glycine residue (corresponding to amino acid residue 258 of SEQ ID No. 8).

7.1 preparation of DNA molecules

A DNA molecule of SEQ ID No.7 was prepared. The DNA shown in SEQ ID No.7 is a fusion protein SrtA-LPETG-OPGR122G gene fused with Sortase A (Sortase A, SrtA) and LPXTG motifs. The gene SrtA-LPETG-OPGR122G expresses the fusion protein SrtA-LPETG-OPGR 122G.

7.2 preparation of expression vectors

The fusion protein SrtA-LPETG-OPGR122G gene is used for replacing a fragment between NcoI recognition sites and XhoI recognition sites of pET28a (+) (a small fragment comprising the NcoI recognition sites and the XhoI recognition sites), and other sequences of pET28a (+) are kept unchanged to obtain a SrtA-LPETG-OPGR122G gene recombinant expression vector which is named as pET28a-SrtA-LPETG-OPGR 122G. pET28a-SrtA-LPETG-OPGR122G contains SrtA-LPETG-OPGR122G gene, and pET28a-SrtA-LPETG-OPGR122G is introduced into E.coli BL21(DE3) to obtain mutant protein OPGR122G of human osteoprotegerin with the amino acid sequence of SEQ ID No. 8.

7.3 mutant protein OPGR122G of human osteoprotegerin having the arginine residue at position 122 of human full-length osteoprotegerin mutated to glycine residue

Referring to step 1.3, the difference from step 1.3 is only two points: the recombinant E.coli containing pET28a-SrtA-LPETG-OPGR122G was named BL21(DE3)/pET28a-SrtA-LPETG-OPGR122G by replacing pET28a-SrtA-LPETG-OPGwt of 1.3 with pET28a-SrtA-LPETG-OPGR 122G; BL21(DE3)/pET28a-SrtA-LPETG-OPGwt of 1.3 was replaced with BL21(DE3)/pET28a-SrtA-LPETG-OPGR122G to obtain human osteoprotegerin mutant protein OPGR 122G.

8. Results of the experiment

The results showed that BL21(DE3)/pET28a-SrtA-LPETG-OPGwt, BL21(DE3)/pET28a-SrtA-LPETG-OPGN139A, BL21(DE3)/pET28a-SrtA-LPETG-OPGN139V, BL21(DE3)/pET28a-SrtA-LPETG-OPGN139G, BL21(DE3)/pET28a-SrtA-LPETG-OPGN139L, BL21(DE3)/pET28a-SrtA-LPETG-OPGE96G and T28a-SrtA-LPETG-OPGR122G were found to exist in the form of expressed protein in the form after pEG induction. This example obtained the corresponding soluble wild-type human osteoprotegerin OPGwt, mutant proteins of human osteoprotegerin OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G by preparation of inclusion bodies, protein denaturation, renaturation and purification by molecular sieve (AKTA Avant, GE Healthcare) (fig. 1).

Affinity determination for RANKL and TRAIL of example 2, wild-type human osteoprotegerin OPGwt and mutant proteins of human osteoprotegerin OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G

Surface Plasmon Resonance (SPR) technology is adopted, instrument equipment is BIAcore 3000(GE Healthcare), RANKL or TRAIL is used as a stationary phase and fixed on a certain channel on the Surface of a CM5 chip (10mM sodium acetate pH5.5 is added for dilution), and the response value is preferably increased by about 2000 RU; TNFRSF9 was immobilized in another channel as an internal control; OPGwt and human osteoprotegerin mutant proteins OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G prepared in example 1 were mobile phase, OPGwt and human osteoprotegerin mutant proteins OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G were formulated in BIacore buffer (10mM HEPES,150mM NaCl, 0.005% Tween 20, pH7.4) at different concentrations (0,3.125,6.25,12.5,25,50,100nM), respectively, flowed through the channel in sequence, the results were recorded as sensorgrams, and fit-dissociation constants (KD) were calculated using BIA evolution 4.1 (biachcart) according to the binding model of 1: 1.

Table 1, affinities of wild-type human osteoprotegerin OPGwt and mutant proteins of human osteoprotegerin to RANKL and TRAIL.

Note: wild type human osteoprotegerin OPGwt, OPGN139G as T1, OPGN139V as T101, OPGN139A as T102, OPGN139L as T103, OPGE96G as T2, and OPGR122G as a positive control as T3.

The results are shown in table 1 and indicate:

affinity of OPGN139G to RANKL (K)_D＝6.52×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Close, 0.80 times that of wild type; but the affinity of the TRAIL is obviously reduced compared with that of the wild type (8.09 multiplied by 10)^-7And 6.55X 10^-8) It is 0.08 times of wild type.

Affinity of OPGN139V to RANKL (K)_D＝5.77×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Is close to, 0.90 times of that of the wild type, but the affinity of the TRAIL is greatly reduced compared with that of the wild type (1.01 multiplied by 10)^-6And 6.55X 10^-8) It is 0.06 times of wild type.

Affinity of OPGN139A to RANKL (K)_D＝5.95×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Close to, 0.88 of wild typeDoubling; but the affinity of the TRAIL is obviously reduced compared with that of the wild type (5.95 multiplied by 10)^-7And 6.55X 10^-8) It is 0.11 times of wild type.

Affinity of OPGN139L to RANKL (K)_D＝5.65×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Close, 0.92 times that of wild type; but the affinity of the TRAIL is obviously reduced compared with that of the wild type (9.35 multiplied by 10)^-7And 6.55X 10^-8) It is 0.07 times of wild type.

Affinity of OPGE96G to RANKL (K)_D＝6.23×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Close to 0.84 times of wild type, but the affinity with TRAIL is also close to that of wild type (7.12X 10)^-8And 6.55X 10^-8) It is 0.92 times of wild type.

Affinity of the positive control OPGR122G to RANKL (K)_D＝6.96×10^-9) Affinity (K) to wild-type OPGwt and RANKL_D＝5.22×10^-9) Close to, 0.75 times of the wild type, but the affinity with TRAIL is obviously reduced compared with the wild type (2.62X 10)^-7And 6.55X 10^-8) It is 0.25 times of wild type.

The above results indicate that the mutant proteins OPGN139A, OPGN139V, OPGN139G, and OPGN139L of human osteoprotegerin of the present application have higher affinity for RANKL than the positive control OPGR122G, and significantly lower affinity for TRAIL than the positive control OPGR 122G. Although OPGE96G has a higher affinity for RANKL than the positive control OPGR122G, the affinity for TRAIL is also significantly higher than the positive control OPGR 122G.

Example 3 biological Activity of wild-type human osteoprotegerin OPGwt and mutant proteins of human osteoprotegerin OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G

1. Experiment for inhibiting RANKL-mediated osteoclast activation

RANKL-induced osteoclast differentiation and maturation is marked by the formation of multinucleated cells and the expression of tartrate-resistant acid phosphatase (TRAP) by the multinucleated cells.

Experiment of cell differentiation Using RAW264.7The ability of wild-type human osteoprotegerin OPGwt and mutant proteins of human osteoprotegerin to inhibit RANKL-mediated osteoclast activation was examined. Mouse monocyte macrophage leukemia cell RAW264.7 differentiated into osteoclasts under RANKL stimulation. The specific test method is as follows: mouse macrophage-derived cell line RAW264.7 was passaged to 24-well plates (1X 10)⁴Cells/well), cultured using α -MEM medium containing 10% FBS. RANKL was added to each well to a final concentration of 50ng/ml to induce differentiation of RAW264.7 cells into osteoclasts, and OPG (OPGwt, OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G prepared in example 1) at different concentrations was added to inhibit the differentiation-promoting effect of RANKL, and the cells were cultured in a constant temperature incubator (37 ℃ C., 5% CO in the incubator)₂) After 4 days of culture, the cells were stained using the tartrate-resistant acid phosphatase (TRAP) kit and multinucleated (number of nuclei) were examined in a literature-reported manner (Polek et al 2003)>3) TRAP staining positive cells were counted. And a negative control group without adding RANKL and OPG is arranged at the same time. The experiment was repeated 4 times and statistically analyzed.

As shown in fig. 2, the TRAP-positive multinucleated cells of the RANKL group (RANKL) were significantly increased compared to the Negative control group (Negative) as a result of quantitative analysis of TRAP staining after differentiation of the RAW264.7 cells, indicating that RANKL successfully stimulated differentiation of the RAW264.7 cells into mature osteoclasts. With the increase of the OPG concentration, the TRAP positive multinucleated cell proportion of each group is continuously reduced, which shows that the OPG plays a role in inhibiting the RANKL activity. Compared with wild OPG (OPGwt), the mutants OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G have no significant difference in the ability of inhibiting the differentiation of the osteoclast induced by RANKL. Thus, the mutation of the N139 site does not reduce the inhibiting effect of OPG on RANKL. This result is in agreement with the affinity assay result of example 2.

2. Experiment for inhibiting TRAIL-induced tumor cell apoptosis

TRAIL-induced apoptosis experiments were performed using the sub-G1DNA content analysis method (Allen et al.2012). That is, in human colon cancer cell Colo205 cell culture medium, TRAIL was added to OPG (OPGwt, OPGN139A, OPGN139V, OPGN139G, OPGN139L, OPGE96G and OPGR122G prepared in example 1) at a concentration of 100ng/ml and at different concentrations, and cells were maintained at constant temperatureWarm incubator (37 ℃, 5% CO content)₂) And culturing for 3 h. The cells were collected by centrifugation, washed and resuspended in 80% ethanol in PBS (pH7.4), centrifuged to discard the supernatant, resuspended again in PBS containing 0.2% Triton X-100 and 0.5mg/ml RNase, and then flow cytometrically tested on a FACStort flow cytometer (BD Biosciences, Mountain View, CA) after staining with 25ug/ml propidium iodide. A Negative control group (Negative) was also provided without TRAIL and OPG.

The results are shown in figure 2, TRAIL successfully promoted apoptosis of 50.25% ± 5.74% colo205 cells. Along with the increase of the OPG concentration, the apoptosis ratio of wild OPG group cells is continuously reduced, which shows that the apoptosis effect of TRAIL induced cells is inhibited; in contrast, the mutant OPGN139A, OPGN139V, OPGN139G, and OPGN139L groups showed less decrease in apoptosis rate with increasing OPG concentration, and in particular, the highest concentration OPGN139L group (OPGN139L concentration 12.5 μ g/ml, TRAIL: OPG ═ 1:125) showed an apoptosis rate of 37.58% ± 4.23%, which was close to the control group (50.25% ± 5.74%) in which TRAIL alone was added to induce expression, and was much higher than the highest concentration wild OPG (OPG wt) (5.25% ± 4.86%). The apoptosis rate of another mutant, OPGE96G, decreased significantly (9.53% + -3.26% at the highest concentration). In addition, OPGR122G as a positive control had a proportion of apoptosis of 21.62 ± 6.53% at its highest concentration. The experimental result shows that the inhibition effect of the mutants OPGN139A, OPGN139V, OPGN139G and OPGN139L on TRAIL is far lower than that of the wild OPG (OPGwt), even lower than that of the positive control OPGR122G, and the reduction degree is even better than that of the positive control OPGR122G site mutation. This result is in agreement with the affinity assay result of example 2.

Combining the above results, the present application successfully finds a new N139 mutation site on OPG that meets the research assumption that RANKL affinity is unchanged but TRAIL affinity is significantly reduced, and obtains more ideal OPGN139A, OPGN139V, OPGN139G and OPGN139L, which are OPG mutant proteins, through multiple amino acid substitutions. The OPG mutant protein can overcome the defect that the wild type can inhibit TRAIL anti-tumor effect, and can more efficiently and safely play a role in preventing and treating bone resorption related diseases.

<110> Beijing Ji complete Biotech, Inc

<120> osteoprotegerin N139 mutant protein, and related product and application thereof

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Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly

100 105 110

Val Val Gln Ala Gly Thr Pro Glu Arg Gly Thr Val Cys Lys Arg Cys

115 120 125

Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg

130 135 140

Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly

145 150 155 160

Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln

165 170 175

Lys Cys Gly Ile Asp Val Thr Leu Leu Glu His His His His His His

180 185 190

<210> 4

<211> 192

<212> PRT

<213> Artificial sequence

<220>

<230>

<400> 4

Gly Gly Gly Ser Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu

1 5 10 15

Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr

20 25 30

Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys

35 40 45

Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu

50 55 60

Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys

65 70 75 80

Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu

85 90 95

Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly

100 105 110

Val Val Gln Ala Gly Thr Pro Glu Arg Ala Thr Val Cys Lys Arg Cys

115 120 125

Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg

130 135 140

Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly

145 150 155 160

Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln

165 170 175

Lys Cys Gly Ile Asp Val Thr Leu Leu Glu His His His His His His

180 185 190

<210> 5

<211> 192

<212> PRT

<213> Artificial sequence

<220>

<230>

<400> 5

Gly Gly Gly Ser Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu

1 5 10 15

Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr

20 25 30

Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys

35 40 45

Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu

50 55 60

Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys

65 70 75 80

Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu

85 90 95

Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly

100 105 110

Val Val Gln Ala Gly Thr Pro Glu Arg Val Thr Val Cys Lys Arg Cys

115 120 125

Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg

130 135 140

Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly

145 150 155 160

Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln

165 170 175

Lys Cys Gly Ile Asp Val Thr Leu Leu Glu His His His His His His

180 185 190

<210> 6

<211> 192

<212> PRT

<213> Artificial sequence

<220>

<230>

<400> 6

Gly Gly Gly Ser Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp Glu

1 5 10 15

Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr Tyr

20 25 30

Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro Cys

35 40 45

Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu

50 55 60

Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys

65 70 75 80

Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu

85 90 95

Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly

100 105 110

Val Val Gln Ala Gly Thr Pro Glu Arg Leu Thr Val Cys Lys Arg Cys

115 120 125

Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys Arg

130 135 140

Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys Gly

145 150 155 160

Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gln

165 170 175

Lys Cys Gly Ile Asp Val Thr Leu Leu Glu His His His His His His

180 185 190

<210> 7

<211> 1019

<212> DNA

<213> Artificial sequence

<220>

<221> CDS

<222> (3)..(1019)

<230>

<400> 7

ccatgggcca agctaaacct caaattccga aagataaatc gaaagtggca ggctatattg 60

aaattccaga tgctgatatt aaagaaccag tatatccagg accagcaaca cctgaacaat 120

taaatagagg tgtaagcttt gcagaagaaa acgaatcact agatgatcaa aatatttcaa 180

ttgcaggaca cactttcatt gaccgtccga actatcaatt tacaaatctt aaagcagcca 240

aaaaaggtag tatggtgtac tttaaagttg gtaatgaaac acgtaagtat aaaatgacaa 300

gtataagaga tgttaagcct acagatgtag aagttctaga tgaacaaaaa ggtaaagata 360

aacaattaac attaattact tgtgatgatt acaatgaaaa gacaggcgtt tgggaaacac 420

gtaaaatctt tgtagctaca gaagtcaaac tgccggaaac cggcggtgga tccgaaacgt 480

ttcctccaaa gtaccttcat tatgacgaag aaacctctca tcagctgttg tgtgacaaat 540

gtcctcctgg tacctaccta aaacaacact gtacagcaaa gtggaagacc gtgtgcgccc 600

cttgccctga ccactactac acagacagct ggcacaccag tgacgagtgt ctatactgca 660

gccccgtgtg caaggagctg cagtacgtca agcaggagtg caatcgcacc cacaaccgcg 720

tgtgcgaatg caaggaaggg cgctaccttg agatagagtt ctgcttgaaa catggtagct 780

gccctcctgg atttggagtg gtgcaagctg gaaccccaga gcgaaataca gtttgcaaaa 840

gatgtccaga tgggttcttc tcaaatgaga cgtcatctaa agcaccctgt agaaaacaca 900

caaattgcag tgtctttggt ctcctgctaa ctcagaaagg aaatgcaaca cacgacaaca 960

tatgttccgg aaacagtgaa tcaactcaaa aactcgagca ccaccaccac caccactga 1019

<210> 8

<211> 338

<212> PRT

<213> Artificial sequence

<220>

<230>

<400> 8

Met Gly Gln Ala Lys Pro Gln Ile Pro Lys Asp Lys Ser Lys Val Ala

1 5 10 15

Gly Tyr Ile Glu Ile Pro Asp Ala Asp Ile Lys Glu Pro Val Tyr Pro

20 25 30

Gly Pro Ala Thr Pro Glu Gln Leu Asn Arg Gly Val Ser Phe Ala Glu

35 40 45

Glu Asn Glu Ser Leu Asp Asp Gln Asn Ile Ser Ile Ala Gly His Thr

50 55 60

Phe Ile Asp Arg Pro Asn Tyr Gln Phe Thr Asn Leu Lys Ala Ala Lys

65 70 75 80

Lys Gly Ser Met Val Tyr Phe Lys Val Gly Asn Glu Thr Arg Lys Tyr

85 90 95

Lys Met Thr Ser Ile Arg Asp Val Lys Pro Thr Asp Val Glu Val Leu

100 105 110

Asp Glu Gln Lys Gly Lys Asp Lys Gln Leu Thr Leu Ile Thr Cys Asp

115 120 125

Asp Tyr Asn Glu Lys Thr Gly Val Trp Glu Thr Arg Lys Ile Phe Val

130 135 140

Ala Thr Glu Val Lys Leu Pro Glu Thr Gly Gly Gly Ser Glu Thr Phe

145 150 155 160

Pro Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gln Leu Leu

165 170 175

Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His Cys Thr Ala

180 185 190

Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr Tyr Thr Asp

195 200 205

Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro Val Cys Lys

210 215 220

Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His Asn Arg Val

225 230 235 240

Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys

245 250 255

His Gly Ser Cys Pro Pro Gly Phe Gly Val Val Gln Ala Gly Thr Pro

260 265 270

Glu Arg Asn Thr Val Cys Lys Arg Cys Pro Asp Gly Phe Phe Ser Asn

275 280 285

Glu Thr Ser Ser Lys Ala Pro Cys Arg Lys His Thr Asn Cys Ser Val

290 295 300

Phe Gly Leu Leu Leu Thr Gln Lys Gly Asn Ala Thr His Asp Asn Ile

305 310 315 320

Cys Ser Gly Asn Ser Glu Ser Thr Gln Lys Leu Glu His His His His

325 330 335

His His

<210> 9

<211> 401

<212> PRT

<213> human

<400> 9

Met Asn Asn Leu Leu Cys Cys Ala Leu Val Phe Leu Asp Ile Ser Ile

1 5 10 15

Lys Trp Thr Thr Gln Glu Thr Phe Pro Pro Lys Tyr Leu His Tyr Asp

20 25 30

Glu Glu Thr Ser His Gln Leu Leu Cys Asp Lys Cys Pro Pro Gly Thr

35 40 45

Tyr Leu Lys Gln His Cys Thr Ala Lys Trp Lys Thr Val Cys Ala Pro

50 55 60

Cys Pro Asp His Tyr Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys

65 70 75 80

Leu Tyr Cys Ser Pro Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu

85 90 95

Cys Asn Arg Thr His Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr

100 105 110

Leu Glu Ile Glu Phe Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe

115 120 125

Gly Val Val Gln Ala Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg

130 135 140

Cys Pro Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys

145 150 155 160

Arg Lys His Thr Asn Cys Ser Val Phe Gly Leu Leu Leu Thr Gln Lys

165 170 175

Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr

180 185 190

Gln Lys Cys Gly Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg

195 200 205

Phe Ala Val Pro Thr Lys Phe Thr Pro Asn Trp Leu Ser Val Leu Val

210 215 220

Asp Asn Leu Pro Gly Thr Lys Val Asn Ala Glu Ser Val Glu Arg Ile

225 230 235 240

Lys Arg Gln His Ser Ser Gln Glu Gln Thr Phe Gln Leu Leu Lys Leu

245 250 255

Trp Lys His Gln Asn Lys Asp Gln Asp Ile Val Lys Lys Ile Ile Gln

260 265 270

Asp Ile Asp Leu Cys Glu Asn Ser Val Gln Arg His Ile Gly His Ala

275 280 285

Asn Leu Thr Phe Glu Gln Leu Arg Ser Leu Met Glu Ser Leu Pro Gly

290 295 300

Lys Lys Val Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys

305 310 315 320

Pro Ser Asp Gln Ile Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn

325 330 335

Gly Asp Gln Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser

340 345 350

Lys Thr Tyr His Phe Pro Lys Thr Val Thr Gln Ser Leu Lys Lys Thr

355 360 365

Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gln Lys Leu

370 375 380

Phe Leu Glu Met Ile Gly Asn Gln Val Gln Ser Val Lys Ile Ser Cys

385 390 395 400

Leu

Claims

1. A mutant protein of wild-type osteoprotegerin, characterized in that: the mutant protein is any one of D1 to D4:

d1, a protein of which the amino acid sequence is SEQ ID No.6, or a protein of which the amino acid sequence is amino acid residues 5 to 184 of SEQ ID No. 6;

d2, a protein of which the amino acid sequence is SEQ ID No.5, or a protein of which the amino acid sequence is amino acid residues 5-184 of SEQ ID No. 5;

d3, a protein of which the amino acid sequence is SEQ ID No.4 or a protein of which the amino acid sequence is amino acid residues 5 to 184 of SEQ ID No. 4;

d4, a protein of which the amino acid sequence is SEQ ID No.3, or a protein of which the amino acid sequence is amino acid residues 5 to 184 of SEQ ID No. 3.

2. A related biomaterial of the mutant protein of claim 1, said related biomaterial being any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the mutant protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector comprising the nucleic acid molecule of B1);

B4) a recombinant vector comprising the expression cassette of B2);

B5) a recombinant microorganism comprising the nucleic acid molecule of B1);

B6) a recombinant microorganism comprising the expression cassette of B2);

B7) a recombinant microorganism containing the recombinant vector of B3);

B8) a recombinant microorganism comprising the recombinant vector of B4).

3. A medicament for inhibiting RANKL-mediated osteoclast activation, characterized by: the medicament comprising the mutant protein of claim 1.

4. Use of a mutant protein according to claim 1 or/and a related biomaterial according to claim 2 for the manufacture of a medicament for inhibiting RANKL-mediated osteoclast activation.