High adhesion polypeptide and application thereof

Abstract

The application discloses a high-adhesion polypeptide and application thereof. The high-adhesion polypeptide comprises Dopa having adhesion performance, and tyrosine and tryptophan having cohesion performance. The high-adhesion polypeptide has high-strength adhesion and high adsorption capacity, and can effectively bond various materials. The cationic polypeptide has the characteristics of good biocompatibility, high safety and strong adhesion, and can be applied to the fields of biological manufacturing and medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomimetic adhesion, and in particular relates to a cationic polypeptide designed based on the structural characteristics of Mfp and its application. Background Art

[0002] Mfp is a multiprotein complex secreted by the marine organism mussel (Mussel sphaeroides). It enables mussels to firmly adhere to various substrates. Mfp-inspired peptides have garnered widespread attention as adhesive materials, and Mfp-inspired peptide analogs are considered ideal materials for mediating adhesion to a variety of substrates.

[0003] The study found that the microscopic adhesion of adhesive proteins based on Mfp and other natural adhesive substances is not particularly strong. In atomic force microscopy (AFM) experiments, it was found that the maximum adhesion force of a colloidal probe with a glass bead with a radius of 5 μm to the tandem polypeptide of mussel adhesive protein 5 (Mfp5) oligomers was 201±36 nNμm. 1 The microscopic adhesion forces of the short model peptide inspired by mussel adhesion protein 3 (Mfp3) and the Mfp3 variant protein were 5.17-12.85 mN m -1 and 37.68~95.53mNm -1 The test substance was combined with the colloidal probe by aminopropyltriethoxysilane and glutaraldehyde. It was found that the adhesion force of the white-spine barnacle cp19k expressed by Escherichia coli to the mica substrate could reach 0.88±0.24mJ m -2 The adhesion of Cell-Tak to its surface can reach 1.54±0.59mJ m -2 Therefore, analyzing the sequence characteristics of Mfp and redesigning a polypeptide complex with stronger adhesion is a difficult problem that needs to be solved by those skilled in the art. Summary of the Invention

[0004] The purpose of the present invention is to provide a high-adhesion polypeptide and its application.

[0005] A high-adhesion polypeptide with the amino acid sequence KYGGKWGGK-Dopa-K-Dopa-K-Dopa-GGKWGGK-Dopa-K-Dopa-K-Dop aGGKWGGKY, where Dopa is L-3,4-dihydroxyphenylalanine. This polypeptide was prepared by Nanjing Peptide Valley Biotechnology Co., Ltd., CAS: TG-LG-11810.

[0006] The polypeptide with high adhesion comprises Dopa with adhesion performance, and tyrosine and tryptophan with cohesion performance.

[0007] The polypeptide with high adhesion has high adhesion strength and high adsorption capacity.

[0008] Application of the high-adhesion polypeptide in preparing medical viscose.

[0009] Application of the high-adhesion polypeptide in preparing biochips.

[0010] The beneficial effects of the present invention are as follows: The highly adhesive polypeptides of the present invention exhibit high adhesion and adsorption capacity, and can adhere to the surfaces of various organic and inorganic substances. The cationic polypeptides of the present invention with these properties are applicable to fields such as biomanufacturing and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Adhesion pressure analysis of high-adhesion peptides with different curing times.

[0012] Figure 2 Atomic force spectrometry analysis of peptides with high adhesion.

[0013] Figure 3 For analysis of the adsorption capacity of peptides with high adhesion. DETAILED DESCRIPTION

[0014] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0015] Example 1: Adhesion pressure analysis of peptides with high adhesion at different curing times

[0016] 1. Prepare the high-adhesion polypeptide sample into a 5% polypeptide solution with ultrapure water and store at 4°C for 24 hours.

[0017] 2. Add 1.4 μl of peptide solution to the surface of a 1 cm × 1.5 cm glass slide, quickly cover it with another glass slide of the same size and fix it with a clamp. Curing was carried out for 0.5 h, 12 h and 24 h respectively.

[0018] 3. The shear pressure at different curing times was measured by a tensile strength tester. The results showed that the adhesion pressure increased with the extension of curing time, and the maximum adhesion pressure could reach 158.87kPa ( Figure 1 ).

[0019] Example 2: Microscopic Adhesion Analysis of Polypeptides with High Adhesion

[0020] The microscopic adhesion of cationic peptides to surfaces was measured using atomic force microscopy. The cationic peptides were prepared as a 0.5% aqueous solution, and the probe applied a pressure of 2 nN to the sample. The surface adhesion test was conducted in air at a room temperature of 25°C and a relative humidity of 28%. The experimental instrument was a German Bruker FASTSCANBIO atomic force microscope (AFM). The probe used was a FASTSCAN-C probe with a triangular Si3N4 tip, a 5 nm radius of curvature, a spring constant of 0.8 Nm-1, and a resonant frequency of 300 kHz.

[0021] The results showed that the average microscopic adhesion force of the peptide with high adhesion can reach 9.32nN, which is higher than most adhesive materials reported in the literature ( Figure 2 ).

[0022] Example 3: Analysis of the adsorption capacity of peptides with high adhesion

[0023] Sample preparation:

[0024] 1. Prepare PBS buffer at pH 6.0 and sterilize by autoclaving.

[0025] 2. Prepare a peptide solution with a high adhesion strength using PBS to a concentration of 1 mg / ml and store at 4°C.

[0026] Measurement steps:

[0027] 1. Place the injection tube in ultrapure water, turn on the peristaltic pump, set the flow rate to 50μl / min, start injection and observe the baseline until it stabilizes.

[0028] 2. Acquisition-restart measurement: After stabilization in air for 5 minutes and good frequency doubling coincidence, pH 6.0 PBS buffer was introduced. The equilibration time was set to 10-15 minutes from the time the liquid response signal was observed.

[0029] 3. Acquisition-restart measurment: If there is no significant difference in the doublet frequency, start the test. The baseline equilibration time must not be less than 10 minutes.

[0030] 4. The peptide solution is adsorbed on the chip until the baseline is stable.

[0031] 5. Acquisition-stop stores the data, stops the pump and adjusts the flow rate to 300 μl / min to flush the system.

[0032] The results showed that the adsorption capacity of peptides with high adhesion on the gold chip can reach 265ng / cm 2 (Figure 3).

[0033] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A high-adhesion polypeptide, characterized in that: Its amino acid sequence is KYGGKWGGK-Dopa-K-Dopa-K-Dopa-GGKWGGK-Dopa-K-Dopa-K-Dopa-GGKWGGKY, where Dopa is L-3,4-dihydroxyphenylalanine. The microscopic adhesion force of the high-adhesion polypeptide is 9.32 nN, and the adsorption capacity on the gold chip is 265 ng / cm 2 .

2. The high-adhesion polypeptide according to claim 1, characterized in that The high-adhesion polypeptide comprises Dopa with adhesive properties, and tyrosine and tryptophan with cohesive properties.

3. Use of the high-adhesion polypeptide according to claim 1 in the preparation of a biochip.

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