CN115403657A

CN115403657A - Affinity polypeptide of TGF-beta 3 growth factor and application thereof

Info

Publication number: CN115403657A
Application number: CN202210724653.9A
Authority: CN
Inventors: 毛传斌; 杨涛; 杨明英
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-11-29

Abstract

The invention discloses an affinity polypeptide for transforming growth factor-beta 3 and application thereof. The amino acid sequences of the affinity polypeptides are respectively ID: MQTPKTM, VPLMFPRT, FLLESSN, LQSVHPL, LMPNSPI, AKYEQPH; can specifically bind TGF-beta 3. The polypeptide and the bioactive fragment containing the polypeptide can form a drug compound with other drugs capable of treating osteoarthritis and can also form a composite scaffold for cartilage regeneration with a scaffold material, the binding force of the polypeptide and TGF-beta 3 is strong, the specificity is high, and a new choice is provided for the research and development of osteoarthritis drugs and cartilage scaffold materials.

Description

Affinity polypeptide of TGF-beta 3 growth factor and application thereof

Technical Field

The invention belongs to a polypeptide in the field of biomedicine and application thereof, and particularly relates to an affinity polypeptide of transforming growth factor-beta 3 (transforming growth factor-beta 3, TGF-beta 3) and application thereof.

Background

Articular cartilage is a special hyaline cartilage formed of non-vascular connective tissue covering the articular surface and is composed mainly of extracellular matrix proteins and a small number of highly differentiated chondrocytes. Articular cartilage lacks supporting tissues such as blood vessels and nerves, and can only obtain nutrition through synovial fluid secreted by joint synovium, so that the self-repair capacity after cartilage injury is very limited. Once the cartilage tissue is damaged due to exercise and other reasons, the disease is not intervened or treated in time, and osteoarthritis is easy to develop. In fact, osteoarthritis patients who develop as a result of cartilage damage are large in number in our country and even worldwide. For cartilage regeneration medicine, mesenchymal Stem Cells (MSCs) are a clinically promising source of stem cells that can differentiate into chondrocytes under stimulation with appropriate growth factors and deposit cartilage-specific cytoplasmic matrix. Among them, transforming growth factor-beta 3 (TGF-. Beta.3) is a growth factor very critical for the differentiation of MSC into cartilage. On the other hand, osteoarthritis treatment and cartilage regeneration through the recruitment of in vivo self-contained MSCs and growth factors are a novel endogenous regenerative medical strategy with great application value. This approach eliminates the need to deliver exogenous stem cells and growth factors into the body, reducing possible immune rejection and secondary damage, and improving the regenerative effect. Therefore, the medicine or the material capable of recruiting the MSC and the key growth factor for chondrogenic differentiation is found, and the medicine or the material has important significance and application value for endogenous cartilage regeneration therapy.

Disclosure of Invention

The invention utilizes the filamentous phage random display library to carry out affinity optimization treatment on the TGF-beta 3 protein, obtains a polypeptide sequence with high affinity with the TGF-beta 3, and has the advantages of safety, practicability, convenience and high efficiency. Meanwhile, the invention provides the application of the affinity polypeptide in osteoarthritis and diseases related to cartilage damage.

The invention adopts the following technical scheme.

1. An affinity polypeptide:

the amino acid sequence of the affinity polypeptide is selected from one of SEQ ID No. 1-SEQ ID No. 6.

The affinity polypeptides are capable of specifically binding transforming growth factor (TGF-beta 3) proteins.

The amino acid sequences of the affinity polypeptides are respectively: MQTPKTM, VPLMFPRT, FLLESSN, LQSVHPL, LMPNSPI, AKYEQPH. As in the table below.

TABLE 1 TGF-. Beta.3 affinity Polypeptides amino acid sequence Listing

2. A biologically active fragment:

the biologically active fragment comprising the affinity polypeptide of claim 1 as part of a functional region of the biologically active fragment;

the biologically active fragment also has a function consistent with that of the affinity polypeptide, i.e., is capable of binding to the growth factor TGF-beta 3.

The use of biologically active fragments in the manufacture of a medicament.

3. A nucleotide sequence of: the nucleotide sequence encodes the affinity polypeptide.

4. A nucleotide sequence of: the nucleotide sequence can encode the bioactive fragment.

5. A pharmaceutical composition for the treatment of osteoarthritis: the pharmaceutical complex comprises the affinity polypeptide or the biologically active fragment and also a drug or a drug carrier for the treatment of osteoarthritis.

The polypeptide or the bioactive fragment is used as a growth factor recruitment agent, is chemically connected with or mixed with a medicament capable of treating osteoarthritis to be used as a medicament compound for treating osteoarthritis, and can also be chemically connected with or mixed with a carrier for coating the medicament to be used as a medicament compound capable of treating osteoarthritis.

The medicine is any one of non-steroidal anti-inflammatory drugs (NSAIDS), cartilage protective agents such as glucosamine and the like, joint cavity injection medicines, antidepressants, opioid analgesics and the like;

the drug carrier is any one of liposome, natural polymer material, artificially synthesized polymer material, inorganic crystal material, carbon material, polymer vesicle and polymer micelle.

6. A composite scaffold for cartilage regeneration:

the composite scaffold simultaneously comprises the affinity polypeptide or the bioactive fragment and a scaffold material capable of being used for cartilage regeneration. The polypeptide or the bioactive fragment is used as a growth factor recruitment element and is chemically connected with or mixed with biological materials capable of being used for cartilage tissue repair to be used as a composite scaffold for cartilage regeneration.

The bracket material is a composite material of any one or more of a natural polymer material, an artificially synthesized polymer material, a metal material, an inorganic non-metal material and a carbon material.

The invention is applied to the preparation and production of medicines and biomedical materials for diseases caused by osteoarthritis and cartilage damage.

The polypeptide of the invention has strong specificity, high affinity and relatively simple and convenient operation, and is suitable for searching the binding polypeptide of transforming growth factor TGF-beta 3.

The polypeptide and the bioactive fragment containing the polypeptide can form a drug compound with other drugs capable of treating osteoarthritis, and can also form a compound scaffold for cartilage regeneration with a cartilage repair material. The polypeptide of the invention has strong binding force with TGF-beta 3 and high specificity, and provides a new choice for the research and development of arthritis drugs and cartilage regeneration scaffolds.

Compared with the prior art, the invention has the following outstanding advantages:

(1) The invention screens the micromolecular polypeptide combined with TGF-beta 3 protein by using phage display technology, has the characteristics of short time consumption, simple and convenient operation and high success rate, and provides a new choice for developing osteoarthritis treatment medicines and cartilage regeneration scaffold materials.

(2) The small molecular polypeptide which can be combined with TGF-beta 3 protein and is screened out by the invention can be synthesized by a standardized chemical synthesis process, compared with recombinant protein or antibody reagents, the production, purification and storage costs are greatly reduced, and animals are not needed, so that compared with an antibody development technology, the small molecular polypeptide is more economic and has no ethical worry;

(3) The specific binding effect of the polypeptide and TGF-beta 3 protein provided by the invention provides reference for further developing new tissue repair materials, researching the interaction of growth factors on stem cell differentiation and cartilage repair and producing more valuable osteoarthritis treatment medicines and cartilage regeneration composite scaffolds.

Drawings

Attached table 2 shows the statistical results of the frequency of obtaining affinity polypeptides by screening phage libraries.

FIG. 1 is a statistical chart of the results of the phage enzyme-linked immunosorbent assay (ELISA) in example 1.

FIG. 2 is a graph of the phage input output statistical experimental results of example 2.

Detailed Description

The present invention is further illustrated by the following examples, which are illustrative of the present invention and are not to be construed as being limited thereto. Any modification and variation made within the spirit and principle of the present invention should be included within the scope of protection of the present invention.

The embodiments of the invention are as follows:

example 1

Phage enzyme-linked immunosorbent assay (ELISA) verifies and compares the binding capacity of the affinity polypeptide specifically binding TGF-beta 3 protein.

1) Phage library screening TGF-beta 3 protein affinity polypeptide sequence statistics: and (3) spreading the affinity phage obtained by screening the 3 rd round phage and the 4 th round phage on a flat plate, selecting monoclonal amplification culture, extracting plasmids, and performing gene sequencing to obtain an affinity polypeptide sequence displayed on the surface of the phage. The polypeptide sequence and frequency statistics for each round are shown in attached table 2.

Attached table 2 3 rd and 4 th round phage library screening to obtain affinity polypeptide frequency statistics

As can be seen in the above table, there are 6 affinity polypeptides with an aggregate repeat count greater than 1 after 3 rd and 4 th rounds of screening, with MQTPKTM repeat counts up to 66, and repeat counts for both vplmfprt and FLLESSN polypeptides greater than 2.

2) The phages referred to in the attached Table 2 were amplified and purified.

The phage is specifically phage containing MQTPKTM, VPLMFPRT, FLLESSN, LQSVHPL, LMPNSPI and AKYEQPH sequences respectively.

3) By means of NaHCO ₃ (0.1M, pH8.6) the TGF-beta 3 protein is diluted by the coating solution, and the final concentration is 1 mu g/mL; dilutions of TGF-. Beta.3 protein were added to 96-well plates, placed in wet chambers, and coated overnight at 4 ℃.

4) The prepared TGF-. Beta.3 protein was blocked with a blocking solution (PBS +0.5mg/ml BSA) for 1 hour.

5) Remove the blocking solution and add the affinity phage (2X 10) with different inserts diluted with blocking solution ⁹ pfu) was incubated at room temperature for 1 hour.

6) Each well was washed 5 times with 5 min each time of PBST (PBS +0.5% (v/v) Tween 20) solution.

7) anti-M13 phage antibody diluted with PBS solution was added and incubated at 37 ℃ for 1 hour. Each well was washed 3 times with PBST for 5 minutes each.

8) Horseradish peroxidase (HRP) labeled secondary antibodies were added and incubated at 37 ℃ for 30 minutes. Each well was washed 3 times with PBST for 5 minutes each.

9) Adding TMB color development solution, incubating at room temperature until full color development, adding 2M sulfuric acid stop solution, reading absorbance value at 450nm with enzyme labeling instrument, and counting to obtain result shown in figure 1.

Example 2

Phage Input (Input) Output (Output) statistical experiments compare the effect of phage concentration on the magnitude of affinity phage binding specifically binding to TGF- β 3 protein.

1) The phage was amplified and purified.

The phage is specifically a phage containing MQTPKTM polypeptide sequence.

2) By means of NaHCO ₃ (0.1M, pH8.6) the TGF-beta 3 protein is diluted by the coating solution, and the final concentration is 1 mu g/mL; dilutions of TGF-. Beta.3 protein were added to 96-well plates, placed in wet boxes, and coated overnight at 4 ℃.

3) The prepared TGF-. Beta.3 protein was blocked with a blocking solution (PBS +0.5mg/ml BSA) for 1 hour.

4) Remove the blocking solution and add different concentrations of affinity phage (2X 10) diluted with blocking solution ¹¹ pfu/mL，2×10 ¹⁰ pfu/mL，2×10 ⁹ pfu/mL，2×10 ⁸ pfu/mL) 50. Mu.L, and incubated at room temperature for 1 hour.

5) Each well was washed 5 times with 5 min each time with PBST (PBS +0.5% (v/v) Tween 20) solution.

6) The wash solution in the well plate was removed and patted dry, glycine eluent (0.2M glycine-hydrochloric acid, pH2.2,1mg/mL BSA) was added, and the affinity phage was eluted by incubation on ice for 10 min.

7) Adding 1M Tris-HCl (pH9.1) to neutralize, and mixing to obtain neutral pH.

8) The collected phage solution was diluted with PBS in a gradient, added to overnight cultured E.coli, incubated at room temperature for 5 minutes, plated on solid LB plates containing IPTG/Xgal after top agar was added, and shaken gently to homogenize. After the liquid was completely solidified, the mixture was placed in an incubator at 37 ℃ for overnight culture while being inverted. The plaques appearing on the plate are counted the next day, and the concentration of the phage is obtained according to the dilution times. And (3) counting and comparing the difference of the output phage concentration under the condition of phage input with different concentrations, and obtaining results shown in figure 2.

The gene and protein sequences related to the invention are as follows:

SEQ ID No.1；

name: amino acid sequence of affinity polypeptide 1

The source is as follows: artificial Sequence (Artificial Sequence)

MQTPKTM

SEQ ID No.2；

Name: amino acid sequence of affinity polypeptide 2

The source is as follows: artificial Sequence (Artificial Sequence)

VPLFPRT

SEQ ID No.3；

Name: amino acid sequence of affinity polypeptide 3

The source is as follows: artificial Sequence (Artificial Sequence)

FLLESSN

SEQ ID No.4；

Name: amino acid sequence of affinity polypeptide 4

The source is as follows: artificial Sequence (Artificial Sequence)

LQSVHPL

SEQ ID No.5；

Name: amino acid sequence of affinity polypeptide 5

The source is as follows: artificial Sequence (Artificial Sequence)

LMPNSPI

SEQ ID No.6；

Name: amino acid sequence of affinity polypeptide 6

The source is as follows: artificial Sequence (Artificial Sequence)

AKYEQPH。

Sequence listing

<110> Zhejiang university

<120> affinity polypeptide of TGF-beta 3 growth factor and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gln Thr Pro Lys Thr Met

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Val Pro Leu Phe Pro Arg Thr

1 5

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Phe Leu Leu Glu Ser Ser Asn

1 5

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Leu Gln Ser Val His Pro Leu

1 5

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Leu Met Pro Asn Ser Pro Ile

1 5

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Lys Tyr Glu Gln Pro His

1 5

Claims

1. An affinity polypeptide characterized by:

2. A biologically active fragment characterized by:

the biologically active fragment comprising the affinity polypeptide of claim 1.

3. A nucleotide sequence characterized in that: the nucleotide sequence encoding the affinity polypeptide of claim 1.

4. A nucleotide sequence characterized in that: the nucleotide sequence encoding the biologically active fragment of claim 2.

5. A pharmaceutical composition for the treatment of osteoarthritis, said pharmaceutical composition comprising the affinity polypeptide of claim 1 or the biologically active fragment of claim 2, and a drug or a pharmaceutical carrier for the treatment of osteoarthritis.

6. A composite scaffold for cartilage regeneration, comprising:

the composite scaffold comprises the affinity polypeptide of claim 1 or the bioactive fragment of claim 2 and a biomaterial that can be used in a cartilage regeneration scaffold.

7. Use of the affinity polypeptide of claim 1, the biologically active fragment of claim 2, the nucleotide sequence of claim 3, the nucleotide sequence of claim 4, the drug complex of claim 5, the composite scaffold of claim 6, wherein: the application in the preparation and production of drugs or stents.

8. Use according to claim 7, characterized in that:

the medicine or the bracket is used for treating diseases caused by osteoarthritis or cartilage damage.