A hydrogel-state human recombinant collagen self-assembly polymer and its preparation method and application

Abstract

The present invention relates to the field of biochemical technology, and more specifically to a hydrogel-state human recombinant collagen self-assembly polymer, a preparation method, and an application thereof. The human recombinant collagen of the present invention is composed of a stacked short sequence and a human collagen short sequence as a repeating unit repeated 8 times to form a specific amino acid sequence structure. The stacked short sequence and calcium ions (Ca 2+ ) form chelates, thereby enabling self-assembly between human recombinant collagen molecules to form stable hydrogel-like human recombinant collagen self-assembly aggregates. The preparation method described herein does not use chemical crosslinkers, avoiding potential toxicity and side effects. The resulting hydrogel-like human recombinant collagen self-assembly aggregates can mimic the body's own collagen to the greatest extent possible, reducing the risk of immune response, increasing biosafety, ensuring biocompatibility, and expanding its application prospects in the field of medical aesthetics.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to a hydrogel-state human recombinant collagen self-assembly polymer and a preparation method and application thereof. Background Art

[0002] Collagen is a structural protein widely found in animal connective tissue, accounting for approximately 30% of the total protein content in the human body. It is a major component of the extracellular matrix, supporting and connecting tissues and found extensively in skin, bone, cartilage, ligaments, and tendons. Collagen's highly regular triple-helical structure gives it excellent mechanical properties and biocompatibility. Collagen began to be used in biomedicine in the 1950s and 1960s, initially primarily in wound dressings and hemostatic materials. Entering the 21st century, collagen's applications have become more extensive and in-depth, with the development and application of new technologies enhancing the performance of collagen materials. The development of collagen as a biomedical material has evolved from initial extraction and purification to initial applications, to tissue engineering and regenerative medicine, and finally to genetic engineering and recombinant collagen, as well as the continuous advancement of new material development and cutting-edge applications. This process demonstrates the tremendous driving force behind scientific and technological progress and the cross-disciplinary integration of multiple disciplines. With continuous technological innovation and the expansion of application areas, the prospects for collagen's application in biomedicine will continue to expand.

[0003] As a versatile biomedical material, collagen is widely used in the biomedical field due to its excellent biocompatibility, biodegradability, and low immunogenicity. Its applications cover a wide range of fields, including tissue engineering, wound dressings, cosmetic surgery, drug delivery, orthopedics and dentistry, neural repair, artificial organs, and medical devices. In particular, in the field of cosmetic surgery, collagen is used as an injectable filler for facial contouring, wrinkle filling, and lip augmentation, with excellent biocompatibility and filling effects. With the continuous advancement of science and technology, the performance and application range of collagen materials will be further expanded, bringing more innovation and development to the medical and health field. However, traditional collagen materials have some significant limitations in their application, which affect their clinical effectiveness and wide application.

[0004] First, collagen's lack of stability is one of the main challenges in its application. Natural collagen molecules are susceptible to enzymatic degradation and degradation in the body, making it difficult to maintain their long-term effectiveness in tissue augmentation. This degradation not only limits the collagen's lifespan but can also trigger adverse reactions in local tissues. Therefore, improving collagen's stability and ensuring its long-term survival in the body has become a key research topic.

[0005] Secondly, the insufficient mechanical properties of collagen also limit its application in high-stress environments. Collagen materials are prone to deformation when subjected to external pressure or mechanical stress, and their insufficient resilience makes it difficult for the material to return to its original shape. This lack of mechanical properties not only affects the mechanical properties of collagen, but may also affect its biological functions in the body, such as affecting cell attachment, growth, and differentiation. Therefore, enhancing the mechanical strength and elastic modulus of collagen so that it can maintain structural stability and functional integrity under high-stress conditions is the key to improving its application performance.

[0006] In response to the above problems, research in recent years has focused on improving the performance of collagen through chemical cross-linking, physical modification and composite materials. The chemical cross-linking method introduces cross-linking agents between collagen molecules to form covalent bonds, thereby improving its stability and mechanical properties. However, the use of chemical cross-linking agents may introduce toxicity and affect the biocompatibility of the material. The physical modification method improves the stability and mechanical properties by changing the physical state of collagen, but these methods often require complex processes and equipment. The composite material method improves its performance by compounding collagen with other biomaterials, such as hyaluronic acid, chitosan, etc. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the existing technology, especially the problems involving insufficient stability and mechanical properties of collagen, and to provide a hydrogel-state human recombinant collagen self-assembly polymer and its preparation method and application.

[0008] In order to solve the above technical problems, the present invention discloses a hydrogel-state human recombinant collagen self-assembly polymer and its preparation method and application. A stacking short sequence (amino acid sequence) is combined with a human collagen amino acid short sequence to form a human recombinant collagen, wherein the stacking short sequence is GKPGTPGPAGAPGKPGTP. The human recombinant collagen is dissolved in pure water, and a solution containing calcium ions is slowly added to the human recombinant collagen solution, wherein the human recombinant collagen and calcium ions (Ca 2+ ) to form a chelate, thereby causing the human recombinant collagen molecules to self-assemble to form a stable human recombinant collagen self-assembled polymer (hydrogel form). The specific technical solution is as follows:

[0009] A human recombinant collagen protein, composed of a stacked short sequence and a human collagen short sequence as a repeating unit, repeated 8 times;

[0010] Wherein, the stacked short sequence is composed of a GXY triplet amino acid sequence in human collagen, and the human collagen is any one or more of type I, type III, type IV, type V, type VII and type XVII human collagen; preferably, the present invention conducts a technical literature survey on the amino acid sequences of type I, type III, type IV, type V, type VII and type XVII human collagen, and digs out four GXY triplet amino acid sequences of GKP, GTP, GPA and GAP from the above six human collagen amino acid sequences, combines them, and combines them with the human collagen amino acid short sequence to carry out actual protein expression and preparation of self-assembled polymers, and finally determines that the stacked short sequence is GKPGTPGPAGAPGKPGTP (SEQID No.4).

[0011] The human collagen short sequence is composed of consecutive (GXY) in the α1 chain of type III human collagen 10 The amino acid sequence of

[0012] Wherein G is a glycine residue, and X or Y represents any amino acid residue.

[0013] Wherein, the amino acid sequence of the human recombinant collagen is shown as SEQ ID No.1.

[0014] In a second aspect, the present invention provides a nucleic acid molecule encoding the human recombinant collagen described in the first aspect.

[0015] Wherein, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.2 or SEQ ID No.3.

[0016] In a third aspect, the present invention provides a method for preparing the human recombinant collagen described in the first aspect, specifically: amplifying the nucleic acid molecule encoding the human recombinant collagen, cloning it into a plasmid vector to obtain a recombinant vector, introducing the recombinant vector into a host strain for expression, and then isolating and purifying it.

[0017] Wherein, the host strain is Pichia pastoris, preferably Pichia pastoris GS115.

[0018] In a third aspect, the present invention provides a hydrogel-state human recombinant collagen comprising the human recombinant collagen described in the first aspect.

[0019] In a fourth aspect, the present invention further provides a method for preparing the hydrogel-state human recombinant collagen described in the third aspect, specifically comprising: preparing the human recombinant collagen into a human recombinant collagen aqueous solution, mixing it with a calcium ion solution, and allowing it to stand and washing to obtain the hydrogel-state human recombinant collagen.

[0020] The concentration of the human recombinant collagen aqueous solution is 20-60 g / L; the calcium ion solution is a 40-120 mM calcium chloride aqueous solution; preferably, the concentration of the human recombinant collagen aqueous solution is 40 g / L; the calcium ion solution is an 80 mM calcium chloride aqueous solution.

[0021] The mixing volume ratio of the human recombinant collagen aqueous solution and the calcium ion solution is 4-7:3-6; preferably, the volume ratio is 1:1.

[0022] In the mixing, preferably, the calcium ion solution is added to the human recombinant collagen aqueous solution at a flow rate of 0.5-5 mL / min; more preferably, the flow rate is 1 mL / min.

[0023] The said standing temperature is 2-10°C and the standing time is 6-18 hours, preferably 4°C for 12 hours.

[0024] More preferably, after standing, the formed hydrogel is soaked in pure water for 3 hours and rinsed with pure water 3 times to obtain the hydrogel.

[0025] More preferably, the preparation method is as follows: without stirring, 2+ ) solution was slowly added to the human recombinant collagen solution along the container wall at a volume ratio of 1:1 at a flow rate of 1 ml per minute. The solution was allowed to stand at 4°C to form a hydrogel. The remaining solution was then poured out of the container and the resulting hydrogel was soaked in pure water for 3 hours and rinsed three times with pure water to remove free calcium ions, resulting in a self-assembled aggregate of human recombinant collagen (hydrogel-like human recombinant collagen).

[0026] In a fifth aspect, the present invention further provides use of the hydrogel-state human recombinant collagen described in the third aspect as a subcutaneous filler.

[0027] Beneficial effects:

[0028] The present invention provides a hydrogel-state human recombinant collagen self-assembly polymer and its preparation method. The human recombinant collagen is composed of stacked short sequences and human collagen amino acid short sequences as repeating units, forming a specific amino acid sequence structure. The stacked short sequences and calcium ions (Ca 2+ ) form a chelate, thereby allowing the human recombinant collagen molecules to self-assemble, and forming a stable recombinant human collagen self-assembled polymer (hydrogel form). No chemical cross-linking agent is used in the preparation process described in the present invention, which avoids the potential toxicity and side effect risks brought by chemical cross-linking agents, increases biosafety, and ensures biocompatibility.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The stacked short sequence is GKPGTPGPAGAPGKPGTP, which is composed of multiple GXY type collagen triplet amino acid sequences. These GXY type triplet amino acid sequences are all taken from the existing GXY type triplet amino acid sequences in the amino acid sequences of type I, type III, type IV, type V, type VII and type XVII human collagen. By repeatedly combining the stacked short sequence with the continuous human collagen amino acid short sequence, a specific human recombinant collagen amino acid sequence is formed, thereby ensuring its high similarity to human collagen.

[0031] (2) High biocompatibility and biosafety. Existing collagen hydrogel preparation technologies usually use naturally extracted collagen, which may cause immune reactions or pathogen contamination. This new method uses human recombinant collagen, which can simulate the body's own collagen to the greatest extent through the combination of continuous human collagen amino acid short sequences and stacked short sequences, reducing the risk of immune reactions and improving biocompatibility and biosafety.

[0032] (3) Spontaneous self-assembly process. In the traditional collagen hydrogel preparation process, it is often difficult to precisely control the self-assembly and polymerization process of collagen, resulting in inconsistent hydrogel performance. By introducing specific short stacking sequences into the recombinant collagen sequence to form chelates with calcium ions, this method enables human recombinant collagen molecules to undergo self-assembly, forming highly stable hydrogels, ensuring the consistency and repeatability of product performance.

[0033] (4) Enhanced mechanical properties and stability. Conventional collagen hydrogels may degrade and experience a decrease in mechanical properties during prolonged use. However, the humanized recombinant collagen hydrogel prepared in the present invention significantly improves the mechanical strength and stability of the collagen hydrogel by optimizing the amino acid sequence and calcium ion chelation, enabling it to maintain its functionality and structural integrity for a longer period of time in the in vivo environment.

[0034] (5) Simplified preparation process and efficient production process. The preparation of traditional collagen hydrogels usually requires complex extraction and purification steps. However, the present invention significantly simplifies the preparation process by directly using human recombinant collagen and combining it with a simple calcium ion solution addition step. This not only improves production efficiency, but also reduces production costs and is easy to promote and apply on an industrial scale.

[0035] (6) Avoid the potential toxicity and side effects of chemical crosslinkers. Traditional collagen hydrogel preparation methods often require the use of chemical crosslinkers (such as glutaraldehyde, BDDE, etc.) to enhance the mechanical properties and stability of the hydrogel. However, chemical crosslinkers may cause potential toxicity and adverse side effects, which may bring biosafety risks. The present invention induces the self-assembly of collagen by stacking short sequences and chelating with calcium ions without using any chemical crosslinkers, thereby avoiding the toxicity risks brought by chemical crosslinkers and improving the biosafety and biocompatibility of the hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0037] Figure 1 This is a simulation diagram of stacked short sequence structures.

[0038] Figure 2 This is a map of the recombinant plasmid of human recombinant collagen OL3.

[0039] Figure 3 This is the protein electrophoresis diagram of human recombinant collagen OL3.

[0040] Figure 4 The morphology of collagen. Figure 4 A is the hydrogel morphology of self-assembled polymers of human recombinant collagen OL3. Figure 4 B is the flocculent form of commercially available type III recombinant human collagen.

[0041] Figure 5 A diagram showing the mechanical properties of self-assembled polymers of human recombinant collagen OL3.

[0042] Figure 6 For mouse abdominal filling experiments.

[0043] Figure 7 This is a cross-section of mouse abdominal skin. DETAILED DESCRIPTION

[0044] The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The human recombinant collagen protein composed of a stacked short sequence (GKPGTPGPAGAPGKPGTP) and a continuous short sequence of human collagen amino acids (taking a short sequence of type III human collagen amino acids as an example) in the present invention is constructed using the pPIC9K expression vector and the Pichia pastoris GS115 strain to construct a recombinant Pichia pastoris genetically engineered bacterium. The genetically engineered bacteria are induced to express by methanol, and the recombinant human collagen is secreted into the fermentation broth. The collected fermentation broth is separated and purified by conventional chromatography, and after freeze-drying, a lyophilized powder of human recombinant collagen is obtained.

[0045] Example 1: Mining Stacked Short Sequences

[0046] The present invention conducted a technical literature survey on the amino acid sequences of type I, type III, type IV, type V, type VII and type XVII human collagen proteins, and mined four GXY triplet amino acid sequences, including GKP, GTP, GPA and GAP, from the above six human collagen protein amino acid sequences. The sequences were combined and combined with the human collagen protein amino acid short sequences in the present invention to perform actual protein expression and prepare self-assembling polymers. The stacked short sequence was finally determined to be GKPGTPGPAGAPGKPGTP (SEQ ID No. 4), and its structural simulation diagram is shown in FIG. Figure 1 shown.

[0047] Example 2 Construction of human recombinant collagen amino acid sequence and recombinant expression vector

[0048] (1) Construction of human recombinant collagen amino acid sequence: Through technical literature research, a continuous human collagen amino acid short sequence in type III human collagen alpha 1 chain protein (GenBank accession number: AGL34959.1; collagen type III alpha 1 [Homosapiens]) was selected as GERGAPGFRGPAGPNGIPGEKGPAGERGAP (SEQID No. 5), and the short sequence GKPGTPGPAGAPGKPGTP was stacked to form a repeating unit with the type III human collagen amino acid short sequence (the amino acid sequence of the repeating unit is GKPGTPGPAGAPGKPGTPGERGAPGFRGPAGPNGIPGEKGPAGERGAP), which was repeated 8 times to form a new human recombinant collagen OL3 (amino acid sequence as SEQ ID No. 1).

[0049] (2) Synthesis of DNA sequence and construction of recombinant expression vector: After optimization based on the codon preference of Pichia pastoris, the DNA fragment of OL3 was synthesized by whole gene synthesis (the nucleotide sequence of OL3 and the optimized nucleotide sequence of OL3 are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively), and EcoRI and Not I recognition sites and signal peptide recognition sites were added to both ends of the gene, respectively. The synthesized gene fragment was cloned into the pPIC9K empty expression vector (purchased from Thermo Fisher Scientific) to obtain the recombinant plasmid pPIC9K-OL3 expressing OL3, as shown in FIG. Figure 2 shown.

[0050] (3) Plasmid linearization: The recombinant plasmid was linearized using Sac I. The enzyme digestion system is shown in Table 1. The reaction system was incubated at 37°C for 4 h. The linearized plasmid fragment was recovered using the same procedure as the commercial kit.

[0051] Table 1 Single enzyme digestion reaction system

[0052]

[0053] Example 3 Construction of recombinant Pichia pastoris GS115 engineered bacteria

[0054] (1) Preparation of competent cells of Pichia pastoris: Use an inoculation needle to pick a small amount of Pichia pastoris GS115 from the plate and inoculate it into a 50 mL Erlenmeyer flask containing 5 mL of YPD liquid medium. Incubate overnight at 30°C and 130 rpm. Take 0.1-0.5 mL of overnight cultured yeast and inoculate it into a 1 L Erlenmeyer flask containing 300 mL of fresh YPD medium. Incubate overnight until the OD 600 =1.3-1.5; centrifuge the cell culture at 4°C, 1500g for 5 min; resuspend the cells with 500 mL of ice-cold sterile water, and centrifuge at 4°C, 1500g for 5 min; resuspend the cells with 250 mL of ice-cold sterile water, and centrifuge at 4°C, 1500g for 5 min; resuspend the cells with 20 mL of ice-cold 1 M sorbitol solution, and centrifuge at 4°C, 1500g for 5 min, and resuspend the cells with 1 mL of ice-cold 1 M sorbitol solution to a final volume of approximately 1.5 mL; store the competent cell on ice.

[0055] (2) Electrotransformation of Pichia pastoris competent cells: 80 μL of the competent cells prepared in step (1) and 5-10 μg of the linearized plasmid DNA prepared in Example 2 (present in 5-10 μL of sterile water) were taken, mixed thoroughly, and transferred to a new ice-cold 0.2 cm electroporation cuvette; the electroporation cuvette was ice-bathed for 5 min; the electroporation instrument was turned on and the sample was electropulsed, wherein the parameters were set as follows: voltage 1500 V, capacitance 25 mV, resistance 200 Ω; after the electric shock, 1 mL of 1 M ice-cold sorbitol was immediately added to the electroporation cuvette, repeatedly aspirated several times with a pipette, and then the mixture was transferred to a new 15 mL EP tube; the EP tube was placed at 30°C for 1-2 h (no shaking was required), and after incubation, the mixture was spread on the corresponding screening plate.

[0056] (3) Screening of strains that highly express human recombinant collagen: After electroporation, take 100 μL of the transformation solution and spread it on the MD plate; after the bacterial solution is fully absorbed, place the plate in a 30°C incubator for 2-3 days to allow the emergence of transformants; draw 1-2 mL of sterile water onto the MD plate containing His+ transformants; use a sterile spreading stick to resuspend the transformants on the agar surface in sterile water; transfer the cell suspension to a sterile 50 mL centrifuge tube and vortex for 5-10 seconds; measure the cell density using a UV spectrophotometer; OD 600 =5×10 7 cells / mL; 10 5 Cells were plated onto YPD plates containing varying concentrations of G418 (G418 solution was applied to YPD plates to achieve final concentrations of 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, and 4.0 g / L, respectively). High-copy positive transformants were selected using a G418 gradient method. Plates were incubated at 30°C for 2-5 days and observed daily to obtain recombinant Pichia pastoris engineered strain OL3.

[0057] Example 4 Induced expression of recombinant Pichia pastoris engineered bacteria

[0058] A single colony of the recombinant Pichia pastoris engineered bacteria OL3 was picked from the plate and inoculated into 25 mL of BMG medium. The medium was shaken at 30°C and 300 rpm until the OD 600 = 2-6; centrifuge at 1500g for 5 min at room temperature, collect the cells, remove the supernatant, and resuspend the cells in BMMY medium to OD 600=1.0, and induce expression; add 20 mL of the above resuspended culture to a 1 L shake flask containing 200 mL of BMMY medium and continue growing on a shaker; add methanol to the culture once every 24 hours, and each time the methanol addition concentration is 0.5% to 1.0% v / v to continue induction; after 96 hours of induction expression, take the culture medium and centrifuge it at 3000g for 5 minutes, take the supernatant and perform SDS-PAGE electrophoresis, the protein electrophoresis pattern is as shown Figure 3 shown.

[0059] Example 5 Purification of recombinant collagen

[0060] The supernatant of the recombinant Pichia pastoris fermentation in Example 4 was collected and separated using an ultrafiltration column membrane with a molecular weight cutoff of 10 kDa to remove low molecular weight proteins and salts. The recombinant collagen was then purified by molecular sieve chromatography (using Superdex 75 prep grade as the filler) using pure water as the mobile phase at a flow rate of 0.5 mL / min. The sample in the elution peak was collected and freeze-dried to obtain recombinant collagen OL3 lyophilized powder.

[0061] Example 6 Preparation of Self-Assembled Polymers of Human Recombinant Collagen

[0062] The recombinant collagen OL3 lyophilized powder prepared in Example 5 was mixed with pure water to prepare a 40 g / L human recombinant collagen OL3 aqueous solution. Without stirring, an aqueous solution containing 80 mM calcium chloride was slowly added to the human recombinant collagen OL3 aqueous solution along the wall of the container at a volume ratio of 1:1 at a flow rate of 1 ml per minute. The solution was allowed to stand at 4°C for 12 hours to form a hydrogel. The remaining solution was then poured out of the container, and the obtained hydrogel was soaked in pure water for 3 hours and rinsed with pure water 3 times to remove free calcium ions, thereby obtaining a self-assembled polymer (hydrogel form) of human recombinant collagen OL3, as shown in FIG. Figure 4 A, its mechanical properties are shown in the figure Figure 5 As shown. Figure 5 It can be seen that the storage modulus (G') and loss modulus (G") of the self-assembled polymer under different shear stress conditions change. In the entire shear stress range, the storage modulus is higher than 4000Pa. The self-assembled polymer can store energy well when subjected to shear stress, indicating that it has good elasticity. At the same time, the loss modulus is lower than 500Pa, and the energy lost when subjected to shear stress is relatively small, indicating that the self-assembled polymer loses less energy when deformed, which manifests as less viscosity. Moreover, with the increase of shear stress, the values ​​of the storage modulus and loss modulus do not change significantly, indicating that the self-assembled polymer has good mechanical stability within a larger shear stress range.

[0063] Example 7 Mouse Abdomen Subcutaneous Filling Experiment

[0064] Eight-week-old male Sprague-Dawley mice (approximately 300 g) were anesthetized with isoflurane, and a 2 cm long skin wound was made on the abdomen using a sterile blade. The self-assembled polymer of human recombinant collagen OL3 prepared in Example 6 (a circular hydrogel with a diameter of approximately 1.5 cm and a thickness of approximately 0.5 cm) was placed in the mouse's abdominal cavity (as shown in FIG. Figure 6 After one week, skin slices were taken from the abdominal wounds of mice to observe the effects of human recombinant collagen OL3 self-assembled polymers as subcutaneous fillers on mice. Figure 7 As shown. Figure 7 It can be seen that the skin structure remains relatively intact, the cells are evenly distributed without obvious abnormal proliferation, there is no obvious inflammation, and the collagen fibers and skin structure in the dermis are not deformed or damaged. This result shows that the self-assembled polymer of recombinant human collagen OL3 has good biosafety as a skin filler.

[0065] Comparative Example 1

[0066] The commercially available type III recombinant collagen lyophilized powder was prepared into a 40 g / L solution with pure water. Without stirring, an aqueous solution containing 80 mM calcium chloride was slowly added to the commercially available type III recombinant human collagen solution along the wall of the container at a volume ratio of 1:1 at a flow rate of 1 ml per minute. It was allowed to stand at 4°C for 12 hours to form a loose flocculent solid. Then, after pouring the remaining solution from the container, the obtained solid was soaked in pure water for 3 hours and rinsed with pure water 3 times to remove free calcium ions, thereby obtaining a commercially available type III recombinant collagen flocculent solid (without forming a formed hydrogel). Figure 4 As shown in B.

[0067] The present invention develops a hydrogel-like self-assembling polymer of human recombinant collagen that can form a stable three-dimensional structure through the self-assembly properties of recombinant collagen without the need for complex chemical crosslinking agents. This is an effective strategy for solving the above-mentioned problems. This self-assembling polymer can not only significantly improve the stability and mechanical properties of recombinant collagen, but also maintain its excellent biocompatibility and biodegradability. The hydrogel-like self-assembling polymer of recombinant collagen mimics the natural assembly process of collagen in the body to form a three-dimensional structure with excellent mechanical properties and stability. This structure can maintain its morphology and function for a long time in the in vivo environment, providing continuous tissue support and filling effects.

[0068] In summary, the self-assembled hydrogel-like human recombinant collagen polymer developed by this invention not only overcomes the shortcomings of traditional collagen materials in terms of stability and mechanical properties, but also provides a novel and effective solution for tissue engineering and soft tissue augmentation. The development of this innovative technology will further broaden the application prospects of collagen materials, expanding their potential in the field of medical aesthetics.

[0069] The present invention provides a self-assembled hydrogel of human recombinant collagen, its preparation method, and its application. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are considered within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. A human recombinant collagen, characterized in that: The repeating unit is composed of stacked short sequences and human collagen short sequences, which are repeated 8 times; in, The stacked short sequence is composed of a GXY triplet amino acid sequence in human collagen, and the human collagen is any one or more of type I, type III, type IV, type V, type VII and type XVII human collagen; The human collagen short sequence is composed of consecutive (GXY) in the α1 chain of type III human collagen. 10 The amino acid sequence of Wherein, G is a glycine residue, and X or Y represents any amino acid residue; The amino acid sequence of the human recombinant collagen is shown in SEQ ID No.

1. 2 . A nucleic acid molecule encoding the human recombinant collagen according to claim 1 .

3. The nucleic acid molecule according to claim 2, characterized in that Its nucleotide sequence is shown as SEQ ID No.2 or SEQ ID No.

3.

4. A method for preparing human recombinant collagen according to claim 1, characterized in that: The nucleic acid molecule encoding the human recombinant collagen is amplified and cloned into a plasmid vector to obtain a recombinant vector, which is then introduced into a host strain for expression and then isolated and purified.

5. The preparation method according to claim 4, characterized in that The host strain is Pichia pastoris.

6. A hydrogel-state human recombinant collagen, characterized in that: Comprising the human recombinant collagen according to claim 1.

7. The method for preparing the hydrogel-state human recombinant collagen according to claim 6, characterized in that: The human recombinant collagen is prepared into a human recombinant collagen aqueous solution, which is then mixed with a calcium ion solution, and the mixture is allowed to stand and washed to obtain the solution.

8. The preparation method according to claim 7, characterized in that The concentration of the human recombinant collagen aqueous solution is 20-60 g / L; the calcium ion solution is a 40-120 mM calcium chloride aqueous solution; The mixing volume ratio of the human recombinant collagen aqueous solution and the calcium ion solution is 4-7:3-6; The standing temperature is 2-10°C and the standing time is 6-18 hours.

9. Use of the hydrogel-state human recombinant collagen according to claim 6 as a subcutaneous filler for non-therapeutic purposes.

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