Hydrogel-state human-derived recombinant collagen self-assembled polymer as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to a hydrogel-state human-derived recombinant collagen self-assembled polymer as well as a preparation method and application thereof. The human-derived recombinant collagen provided by the invention is formed by repeating a stacked short sequence and a human-derived collagen short sequence as repetitive units for eight times, and a specific amino acid sequence structure is formed. The stacked short sequence and calcium ions (Ca < 2 + >) form a chelate, so that the human recombinant collagen molecules are self-assembled, and a stable hydrogel-form human recombinant collagen self-assembled polymer can be formed. According to the preparation method, a chemical cross-linking agent is not used, potential toxicity and side effect risks are avoided, the prepared hydrogel-state human-derived recombinant collagen self-assembly polymer can simulate collagen of a human body to the maximum extent, the risk of immunoreaction is reduced, the biological safety is improved, the biocompatibility is ensured, and the hydrogel-state human-derived recombinant collagen self-assembly polymer is suitable for being used as a self-assembly material for preparing the hydrogel-state human-derived recombinant collagen self-assembly polymer and the preparation method of the hydrogel-state human-derived recombinant collagen self-assembly polymer. The application prospect in the field of medical beauty is expanded.

Description

Technical Field

[0001] The 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 about 30% of the total protein in the human body. It is the main component of the extracellular matrix, has the function of supporting and connecting tissues, and is widely found in skin, bones, cartilage, ligaments and tendons. Collagen has a highly regular triple helix structure, which gives it excellent mechanical properties and biocompatibility. In the 1950s and 1960s, collagen began to be used in the biomedical field, and was initially mainly concentrated in wound dressings and hemostatic materials. Entering the 21st century, the application of collagen has become more extensive and in-depth. With the development and application of new technologies, the performance of collagen materials has been improved. The development of collagen as a biomedical material has gone through the initial extraction and purification and preliminary application, to tissue engineering and regenerative medicine, to genetic engineering and recombinant collagen, as well as the continuous advancement of new material development and cutting-edge applications. This process demonstrates the huge driving force of scientific and technological progress and multidisciplinary cross-integration. With the continuous innovation of technology and the expansion of application fields, the application prospects of collagen in the biomedical field will be broader.

[0003] As a multifunctional biomedical material, collagen is widely used in the biomedical field due to its excellent biocompatibility, biodegradability and low immunogenicity. Its applications cover many fields such as tissue engineering, wound dressings, cosmetic surgery, drug delivery, orthopedics and dentistry, nerve repair, artificial organs and medical devices. Especially in the field of cosmetic surgery, collagen is used as an injectable filler for facial contour modification, wrinkle filling and lip plumping, with good biocompatibility and filling effect. 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 limiting problems in the application process, which affects their clinical effects and wide application.

[0004] First, the lack of stability of collagen is one of the main challenges in its application. Natural collagen molecules are susceptible to enzymatic hydrolysis and degradation in the body environment, making it difficult to maintain its effect in tissue filling for a long time. This degradation not only limits the service life of collagen, but may also cause adverse reactions in local tissues. Therefore, how to improve the stability of collagen so that it can exist for a long time in the body environment has become an important direction of current research.

[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 provide a hydrogel-state human recombinant collagen self-assembly polymer and its preparation method and application in view of the shortcomings of the prior art, especially the problems involving the stability and mechanical properties of collagen.

[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, which is composed of stacked short sequences and human collagen short sequences as repeating units, repeated 8 times;

[0010] Among them, 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 type I, type III, type IV, type V, type VII and type XVII human collagen amino acid sequences, and mines 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, performs 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 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: configuring the human recombinant collagen into a human recombinant collagen aqueous solution and then mixing it with a calcium ion solution, and then standing and washing to obtain the hydrogel-state human recombinant collagen.

[0020] Wherein, the concentration of the human recombinant collagen aqueous solution is 20-60g / L; the calcium ion solution is a 40-120mM calcium chloride aqueous solution; preferably, the concentration of the human recombinant collagen aqueous solution is 40g / L; the calcium ion solution is an 80mM 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 6-18h, preferably 4°C for 12h.

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

[0025] More preferably, the preparation method is as follows: without stirring, 2+ ) solution, with a volume ratio of 1:1, at a flow rate of 1 ml per minute, and slowly added to the human recombinant collagen solution along the container wall. Let it stand at 4°C to form a hydrogel. Then, after pouring the remaining solution from the container, 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 of human recombinant collagen (hydrogel-state human recombinant collagen).

[0026] In a fifth aspect, the present invention further provides use of the hydrogel 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 a preparation method thereof. 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 to form a stable recombinant human collagen self-assembled polymer (hydrogel form). No chemical cross-linking agent is used in the preparation process of the present invention, thereby avoiding the potential toxicity and side effect risks brought by chemical cross-linking agents, increasing biosafety, and ensuring 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 with human collagen.

[0031] (2) High biocompatibility and biosafety. Existing collagen hydrogel preparation technologies usually use naturally extracted collagen, which may cause immune responses 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 response and improving biocompatibility and biosafety.

[0032] (3) Spontaneous self-assembly process. In the traditional preparation process of collagen hydrogel, it is often difficult to accurately control the self-assembly and polymerization process of collagen, resulting in inconsistent performance of hydrogel. 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 process to form highly stable hydrogels, ensuring the consistency and repeatability of product performance.

[0033] (4) Enhanced mechanical properties and stability. Conventional collagen hydrogels may degrade and have reduced mechanical properties during long-term use. The humanized recombinant collagen hydrogel prepared by the present invention significantly improves the mechanical strength and stability of the collagen hydrogel by optimizing the amino acid sequence and calcium ion chelation, so that it can maintain its functionality and structural integrity for a long time in the in vivo environment.

[0034] (5) Simplified preparation process and efficient production process. The preparation of traditional collagen hydrogel usually requires complicated 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 methods for preparing collagen hydrogels 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 calcium ions without using any chemical crosslinkers, thereby avoiding the toxicity risks caused 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 in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

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

[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 the self-assembled polymer of human recombinant collagen OL3. Figure 4 B is the flocculent morphology of commercially available type III recombinant human collagen.

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

[0042] Figure 6 For abdominal filling experiments in mice.

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

[0044] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are 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 a pPIC9K expression vector and 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 human recombinant collagen lyophilized powder is obtained.

[0045] Example 1 Mining stacked short sequences

[0046] 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 type triplet amino acid sequences, namely GKP, GTP, GPA and GAP, from the above six human collagen amino acid sequences, combines them, and combines them with human collagen amino acid short sequences in the present invention to perform actual protein expression and preparation of self-assembling polymers, and finally determines that the stacking short sequence is GKPGTPGPAGAPGKPGTP (SEQ ID No. 4), and its structural simulation diagram is shown in the figure Figure 1 shown.

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

[0048] (1) Constructing the amino acid sequence of human recombinant collagen: Through technical literature research, a continuous short sequence of human collagen amino acids 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 a 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 optimizing 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 placed at 37°C for 4 hours. The plasmid linearized fragment was recovered. The steps are shown in 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 culture 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 YPD liquid medium, and culture it 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 fresh YPD medium, and culture it overnight until OD 600 =1.3-1.5; centrifuge the cell culture at 4°C, 1500g for 5 min; resuspend the cells with 500mL of ice-cold sterile water, and centrifuge at 4°C, 1500g for 5 min; resuspend the cells with 250mL of ice-cold sterile water, and centrifuge at 4°C, 1500g for 5 min; resuspend the cells with 20mL of ice-cold 1M sorbitol solution, and centrifuge at 4°C, 1500g for 5 min, and resuspend the cells with 1mL of ice-cold 1M sorbitol solution, the final volume is about 1.5mL; store the competent state 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-precooled 0.2 cm electroporation cuvette; the electroporation cuvette was placed in an ice bath for 5 min; the electroporation instrument was turned on and the sample was subjected to an electric pulse, 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-precooled sorbitol was immediately added to the electroporation cuvette, the mixture was repeatedly pipetted several times, and then the mixture was transferred to a new 15 mL EP tube; the EP tube was incubated 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 transformants to appear; 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 The cells were spread on YPD plates containing different concentrations of G418 (a certain amount of G418 solution was spread on the YPD plate to make the 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), and high-copy positive transformants were selected by the G418 gradient method. The plates were placed at 30°C for 2-5 days and observed every day to obtain the recombinant Pichia pastoris engineered bacteria OL3.

[0057] Example 4 Inducible expression of recombinant Pichia pastoris engineering 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 culture 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 cells, remove supernatant, and resuspend cells in BMMY medium to OD 600=1.0, to induce expression; add 20 mL of the above resuspended culture to a 1L shaking flask containing 200 mL of BMMY medium, and place it in a shaking table to continue growing; add methanol to the culture once every 24 hours, and the concentration of methanol added each time is 0.5% to 1.0% v / v to continue induction; induce expression for 96 hours, take the culture solution and centrifuge it at 3000g for 5 minutes, take the supernatant, and perform SDS-PAGE electrophoresis. The protein electrophoresis diagram is as follows Figure 3 shown.

[0059] Example 5 Purification of recombinant collagen

[0060] The recombinant Pichia fermentation supernatant in Example 4 was collected, and the supernatant was separated using an ultrafiltration column membrane with a molecular weight cutoff of 10 kDa to remove small molecular weight proteins and salts, and then the recombinant collagen was purified by molecular sieve chromatography (filler: Superdex 75prep grade), using pure water as the mobile phase at a flow rate of 0.5 mL / min for elution, and 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 container wall 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 hydrogel. Then, after pouring the remaining solution from the container, 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 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 has less energy loss when deformed, which is manifested as less viscosity. Moreover, with the increase of shear stress, the values ​​of storage modulus and loss modulus do not change significantly, indicating that the self-assembled polymer has good mechanical stability in a larger shear stress range.

[0063] Example 7: Subcutaneous filling experiment in the abdomen of mice

[0064] Eight-week-old male Sprague-Dawley mice (about 300 g) were anesthetized with isoflurane, and a skin wound about 2 cm long was cut on the abdomen with a sterile small blade. The self-assembled polymer of human recombinant collagen OL3 prepared in Example 6 (circular hydrogel with a diameter of about 1.5 cm and a thickness of about 0.5 cm) was placed in the abdominal cavity of the mouse (as shown in FIG. Figure 6 After one week, skin slices were made on the wounds of the abdomen 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 polymers of recombinant human collagen OL3 have good biosafety as skin fillers.

[0065] Comparative Example 1

[0066] Prepare a 40g / L solution of commercially available type III recombinant collagen lyophilized powder with pure water. Without stirring, slowly add an aqueous solution containing 80mM calcium chloride to the commercially available type III recombinant human collagen solution along the container wall at a volume ratio of 1:1 at a flow rate of 1 ml per minute. Let it stand at 4°C for 12 hours to form a loose flocculent solid. Then, pour the remaining solution from the container, soak the obtained solid in pure water for 3 hours, and rinse it with pure water 3 times to remove free calcium ions, and obtain a commercially available type III recombinant collagen flocculent solid (without forming a shaped hydrogel). Figure 4 As shown in B.

[0067] The present invention develops a hydrogel-state human recombinant collagen self-assembly polymer, which can form a stable three-dimensional structure through the self-assembly characteristics of recombinant collagen itself without the need for complex chemical cross-linking agents, which is an effective strategy to solve the above problems. This self-assembly polymer can not only significantly improve the stability and mechanical properties of recombinant collagen, but also maintain its excellent biocompatibility and biodegradability. The hydrogel-state recombinant collagen self-assembly polymer forms a three-dimensional structure with good mechanical properties and stability by simulating the natural assembly process of collagen in the body. The 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 hydrogel-state human recombinant collagen self-assembly polymer developed by the present invention can not only overcome the deficiencies of traditional collagen materials in stability and mechanical properties, but also provide a new and effective solution for tissue engineering and soft tissue filling. Through the development of this innovative technology, the application prospects of collagen materials will be broader, expanding their application prospects in the field of medical beauty.

[0069] The present invention provides a hydrogel-state human recombinant collagen self-assembly polymer and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by 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.

2. The human recombinant collagen according to claim 1, characterized in that: The amino acid sequence of the human recombinant collagen is shown in SEQ ID No.

1.

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

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

3.

5. A method for preparing human recombinant collagen according to claim 1 or 2, 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 separated and purified.

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

7. A hydrogel human recombinant collagen, characterized in that: Contains the human recombinant collagen according to claim 1 or 2.

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

9. The preparation method according to claim 8, 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 6-18h.

10. Use of the hydrogel human recombinant collagen according to claim 7 as a subcutaneous filler.

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