Novel collagen functional variant amino acid and functional application thereof

Abstract

The invention discloses a novel collagen functional variant amino acid and functional application thereof, and relates to the technical field of collagen variants, the amino acid of the collagen functional variant is a COL fragment which is extracted from full-length human COL1 and has the length of 30 amino acids, and a collagen variant C109 synthesized by polypeptide has the structural formula shown in the description. It is verified that the variant has a self-assembly structural property and a good biological activity function, and the amino acid sequence of the collagen functional variant is GLPGPSGEPGKQGPSGASGERGPPGPMGPP (SEQ ID No. 1); the polypeptide formed by the collagen functional variant provided by the invention has the same self-assembly capability as full-length collagen, has better biological activity, is non-toxic, can promote osteoblast differentiation, and supports tissue regeneration at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen variants, in particular to a novel collagen functional variant amino acid and its functional application. Background Art

[0002] Collagen is an extracellular matrix protein present throughout the human body and has multiple important biological functions in normal physiology. Collagen can self-assemble into striated fibrils, which not only provide support for cell growth but are also responsible for the mechanical elasticity of connective tissue. Based on differences in amino acid sequence, function, and protein structure, 29 different collagens (I–XXIX) have been identified. Despite the expanding market demand for collagen in the fields of biomedicine, cosmetics, and health foods, the traditional method of extracting collagen from animals is expensive, inefficient, and unsuitable to meet the growing industrial demand. Therefore, various expression systems (including insects, animal cells, plants, bacteria, and yeast) have been developed to synthesize recombinant collagen. With increasing usage, the collagen market is expected to reach US$6.63 billion by 20254. Although the synthesis of recombinant collagen has achieved good commercialization, further research and expansion are needed to improve biosafety, yield, and functionality. In addition, full-length collagen is poorly soluble and has low yields, so it is necessary to identify new functional collagen fragments that can be commercially produced for use as biomaterials.

[0003] Existing collagen has not been used in the field of osteoblast differentiation. When synthesizing collagen variants, the inventors found that the amino acids of the collagen variants synthesized by polypeptides can significantly increase the rate of osteoblast differentiation and are non-toxic. Therefore, it is necessary to propose a new type of amino acids of collagen functional variants to expand the application of collagen variants in the field of osteoblast differentiation. Summary of the invention

[0004] The purpose of the present invention is to provide a novel collagen functional variant amino acid and its functional application to solve the problems raised in the above background technology.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions: amino acids of a novel collagen functional variant, the amino acids of the collagen functional variant are collagen variant C109 synthesized by polypeptide, the amino acids are COL fragments with a length of 30 amino acids extracted from full-length human COL1, and it has been verified that it can have a triple helix structure, can be observed to form fibers under a transmission electron microscope, and can be observed to form a hydrogel at low temperature. And it has good biological activity, and the amino acid sequence of the collagen functional variant is GLPGPSGEPGKQGPSGASGERGPPGPMGPP (SEQ ID No. 1).

[0006] The peptide synthesis step of the collagen variant generally adopts solid-phase peptide synthesis (SPPS), which is a commonly used method for synthesizing peptides and generally includes the following main steps: S1. Prepare synthetic resin: Select a suitable synthetic resin as the solid phase support. The resin usually contains a protecting group that can react with the amino group of the amino acid.

[0007] S2. Removal of protecting groups: Prior to synthesis, the protecting groups on the resin are removed to enable binding to the first amino acid.

[0008] S3. Accession of the first amino acid: The first amino acid is reacted with the resin to form a peptide bond. Usually, the reaction is carried out under certain conditions (such as appropriate temperature and time).

[0009] S4. Addition of protecting groups: protect the inserted amino acid. Commonly used protecting groups are Fmoc (fluorinated methylamino) or Boc (tert-butyloxycarbonyl).

[0010] S5. Repeated insertion of amino acids: Repeat steps S2 to S4 to insert subsequent amino acids into the polypeptide chain one by one until the synthesis is completed.

[0011] S6. Removal of protecting groups: After the synthesis is completed, all amino acid protecting groups are removed, usually using acid or alkaline solutions.

[0012] S7. Release the polypeptide from the resin: Cut the synthesized polypeptide from the resin by appropriate chemical methods. A commonly used method is acid hydrolysis.

[0013] S8. Purification and analysis: The synthesized peptides are purified using methods such as high performance liquid chromatography (HPLC), and their structures are analyzed and confirmed using techniques such as mass spectrometry.

[0014] S9. Validation and storage: The purified peptide is further validated and analyzed to ensure its correctness and purity, and finally stored in an appropriate environment.

[0015] This method is widely used in peptide synthesis due to its high efficiency and flexibility, especially in fields such as biochemistry and drug development.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are: the amino acids of the collagen functional variant proposed by the present invention have better biological activity, are non-toxic, can promote osteoblast differentiation, and support tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 , Graph showing the cell proliferation rate of L929 cells supplemented with 2 mg / mL collagen-like samples and commercial rat tail collagen I.

[0018] Figure 2 , Schematic representation of VCL mRNA levels in MC-3T3 cells cultured with C109 variants, commercial rat tail COL1, and BSA.

[0019] Figure 3 , Schematic representation of ACTN mRNA levels in MC-3T3 cells cultured with C109 variants, commercial rat tail COL1, and BSA.

[0020] Figure 4 , CD spectrum of C109 sample obtained at 4°C.

[0021] Figure 5 , By heating the system from 0 to 70°C, the melting curve of the C109 sample was obtained, and the wavelength schematic diagram of 220nm was recorded.

[0022] Figure 6 , C109 sample observed under transmission electron microscope TEM.

[0023] Figure 7 , C109 sample observed under scanning electron microscope (SEM).

[0024] Figure 8 The samples were prepared to 1 mg / mL and 5 mg / mL, respectively, and incubated at 4°C, showing a schematic diagram of hydrogel formation. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The synthesized collagen variant C109 solution was subjected to CD analysis at low temperature to detect the formation of the triple helix structure of collagen, which can further support higher-order assembly. It was then observed under transmission electron microscopy and scanning electron microscopy to see whether higher-order fibrous structures and collagen sponge structures were formed. Finally, it was incubated at 4°C to see whether it could present a hydrogel state.

[0027] The collagen variant C109 sample was freeze-dried to prepare COL sponge and supplemented with 5 mg / mL MC3T3-E1 (mouse embryonic osteoblasts, a multipotent stem cell that can differentiate into a variety of cell types, such as osteoblasts, chondrocytes, muscle cells, etc. Under osteogenic differentiation conditions, MC3T3-E1 mouse embryonic osteoblasts can express osteoblast-specific genes and form calcified bone-like structures). Cell proliferation and differentiation rates were detected and compared with commercial rat tail COL1 (rat tail type I collagen, which can be used as a thin layer coating on the surface of tissue culture to promote cell adhesion and proliferation, and can also be used to prepare gels, which helps in vitro culture and enhances the expression of cell-specific morphology and function) as a positive control and BSA (bovine serum albumin, also known as the fifth component, is an albumin in bovine serum. Bovine serum albumin can be used as a protein concentration standard for protein quantification in the Bradford assay) as a negative control.

[0028] Example 1 Characterization of structural properties of collagen variants The prepared samples were tested as follows: (1) Circular dichroism (CD) detection The collagen variant prepared in Example 3 was prepared into a collagen-like solution with a concentration of 0.2 mg / mL in PBS (pH 7.5) and incubated at 4°C for 12 h before CD measurement. CD spectra were recorded at 4°C using a Photophysics Chirascan instrument equipped with a Peltier temperature controller (Model 110-OS; Hellma, Shanghai, China) and a quartz cuvette with an optical path length of 1 mm (Model 110-OS; Hellma). The scanning wavelength range was 190 - 250 nm. The change in ellipticity at 220 nm during heating at 0 ~ 70°C and 1°C / min was recorded, and the Tm value was calculated based on the melting curve.

[0029] CD was performed on the expressed protein to confirm triple helix formation. The sample purified from E. coli showed a positive peak at 220-222 nm, proving that it can self-assemble into a triple helix structure.

[0030] (2) Transmission electron microscopy (TEM) detection The collagen-like PBS (pH 7.5) prepared in Example 3 was formulated into a collagen-like solution at a concentration of 1 mg / mL and incubated at 4°C. The obtained solution was loaded onto a copper grid, allowed to absorb for 1 minute, and the excess solvent was removed using filter paper. The samples were negatively stained with 0.75% phosphotungstic acid before imaging using a Hitachi H-7650 electron microscope (Hitachi, Tokyo, Japan). The sample width was evaluated using ImageJ (https: / / imagej.net / downloads). Sample E3 was able to form nanofibers detected at 100 to 200 nm by transmission electron microscopy (TEM) after 12 hours of incubation.

[0031] (3) Scanning electron microscope (SEM) detection The morphology and microstructure of the collagen-like sponges were characterized using a SEM SU8220 (Hitachi). The collagen-like gel was prepared into a sponge-like form by freeze-drying. The dried sample was loaded onto a SEM disk and imaged.

[0032] Sample E3 was found to be porous by scanning electron microscopy (SEM) from a collagen-like sponge prepared by freeze-drying.

[0033] Example 2 Preparation of hydrogel using collagen The formation of collagen hydrogels is very important for the development of biocompatible materials for cell adhesion and proliferation. The formation of a triple helix structure is the basis, followed by the formation of nanofibers and finally the formation of a gel. Here, the gelation test was carried out on the samples that could form a triple helix, and the prepared samples were dissolved in PBS solution at different concentrations (e.g., 1 mg / mL, 5 mg / mL) and incubated at 4°C. After incubation at 4°C for 8 hours, the samples could form a hydrogel. The storage modulus G' and loss modulus G" at low deformation amplitude were measured to determine the elasticity and viscosity of the hydrogel. For the above samples, the storage modulus was more than twice that before treatment, indicating that all tested samples formed a hydrogel. The hydrogel is fragile and can be easily converted into a liquid phase by shaking or increasing the temperature. Example

[0034] Cytotoxicity assay: used to detect the toxicity of collagen variants, including the following steps: Step 1: Collagen-like and commercial rat collagen I (Merck, Shanghai, China) with a concentration of 1 mg / mL were treated by freeze-drying technology at -60 °C and a pressure of 0.01-0.1 MPa for 24 h to prepare collagen sponges for L929 fibroblast (ATCC, China) culture to evaluate their toxicity.

[0035] Step 2: The cell culture medium contained 10% (v / v) fetal bovine serum (Ausbian, Australia) and 1% (v / v) penicillin-streptomycin.

[0036] Step 3: L929 cells were cultured for 48 hours and transferred to a new culture plate at a cell density of 104 cells per well.

[0037] Step 4: Allow cells to adhere for 12 hours, then add 1 mL of cell culture medium containing 2 mg / mL collagen sponge.

[0038] Step 5: The cells were cultured for 48 h, centrifuged, the solution was discarded, and the cell number was measured using a CCK-8 kit (Sangon).

[0039] Step 6: The proliferation rate of L929 cells was evaluated by using the following cell counting formula: Proliferation rate (%) = (total number of cells after culture) / (total number of cells before culture). The cell proliferation rate ranged from 306% to 347% ( Figure 1 ), indicating that all tested samples were non-toxic. Example

[0040] Cell differentiation experiment: used to detect the promoting effect of collagen variants on cell differentiation. The ability of collagen functional variants in promoting osteoblast differentiation is tested according to the following method, including the following steps: Step 1: Commercial rat tail collagen I (Merck, Shanghai, China) was used as a positive control. Gene expression levels were measured by real-time quantitative PCR (RT-qPCR).

[0041] Step 2: Total RNA was extracted from L929 cells using TRIzol reagent (Invitrogen).

[0042] Step 3: RNA was reverse transcribed into cDNA using the One Step RT-qPCR kit (Sangon) and the QuantStudio 3 RT-qPCR instrument (Thermo Fisher, Shanghai, China).

[0043] Step 4: Gene expression level GAPDH was used as a reference, and the results showed that the collagen variants showed the ability to upregulate VEGF for collagen I, and the mRNA levels of the three given collagen variants were higher than the mRNA levels using BSA. These results indicate that the designed type II collagen variants can promote cell differentiation.

[0044] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An amino acid of a novel collagen functional variant, characterized in that: The amino acids of the collagen functional variant include the sequence shown in SEQ ID No.

1.

2. The amino acid of a novel collagen functional variant according to claim 1, characterized in that: The amino acids of the collagen functional variant are collagen variants synthesized by polypeptides.

3. A method for preparing amino acids of a novel collagen functional variant according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Prepare synthetic resin: Select a suitable synthetic resin as the solid phase support. The resin contains a protective group that can react with the amino group of the amino acid; S2. Removal of protecting groups: Before synthesis, the protecting groups on the resin are removed to enable binding to the first amino acid; S3, access to the first amino acid: react the first amino acid with the resin to form a peptide bond; S4, adding protecting groups: protecting the inserted amino acids; S5, repeated insertion of amino acids: repeating steps S2 to S4, inserting subsequent amino acids into the polypeptide chain one by one until the synthesis is completed; S6. Removal of protecting groups: After the synthesis is completed, the protecting groups of all amino acids are removed; S7. Release the peptide from the resin: cleave the synthesized peptide from the resin by appropriate chemical methods; S8. Purification and analysis: Purify the synthesized peptide using high performance liquid chromatography (HPLC) and analyze and confirm its structure using mass spectrometry; S9. Validation and storage: The purified peptide is further validated and analyzed to ensure its correctness and purity, and finally stored.

4. The method for preparing amino acids of a novel collagen functional variant according to claim 3, characterized in that: In S4, the protecting group is Fmoc fluorinated methylamino or Boc tert-butyloxycarbonyl.

5. The method for preparing amino acids of a novel collagen functional variant according to claim 3, characterized in that: In the above S6, an acidic or alkaline solution is used to remove the protecting group.

6. The method for preparing amino acids of a novel collagen functional variant according to claim 3, characterized in that: In the above S7, the method for releasing the polypeptide is acid hydrolysis.

7. Use of the amino acid according to any one of claims 1-2 in the fields of food, biomedicine and tissue engineering.

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