CN112375135B - Artificial epidermal growth factor, designed polypeptide and application thereof - Google Patents

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 artificial EGF capable of simulating the function of the EGF is successfully prepared by the method of 'conformation engineering'. The change of the binding strength of the artificial EGF and the sEGFR and the change of the binding kinetics are measured by an SPR technology, and the strong binding between 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. The promotion effect of the artificial EGF on the proliferation of HeLa cells and the inhibition effect of the artificial EGF on the proliferation of MDA-MB-468 cells 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

A kind of artificial epidermal growth factor and design polypeptide and application thereof

technical field

The present invention relates to a kind of polypeptide and artificial epidermal growth factor (EGF) and application thereof.

Background technique

The history of discovery and research of EGF is very short, but its special biological effect determines its wide application. With the in-depth research and wide clinical application, some difficult diseases, such as severe burns, large-area trauma, peptic ulcer, severe corneal injury, etc., are expected to be cured quickly. Even some currently incurable diseases such as nerve damage, malignant tumors, AIDS, etc., may also be alleviated and restored through the use of EGF. It can be predicted that the application of EGF will bring a great leap forward in the research of life science in the future.

In recent years, research on EGF and its receptor (EGFR) has become one of the hotspots in biomedical research. For EGFR, an important target in tumor therapy, nanoparticles can be targeted by coupling the natural ligand EGF. EGF is a protein consisting of 53 amino acids, which has three disulfide bonds and many hydrophobic residues, all suitable for interacting with nanoparticles. Compared with antibodies or even antibody fragments, EGF is smaller in size, specific natural ligands, and less likely to trigger immune responses. Unfortunately, its use also has disadvantages, such as EGF is less available from human sources, it is expensive, it is difficult to control the orientation of EGF on the surface of nanoparticles by covalent linkage, and the EGF recognition ability caused by nonspecific adsorption of nanoparticles to EGF The drop of EGF may change the size and related characteristics of the whole nanoparticle after connecting with EGF.

The patent document with the publication number CN105884888A discloses the design method of "conformation engineering". One example is to select complementarity determining region (CDR) polypeptides from known natural antibodies and covalently link their two ends to gold nanoparticles , successfully reproducing the function of natural antibodies.

Contents of the invention

One of the objectives of the present invention is to overcome the limitations of the prior art that are limited to the grafting of natural antibody CDR polypeptide loop regions, and provide a polypeptide selected from a β-hairpin structural fragment derived from the natural ligand EGF.

The second object of the present invention is to provide grafting of the polypeptide fragment on the surface of gold nanoparticles, and by adjusting the density of the polypeptide on the surface of gold nanoparticles to realize the regulation of the correct spatial conformation of the polypeptide, to prepare a protein that can specifically bind EGFR artificial epidermal growth factor EGF.

The third object of the present invention is to provide the application of the artificial epidermal growth factor in the preparation of wound repairing medicine.

The fourth object of the present invention is to provide the application of the artificial epidermal growth factor in the preparation of medicaments for preventing pigmentation.

The fifth object of the present invention is to provide the application of the artificial epidermal growth factor in the preparation of reagents for specific detection of EGFR.

The sixth object of the present invention is to provide the artificial epidermal growth factor and the designed polypeptide as a functional unit targeting EGFR protein for application in imaging localization and drug delivery.

The seventh object of the present invention is to provide the application of artificial epidermal growth factor to specifically promote the proliferation activity of cells with medium and low expression (such as HeLa cells).

The eighth object of the present invention is to provide the application of the artificial epidermal growth factor to specifically inhibit the proliferation activity of cells with high expression of EGFR (such as MDA-MB-468 cells).

In order to achieve the above invention creation purpose, the present invention adopts the following technical solutions:

A polypeptide, characterized in that the polypeptide is any one of the following (A) or (B):

(A) polypeptide EBeta1 containing the amino acid sequence shown in Sequence 1;

(B) Under the condition that the key binding residues are not changed, the amino acid residue sequence shown in sequence 1 is replaced and/or deleted and/or added and/or extended by one or several amino acid residues (A ) derived polypeptides.

The above polypeptide is the amino acid sequence shown in SEQ ID NO.1.

An artificial epidermal growth factor, characterized in that the artificial epidermal growth factor utilizes two cysteines designed in the above-mentioned polypeptide to form two Au-S bonds on the surface of gold nanoparticles for anchoring, and adjusts the polypeptide The density on the surface of gold nanoparticles makes the polypeptide form a specific hairpin (β-hairpin) conformation. It is obtained by making the polypeptide form a specific β-hairpin conformation on the surface of gold nanoparticles.

The density range of the above-mentioned polypeptides on the surface of gold nanoparticles is: 0.2-2 strips/square nanometer surface area.

An application of the above-mentioned artificial epidermal growth factor in the preparation of wound repairing medicine.

An application of the above-mentioned artificial epidermal growth factor in the preparation of a medicament for preventing pigmentation.

An application of the aforementioned artificial epidermal growth factor in the preparation of reagents for specific detection of EGFR.

A kind of above-mentioned designed polypeptide and artificial epidermal growth factor are used as the functional unit targeting EGFR protein,

It is used in imaging positioning and drug delivery.

An application of the aforementioned artificial epidermal growth factor in specifically promoting the proliferation of HeLa cells.

An application of the aforementioned artificial epidermal growth factor in specifically inhibiting the proliferation of MDA-MB-468 cells.

Compared with the prior art, the present invention has the following obvious outstanding substantive features and significant advantages:

1. Breaking through the limitation that the original technology was only used for natural antibody fragments, the simulation of artificial ligand protein was carried out for the first time, and artificial EGF with normal physiological functions was successfully synthesized.

2. Breaking through the limitation that the original technology is only used for grafting loop fragments, the conformational engineering method is used for the first time to reconstruct the β-hairpin structure on the surface of nanoparticles.

3. The artificial EGF of the present invention can be used as a drug for repairing wounds and preventing pigmentation and other related diseases; artificial EGF can be used as a reagent for specific detection of EGFR; targeted functional units.

Description of drawings

Fig. 1 is the ultraviolet characterization of gold nanoparticles and artificial EGF in the preferred embodiment of the present invention.

Fig. 2 is the hydration particle size characterization of gold nanoparticles and artificial EGF in a preferred embodiment of the present invention.

Fig. 3 is a preferred embodiment of the present invention to regulate the conformation of the polypeptide and the activity of artificial EGF by adjusting the density of the polypeptide.

Fig. 4 is the hydration particle size characterization of the preferred embodiment of the present invention after the artificial EGF is uniformly mixed with sEGFR or BSA respectively.

Fig. 5 is a characterization of the specific binding performance between artificial EGF and sEGFR in a preferred embodiment of the present invention.

Fig. 6 shows the binding kinetics of artificial EGF and sEGFR measured by SPR in a preferred embodiment of the present invention.

Fig. 7 is a preferred embodiment of the present invention artificial EGF specifically promotes the proliferation of HeLa cells.

Fig. 8 is a preferred embodiment of the present invention artificial EGF specifically inhibits the proliferation of MDA-MB-468 cells.

Fig. 9 is the electron microscope characterization of the negatively stained sample of the combination of artificial EGF and sEGFR in Example 2 of the present invention.

Example 1

Below in conjunction with specific implementation example, above-mentioned scheme is described further, and preferred embodiment of the present invention is described in detail as follows:

In this embodiment, the design of polypeptide and artificial EGF is as follows

1. Design and synthesis of polypeptide fragments

Designed and synthesized polypeptide EBeta1, its 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 gold nanoparticles through two cysteines in the sequence, thus forming a specific β-hairpin conformation.

2. Preparation of artificial EGF

Referring to our previous "conformational engineering" method, the designed polypeptide sequence was grafted on the surface of gold nanoparticles to prepare artificial EGF. Firstly, 20 μL of 0.2 M trisodium citrate solution was added to the aqueous solution (6 mL) of gold nanoparticles to enhance the stability of gold nanoparticles. 2 mL of polypeptide solution (containing 2 mM sodium hydroxide) was added dropwise into the stirring gold nanoparticle aqueous solution, and stirring was continued at room temperature for 1 h. The polypeptide is grafted onto gold nanoparticles in the form of S-Au bonds through the sulfhydryl groups at both ends of its own sequence, thereby preparing artificial EGF.

3. Size characterization of gold nanoparticles before and after polypeptide grafting

Figure 1 is the ultraviolet spectrum characterization of gold nanoparticles and artificial EGF. The results show that after peptide modification of gold nanoparticles, the surface plasmon resonance absorption peak of gold nanoparticles has a red shift, which proves the successful grafting of peptides on the surface of gold nanoparticles. Figure 2 is the characterization of the hydrated particle size of gold nanoparticles and artificial EGF. The results show that the polypeptide was successfully grafted on the surface of gold nanoparticles, which significantly increased the hydrated particle size of gold nanoparticles.

4. Peptide density affects the biological activity of artificial EGF

We grafted different amounts of polypeptide EBeta1 on the surface of gold nanoparticles through gradient screening, and measured the change in the binding strength between artificial EGF and sEGFR by SPR technology, as shown in Figure 3. Grafting the polypeptide EBeta1 on the surface of the gold nanoparticles, the preferred density is: 0.2-2 strips/square nanometer surface area of the gold nanoparticles, and the polypeptide fragment is in a suitable conformation at this time, which endows the artificial EGF with a stronger ability to bind sEGFR.

5. Characterization of specific binding of artificial EGF (hereinafter referred to as AuNP-EBeta1, in order to compare with AuNP-EBeta1m which has only one cysteine but no activity) and sEGFR

We selected AuNP-EBeta1 to mix uniformly with sEGFR or BSA respectively, and determined whether artificial EGF specifically binds to sEGFR by measuring the hydrated particle size distribution of the two mixed samples. As shown in Figure 4, the hydrated particle size distribution of AuNP-EBeta1 mixed with BSA was not significantly different from that before mixing, indicating that artificial EGF did not combine with BSA. The hydrated particle size distribution of AuNP-EBeta1 mixed with sEGFR changed significantly, and the measured hydrated particle size increased. It preliminarily shows that artificial EGF can specifically combine with sEGFR. In order to prove more strongly that the ability of artificial EGF to specifically bind sEGFR is based on the folding of the polypeptide EBeta1 on the surface of gold nanoparticles into a specific conformation, we selected three proteins BSA, IgG and Fib that are abundant in serum as As a control protein, AuNP-EBeta1, AuNP-EBeta1m, and corresponding concentrations of free polypeptides (EBeta1, EBeta1m (the difference between the sequence and EBeta1 is only one cysteine)) were compared with sEGFR, AuNP-EBeta1m, and sEGFR immobilized on the CM5 chip channel by SPR technology. Binding strength of BSA, IgG and Fib. As shown in Figure 5, the artificial EGF did not bind strongly to the three proteins that are abundant in the blood, indicating that the binding between the artificial EGF and sEGFR is specific.

6. Characterization of the binding strength between artificial EGF and sEGFR

In order to prove that the synthetic artificial EGF and sEGFR have strong specific binding, we measured the binding kinetics of artificial EGF and sEGFR. sEGFR maintained a low coupling level, and the binding kinetics data of artificial EGF and sEGFR were suitable for fitting with a 1:1 model. The kinetic binding constant K _D =6.7×10 ^{- 11} M, k _on =2.2×10 ⁶ M ^-1 s ^-1 and k _off =1.5×10 ⁴ s ^-1 after fitting the interaction between artificial EGF and sEGFR. It can also be seen from Figure 6 that the artificial EGF can hardly be eluted from the chip during the elution stage, so the signal of the SPR spectrum in the elution stage is almost a flat line. This also shows that artificial EGF has a strong ability to bind sEGFR.

7. Biological activity of artificial EGF at the cellular level

A certain concentration of artificial EGF can also cause proliferation or apoptosis of corresponding tumor cells. We selected HeLa cells with medium and low expression of EGFR and MDA-MB-468 cells with high expression of EGFR as the research objects to explore whether artificial EGF can successfully replace the function of EGF at the cellular level. Figure 7 shows that adding 20nMEGF or 40nMAuNP-EBeta1 has basically the same effect on the proliferation of HeLa cells; Figure 8 shows that adding 20nMEGF or 40nMAuNP-EBeta1 has basically the same effect on the proliferation of MDA-MB-468 cells. These two results strongly proved that artificial EGF successfully replaced the biological function of EGF.

Example 2

In this example, without changing the key binding residues, the polypeptide EBeta1 was simply modified to obtain the polypeptide EBeta2, and an artificial EGF (AuNP-EBeta2) with EGF function was synthesized.

1. Design and synthesis of polypeptide fragments

Designed and synthesized polypeptide EBeta2, its sequence is as follows:

Ser-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Val-Gly

The synthesis and preparation of artificial EGF (AuNP-EBeta2) is the same as that of Example 1.

2. Characterization of specific binding between artificial EGF (AuNP-EBeta2) and sEGFR

In this implementation case, we directly characterized the specific binding function of artificial EGF and sEGFR through the electron microscope pictures of the combination of AuNP-EBeta2 and sEGFR. We negatively stained the mixed samples of artificial EGF and sEGFR, and further used transmission electron microscopy to observe the combination of artificial EGF and sEGFR to determine the binding ratio between the two. As shown in Figure 9, we can see from the figure that the particle size of sEGFR is about 20nm (the molecular weight of sEGFR is 110kDa, the size of sEGFR is about 20nm in theory), and there is an artificial EGF around almost every protein. It perfectly reflects the phenomenon that artificial EGF binds specifically to sEGFR strictly according to the ratio of 1:1. The specific combination with sEGFR is the molecular basis for EGF to exert cell activity and related biomedical applications.

In summary, at the molecular level, the changes in the binding strength and binding kinetics of artificial EGF and sEGFR were measured by SPR technology, which proved that artificial EGF and sEGFR had a very strong specific binding. At the cellular level, the ability of artificial EGF to bind tumor cell epidermal growth factor receptors has been proved, and it has the same cell biological activity as natural EGF, indicating that artificial EGF can replace natural EGF in the biomedical field. The molecular mechanism of artificial EGF cell activity and its biomedical application is its specific combination with sEGFR. Negative staining electron microscope photos prove that artificial EGF that can specifically bind sEGFR in a 1:1 model can also be synthesized by modifying the polypeptide without changing the key residues of the polypeptide we designed.

The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and various changes can also be made according to the purpose and principle of the present invention. The changes, modifications, substitutions, combinations or simplifications should be equivalent replacement methods, as long as they meet the purpose of the invention, as long as they do not deviate from the technical principles and inventive concept of the polypeptide, artificial EGF and their application of the present invention, they all belong to this invention. protection scope of the invention.

sequence listing

<110> Shanghai University

<120>An artificial epidermal growth factor and designed polypeptide and its application

<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 (3)

1. An artificial epidermal growth factor, characterized in that, utilizes two cysteines designed in the polypeptide to form two Au-S bonds on the surface of the gold nanoparticle to anchor, and by adjusting the polypeptide to form two Au-S bonds on the surface of the gold nanoparticle The density of the polypeptide is obtained by forming a specific β hairpin, that is, β-hairpin conformation; The polypeptide is the polypeptide EBeta1, and its sequence is as follows: Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Val The designed polypeptide EBeta1 is immobilized on the surface of gold nanoparticles through two cysteines in the sequence; the polypeptide is the amino acid sequence shown in SEQID NO.1; Or, the polypeptide is the polypeptide EBeta2, and its sequence is as follows: Ser-Val-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Val-Gly. 2. The artificial epidermal growth factor according to claim 1, characterized in that the density range of the polypeptide on the surface of gold nanoparticles is: 0.2-2 strips/square nanometer surface area. 3. an application of artificial epidermal growth factor according to claim 1, comprising at least one purposes in following a-d: a. Application in preparation of repairing wound medicine; b. Application in the preparation of anti-pigmentation medicaments; c. Application in the preparation of specific detection EGFR reagents; d. Application in the preparation of carriers for imaging localization and drug delivery targeting EGFR.

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