CN112375135A - Artificial epidermal growth factor, designed polypeptide and application thereof - Google Patents
Artificial epidermal growth factor, designed polypeptide and application thereof Download PDFInfo
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- CN112375135A CN112375135A CN202010639721.2A CN202010639721A CN112375135A CN 112375135 A CN112375135 A CN 112375135A CN 202010639721 A CN202010639721 A CN 202010639721A CN 112375135 A CN112375135 A CN 112375135A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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Abstract
According to the polypeptide fragment of a natural ligand EGF of an epidermal growth factor receptor, a polypeptide fragment EBeta1 with a specific sequence and containing two cysteines is designed, gold nanoparticles are used as a protein framework for supporting the conformation of the polypeptide fragment, and the method of conformation engineering is used for successfully preparing the artificial EGF capable of simulating the function of the EGF. The combination strength change and combination kinetic change of the artificial EGF and the sEGFR are measured by an SPR technology, and the strong combination of the artificial EGF and the sEGFR is proved. Negative staining electron micrograph characterization demonstrated that artificial EGF was able to specifically bind to egfr in a 1:1 model (see abstract figure). The promotion effect of the artificial EGF on HeLa cell proliferation and the inhibition effect on MDA-MB-468 cell proliferation indicate that the artificial EGF can replace natural EGF to play a high-level biological function. The gold-combined nano particle has high stability, and the artificial EGF can be widely used for replacing natural EGF in the field of biomedicine, can be used as a functional unit of targeting EGFR and is used for in vivo imaging positioning and drug targeting delivery.
Description
Technical Field
The invention relates to a polypeptide, an artificial Epidermal Growth Factor (EGF) generated by the polypeptide and application of the polypeptide.
Background
The discovery and study of EGF has a very short history, but its wide application is determined by its particular biological effects. With the intensive research and wide clinical application, some difficult and complicated diseases such as severe burn, large-area wound, digestive tract ulcer, severe corneal injury and the like are expected to be cured quickly. Even some of the current incurable diseases: such as nerve damage, malignancy, aids, etc., may also be alleviated and restored by the use of EGF. It can be predicted that the application of EGF will bring about a great leap for future life science research.
In recent years, the research on EGF and its receptor (EGFR) has become one of the hot spots in biomedical research, and nanoparticles can achieve targeting by coupling with natural ligand EGF for EGFR, an important target in tumor treatment. EGF is a protein consisting of 53 amino acids with three disulfide bonds and many hydrophobic residues, all suitable for interaction with nanoparticles. EGF is a natural ligand with small size and specificity compared to antibodies and even antibody fragments, and does not readily elicit an immune response. Unfortunately, its use has disadvantages such as low availability of EGF from human source, high price, difficulty in controlling the orientation of EGF on the surface of the nanoparticle by covalent attachment and decrease in EGF recognition ability due to non-specific adsorption of the nanoparticle to EGF, possibility of changing the size and related characteristics of the entire nanoparticle after attachment of EGF, etc.
Patent publication No. CN105884888A discloses a design method of "conformation engineering", in which a Complementarity Determining Region (CDR) polypeptide is selected from known natural antibodies, and both ends of the CDR polypeptide are covalently linked to gold nanoparticles, so as to successfully reproduce the functions of the natural antibodies.
Disclosure of Invention
One of the objectives of the present invention is to overcome the limitation of the prior art limited to the grafting of the CDR polypeptide loop region of the natural antibody, and to provide a polypeptide selected from a beta-hairpin structural fragment derived from EGF, a natural ligand.
The second purpose of the invention is to graft the polypeptide fragment on the surface of the gold nanoparticle, and to adjust and control the correct spatial conformation of the polypeptide by adjusting the density of the polypeptide on the surface of the gold nanoparticle, so as to prepare the artificial epidermal growth factor EGF capable of specifically binding to the EGFR.
The invention also aims to provide the application of the artificial epidermal growth factor in preparing a medicament for repairing wounds.
The fourth purpose of the invention is to provide the application of the artificial epidermal growth factor in preparing the medicament for preventing color spots.
The fifth purpose of the invention is to provide the application of the artificial epidermal growth factor in the preparation of a reagent for specifically detecting EGFR.
The sixth purpose of the invention is to provide the artificial epidermal growth factor and the designed polypeptide as the functional unit of the targeting EGFR protein, and the artificial epidermal growth factor and the designed polypeptide are applied to imaging localization and drug delivery.
The seventh purpose of the invention is to provide the application of the artificial epidermal growth factor in specifically promoting the proliferation activity of low-expression cells (such as HeLa cells).
The eighth purpose of the present invention is to provide the application of the artificial epidermal growth factor in specifically inhibiting the proliferation activity of EGFR high expressing cells (such as MDA-MB-468 cells).
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a polypeptide, wherein the polypeptide is any one of the following (A) or (B):
(A) a polypeptide EBeta1 containing an amino acid sequence shown in sequence 1;
(B) and (b) the polypeptide derived from the (A) by substituting and/or deleting and/or adding and/or prolonging the amino acid residue sequence shown in the sequence 1 by one or more amino acid residues under the condition of not changing the key binding residues.
The polypeptide is an amino acid sequence shown in SEQ ID NO. 1.
The artificial epidermal growth factor is characterized in that the artificial epidermal growth factor is obtained by anchoring two Au-S bonds formed by two cysteine designed in the polypeptide and the surface of a gold nanoparticle and adjusting the density of the polypeptide on the surface of the gold nanoparticle to enable the polypeptide to form a specific hairpin (beta-hairpin) conformation. The polypeptide is obtained by forming specific beta hairpin (beta-hairpin) conformation on the surface of the gold nano particle.
The density range of the polypeptide on the surface of the gold nanoparticle is as follows: 0.2-2 stripes per square nanometer surface area.
An application of the artificial epidermal growth factor in preparing a medicament for repairing wounds.
An application of the artificial epidermal growth factor in preparing a medicament for preventing color spots.
An application of the artificial epidermal growth factor in preparing a reagent for specifically detecting EGFR.
The designed polypeptide and the artificial epidermal growth factor are used as functional units of the targeting EGFR protein,
the application in imaging localization and drug delivery.
The application of the artificial epidermal growth factor in specifically promoting HeLa cell proliferation.
The application of the artificial epidermal growth factor in specifically inhibiting MDA-MB-468 cell proliferation.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. breaks through the limitation that the original technology is only used for natural antibody fragments, carries out the simulation of artificial ligand protein for the first time, and successfully synthesizes the artificial EGF with normal physiological function.
2. Breaks through the limitation that the prior art is only used for loop fragment grafting, and reconstructs a beta-hairpin structure on the surface of the nano particle by using a conformation engineering method for the first time.
3. The artificial EGF can be used as a medicament for repairing wounds, preventing related diseases such as color spots and the like; the artificial EGF can be used as a reagent for specifically detecting the EGFR; the artificial EGF can be used as a targeting functional unit for targeting EGFR protein and used for in vivo imaging localization and drug delivery.
Drawings
FIG. 1 is a UV characterization of gold nanoparticles and artificial EGF according to a preferred embodiment of the present invention.
FIG. 2 is a hydrated particle size characterization of gold nanoparticles and artificial EGF according to a preferred embodiment of the present invention.
FIG. 3 shows the regulation of polypeptide conformation and artificial EGF activity by adjusting polypeptide density in a preferred embodiment of the invention.
Fig. 4 is a representation of hydrated particle size of artificial EGF after being homogeneously mixed with egfr or BSA, respectively, according to a preferred embodiment of the present invention.
FIG. 5 is a graph showing the specific binding properties of artificial EGF to sEGFR in accordance with a preferred embodiment of the present invention.
FIG. 6 shows the binding kinetics of the artificial EGF to sEGFR measured by SPR, which is a preferred embodiment of the present invention.
FIG. 7 is a diagram illustrating that the artificial EGF specifically promotes HeLa cell proliferation in accordance with a preferred embodiment of the present invention.
FIG. 8 shows that the artificial EGF specifically inhibits MDA-MB-468 cell proliferation in accordance with a preferred embodiment of the present invention.
FIG. 9 is electron microscopy characterization of negatively stained samples of artificial EGF binding to sEGFR in example 2 of the present invention.
Example 1
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
in this example, the design of the polypeptide and the artificial EGF was performed as follows
1. Design synthesis of polypeptide fragments
The polypeptide EBeta1 is designed and synthesized, and the sequence is as follows:
Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Val
the designed polypeptide EBeta1 can be fixed on the surface of the gold nanoparticle through two cysteines in the sequence, so that a specific beta-hairpin conformation is formed.
2. Preparation of artificial EGF
Referring to the previous 'conformation engineering' method, the designed polypeptide sequence is grafted on the surface of the gold nano-particle to prepare the artificial EGF. First, 20. mu.L of 0.2M trisodium citrate solution was added to an aqueous solution of gold nanoparticles (6mL) to enhance the stability of the gold nanoparticles. 2mL of the polypeptide solution (containing 2mM sodium hydroxide) was added dropwise to the stirring aqueous gold nanoparticle solution, and stirring was continued at room temperature for 1 h. The polypeptide is grafted on the gold nano-particles in the form of S-Au bonds through sulfydryl groups contained at two ends of a self sequence, so that the artificial EGF is prepared.
3. Particle size characterization of gold nanoparticles before and after polypeptide grafting
FIG. 1 shows ultraviolet spectrum characterization of gold nanoparticles and artificial EGF, and results show that after the gold nanoparticles are modified by the polypeptide, the surface plasma resonance absorption peak of the gold nanoparticles is red-shifted, which proves successful grafting of the polypeptide on the surface of the gold nanoparticles. FIG. 2 is a representation of the hydrated particle size of gold nanoparticles and artificial EGF, and the results show that the polypeptide is successfully grafted on the surface of the gold nanoparticles, so that the hydrated particle size of the gold nanoparticles is obviously increased.
4. Polypeptide density affecting biological activity of artificial EGF
We grafted different amount of polypeptide EBeta1 on the surface of gold nanoparticles by means of gradient screening, and determined the binding strength change of the artificial EGF and sEGFR by SPR technique, as shown in FIG. 3. The polypeptide EBeta1 is grafted on the surface of the gold nano particle, and the preferred density is as follows: 0.2-2 per square of the surface area of the nano-gold nanoparticle, the polypeptide fragment is in the appropriate conformation at the moment, so that the artificial EGF has stronger capability of binding to the sEGFR.
5. Characterization of specific binding of artificial EGF (i.e., AuNP-EBeta1, infra, for comparison to AuNP-EBeta1m with only one cysteine but no activity) to sEGFR
AuNP-EBeta1 is selected to be respectively and uniformly mixed with sEGFR or BSA, and whether the artificial EGF is specifically combined with the sEGFR or not is preliminarily judged by measuring the hydrated particle size distribution of the two mixed samples. As shown in fig. 4, the hydrated particle size distribution of AuNP-EBeta1 after mixing with BSA was not significantly different from that before mixing, indicating that artificial EGF was not bound to BSA. The hydrated particle size distribution of AuNP-EBeta1 after mixing with sEGFR changed significantly and the measured hydrated particle size increased. Preliminarily shows that the artificial EGF can be specifically combined with the sEGFR. In order to more powerfully prove the capability of the artificial EGF to specifically bind to the sEGFR, which is based on the fact that the polypeptide EBeta1 on the surface of the gold nanoparticles is folded to have a specific conformation, three proteins BSA, IgG and Fib with relatively high content in serum are selected as control proteins, and the binding strength of AuNP-EBeta1, AuNP-EBeta1m and free polypeptides (EBeta1 and EBeta1m (the sequence is different from EBeta1 by only one cysteine)) with corresponding concentration and the sEGFR, BSA, IgG and Fib fixed on a CM5 chip channel are respectively measured by an SPR technology. As shown in fig. 5, the artificial EGF did not produce strong binding to three proteins abundant in blood, indicating that the binding of the artificial EGF to the egfr is specific.
6. Characterization of binding Strength of Artificial EGF to sEGFR
To demonstrate that synthetic artificial EGF binds very strongly specifically to the egfr, we measured the binding kinetics of artificial EGF to the egfr. The coupling level of sEGFR is kept low and the binding kinetics data of artificial EGF to sEGFR are suitable for fitting in a 1:1 model. Kinetic binding constant K after fitting of interaction of artificial EGF and sEGFRD＝6.7×10- 11M，kon＝2.2×106M-1s-1And koff＝1.5×104s-1. It can also be seen from FIG. 6 that the artificial EGF is hardly eluted from the chip at the elution stage, so that the signal of the SPR spectrum at the elution stage is almost a flat line. This also indicates that artificial EGF has a very strong ability to bind to egfr.
7. Biological Activity of Artificial EGF at cellular level
Artificial EGF at a certain concentration can also cause proliferation or apoptosis of corresponding tumor cells. Two cell models, namely HeLa cells with medium-low expression of EGFR and MDA-MB-468 cells with high expression of EGFR are selected as research objects, and whether the artificial EGF can successfully replace the EGF at a cellular level is researched. FIG. 7 shows that the addition of 20 nMAGF or 40 nMAGNP-EBeta 1 has substantially the same effect on the proliferation of HeLa cells; FIG. 8 shows that the addition of 20 nMAGF or 40 nMAGNP-EBeta 1 has substantially the same effect on MDA-MB-468 cell proliferation. These two results strongly demonstrate the successful biological function of artificial EGF in replacing EGF.
Example 2
In this embodiment, the polypeptide EBeta1 is simply modified without changing the key binding residues to obtain the polypeptide EBeta2, and the artificial EGF with EGF function (AuNP-EBeta2) is synthesized.
1. Design synthesis of polypeptide fragments
The polypeptide EBeta2 is designed and synthesized, and the sequence is as follows:
Ser-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Val-Gly
synthesis of artificial EGF (AuNP-EBeta2) was prepared as in example 1.
2. Characterization of specific binding of artificial EGF (AuNP-EBeta2) to sEGFR
In the embodiment, the specific binding function of the artificial EGF and the sEGFR is directly represented by an electron microscope picture of the binding of AuNP-EBeta2 and the sEGFR. We stained the artificial EGF and the egfr mixture negatively and observed the binding of the artificial EGF to the egfr further using a transmission electron microscope to determine the binding ratio between the two. As shown in fig. 9, we can see that the particle size of the sfegfr is about 20nm (the molecular weight of the sfegfr is 110kDa, and theoretically the size of the sfegfr is about 20 nm), and almost every protein is surrounded by an artificial EGF, which truly reflects the phenomenon that the artificial EGF specifically binds to the sfegfr strictly in a ratio of 1: 1. Specific binding to sEGFR is the molecular basis for EGF to exert cellular activity and related biomedical applications.
In conclusion, the changes of the binding strength of the artificial EGF and the sEGFR and the changes of the binding kinetics are measured by the SPR technology on a molecular level, and the strong specific binding of the artificial EGF and the sEGFR is proved. At cellular level, the capability of the artificial EGF binding to the epidermal growth factor receptor of tumor cells is proved, and the artificial EGF has the same cellular biological activity as the natural EGF, which indicates that the artificial EGF can replace the natural EGF to be used in the biomedical field. The molecular mechanism of artificial EGF cell activity and its biomedical applications is its specific binding to the egfr. Negative-staining electron micrographs demonstrate that artificial EGF capable of specifically binding to egfr in a 1:1 model can also be synthesized by engineering polypeptides without altering the key residues of the polypeptides we designed.
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made according to the purpose and principle of the present invention, and any changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the technical scheme of the present invention shall be equivalent substitutions, as long as the purpose of the present invention is met, and the present invention shall fall within the protection scope of the present invention as long as the technical principle and the inventive concept of the polypeptide, the artificial EGF and its application are not deviated.
Sequence listing
<110> university at Shanghai
<120> artificial epidermal growth factor, designed polypeptide and application thereof
<160> 1
<210> 1
<211> 15
<212> RNA
<213> Acinetobacter sp
<400> 1
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Val
1 5 10 15
Claims (11)
1. A polypeptide, wherein the polypeptide is any one of the following (A) or (B):
(A) a polypeptide EBeta1 containing an amino acid sequence shown in sequence 1;
(B) and (b) the polypeptide derived from the (A) by substituting and/or deleting and/or adding and/or prolonging the amino acid residue sequence shown in the sequence 1 by one or more amino acid residues under the condition of not changing the key binding residues.
2. The polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID No. 1.
3. An artificial epidermal growth factor, characterized in that the artificial epidermal growth factor is obtained by anchoring two Au-S bonds formed on the surface of gold nanoparticles by two cysteines designed in the polypeptide according to claim 1 or 2, and forming a specific beta hairpin (beta-hairpin) conformation by the polypeptide by adjusting the density of the polypeptide on the surface of the gold nanoparticles.
4. The artificial epidermal growth factor of claim 3, wherein the polypeptide has a density on the surface of the gold nanoparticle in the range of: 0.2-2 stripes per square nanometer surface area.
5. Use of the artificial epidermal growth factor of claim 3 or 4 in the preparation of a medicament for repairing a wound.
6. Use of the artificial epidermal growth factor of claim 3 or 4 in the preparation of a medicament for preventing color spots.
7. Use of the artificial epidermal growth factor of claim 3 or 4 in the preparation of a reagent for specifically detecting EGFR.
8. The use of a polypeptide according to claim 1 or 2 as a functional unit targeting an EGFR protein for imaging localization and drug delivery.
9. The use of the artificial epidermal growth factor of claim 3 or 4 as a functional unit targeting EGFR protein for imaging localization and drug delivery.
10. Use of the artificial epidermal growth factor of claim 3 or 4 for specifically promoting the proliferative activity of low-expressing cells (such as HeLa cells) of EGFR.
11. Use of the artificial epidermal growth factor of claim 3 or 4 for specifically inhibiting the proliferative activity of a cell with high expression of EGFR (such as MDA-MB-468 cell).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1853731A (en) * | 2005-04-26 | 2006-11-01 | 中国人民解放军军事医学科学院生物工程研究所 | Use of staphylococcal enterotoxin A gene and its coded protein |
| CN101321784A (en) * | 2005-10-11 | 2008-12-10 | 埃博灵克斯股份有限公司 | Nanobodies and polypeptides against EGFR and IGF-IR |
| US20110318380A1 (en) * | 2008-10-01 | 2011-12-29 | Dako Denmark A/S | MHC Multimers in Cancer Vaccines and Immune Monitoring |
| CN106699890A (en) * | 2016-10-31 | 2017-05-24 | 上海大学 | Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof |
-
2020
- 2020-07-06 CN CN202010639721.2A patent/CN112375135B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1853731A (en) * | 2005-04-26 | 2006-11-01 | 中国人民解放军军事医学科学院生物工程研究所 | Use of staphylococcal enterotoxin A gene and its coded protein |
| CN101321784A (en) * | 2005-10-11 | 2008-12-10 | 埃博灵克斯股份有限公司 | Nanobodies and polypeptides against EGFR and IGF-IR |
| US20110318380A1 (en) * | 2008-10-01 | 2011-12-29 | Dako Denmark A/S | MHC Multimers in Cancer Vaccines and Immune Monitoring |
| CN106699890A (en) * | 2016-10-31 | 2017-05-24 | 上海大学 | Artificial antibody for targeting EGFR (epidermal growth factor receptor) based on gold nanoparticles and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| HIDEO OGISO等: "Crystal Structure of the Complex of Human Epidermal Growth Factor and Receptor Extracellular Domains", 《CELL》, vol. 110, no. 6, 20 September 2002 (2002-09-20), pages 776 - 787, XP002961395, DOI: 10.1016/S0092-8674(02)00963-7 * |
| HIROSHI KOIDE等: "Recognition of an antiparallel beta-sheet structure of human epidermal growth factor by its receptor. Site-directed mutagenesis studies of Ala-30 and Asn-32", 《FEBS》, vol. 302, no. 1, 4 May 1992 (1992-05-04), pages 39 - 42 * |
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