WO2023143394A1

WO2023143394A1 - Injection containing stable macromolecular i-type recombinant collagen

Info

Publication number: WO2023143394A1
Application number: PCT/CN2023/073218
Authority: WO
Inventors: 范代娣; 王小军; 罗培培; 严建亚; 刘琳
Original assignee: 陕西巨子生物技术有限公司
Priority date: 2022-01-27
Filing date: 2023-01-19
Publication date: 2023-08-03
Also published as: CN114369156A; CN114369156B

Abstract

The present invention relates to an injection containing stable macromolecular I-type recombinant collagen. The collagen injection contains macromolecular I-type recombinant collagen, injection water and excipients, wherein the macromolecular I-type recombinant collagen is composed of multiple repetitions of a short amino acid sequence (GAPGAPGSQGAPGLQ) from natural human I-type collagen as a repeating unit, and the number of repetitions is 75-110 times. The collagen injection of the present invention can be subjected to damp-heat sterilization under the condition that the macromolecular I-type recombinant collagen is not crosslinked using a cross-linking agent, so that the toxicity of a residue in a cross-linking agent in a collagen injection product to a human body can be effectively avoided.

Description

Injection containing stable macromolecule type I recombinant collagen

technical field

The invention belongs to the field of biotechnology, and relates to a stable macromolecule type I recombinant collagen, the use of the recombinant collagen and an injection containing the recombinant collagen.

Background technique

Collagen is a biological polymer protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. It is used in medical materials, cosmetics , Food industry are widely used.

The source of natural human collagen is limited. The natural collagen currently used in industry is mainly extracted from animal skin or bone by acid, alkali or enzymatic methods, and its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases, and at the same time, large-scale preparations have caused huge pressure on animal feeding on the supply side.

With the widespread application of genetic engineering technology, the bottleneck problem of large-scale production of human collagen has been successfully solved by using suitable engineering strains (Escherichia coli, Pichia pastoris, etc.) to express human collagen exogenously. However, when engineering strains such as Pichia pastoris genetically engineered strains are used to express human collagen, human collagen will be degraded during fermentation, purification and storage, which increases production costs and affects the human collagen produced by this method. performance.

It is speculated that the reason for the degradation of human collagen may be that its amino acid sequence contains more sites that are prone to hydrolysis. Therefore, technicians construct recombinant collagen by selecting and repeating short amino acid sequences from natural human collagen in order to avoid sites prone to hydrolysis, thereby improving the stability of collagen while maintaining the excellent properties of natural human collagen. performance. However, the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen has a relatively monotonous amino acid composition and distribution. In theory, this will cause a large charge load on its surface, and it is difficult to achieve a stable equilibrium state as a whole. , so it is easy to hydrolyze and denature in aqueous solution, and there is a tendency that the shorter the repeating unit of the short amino acid sequence and the more the number of repeats, the more unstable the recombinant collagen molecule is in aqueous solution.

One can attempt to obtain recombinant collagens that are more resistant to degradation by mutating short amino acid sequences derived from native collagen as repeating units. However, the homology between the recombinant collagen obtained by such mutation and natural human collagen is reduced, and immunogenicity problems may arise, so it is not suitable for use in biomaterials that need to be in long-term contact with the human body.

On the other hand, tissue engineering materials such as dermal fillers are an important application direction of human collagen. As tissue engineering materials, collagen is required to have good mechanical strength and stability in aqueous solution (it can be stored in aqueous solution for a long time) . In general, the greater the molecular weight of collagen, the better its mechanical strength, and the poorer its stability in aqueous solution, especially for recombinant collagen constructed by repeating short amino acid sequences from natural human collagen. in this way.

The single-chain molecular weight of natural human collagen is about 110-130kD. From a practical point of view, technical personnel generally believe that the molecular weight of collagen suitable for replacing natural human collagen as tissue engineering materials needs to reach more than 100kD. However, as mentioned above, the source of natural human collagen is limited, animal-derived collagen has the risk of spreading diseases, and human collagen expressed by genetic engineering is prone to degradation during fermentation, purification and storage. short amino acid sequence Repeatedly constructed recombinant collagen is unstable in aqueous solution, and the recombinant collagen obtained through mutation transformation has immunogenicity problems. Therefore, how to obtain collagen suitable for replacing natural human collagen as tissue engineering materials has become a technical field. restrictive issues.

In addition, since the 1970s, people began to study injectable bovine collagen, and in 1981 passed the FDA certification for soft tissue filling, which restores the volume and elasticity of soft tissue by injecting or filling to achieve beauty. Effect. Collagen has high safety and degradability, and can be used to restore lip edges, repair facial wrinkles and other soft tissue contour loss and other treatments.

Up to now, collagen injection has been widely used in the field of medical cosmetology. Based on the safety requirements for the use of this product, the product needs to reach a sterile level, so various forms of collagen injections must be sterilized after packaging to kill the (pathogenic) microorganisms present therein. In view of the characteristics of collagen macromolecules and variability, from the perspective of sterilization effect and product performance after sterilization, moist heat sterilization is more suitable for the sterilization of this type of product, and it is the most effective in killing pathogenic microorganisms, but , Collagen is not heat-resistant, and the temperature conditions of moist heat sterilization (usually 121°C) will cause its thermal degradation, the overall structure is fragmented, and it changes from a viscous liquid to a watery substance, losing its biological activity and mechanical properties.

It is known that cross-linking is beneficial for collagen to resist thermal degradation, but for collagen injection products, the cross-linking agent (such as glutaraldehyde, etc.) used for cross-linking will eventually enter with collagen and exist in the human body for a long time, These cross-linking agents can be toxic to humans. Therefore, those skilled in the art have long desired to find recombinant collagen that can withstand moist heat sterilization without cross-linking.

Contents of the invention

In order to solve the above-mentioned technical problems existing in the prior art, the inventors conducted in-depth research. The inventors first conducted a technical literature survey on recombinant collagens constructed by repeating short amino acid sequences from natural human collagen, and selected some of the existing technologies from natural human type I collagen. White (the most widely used tissue engineering material) short amino acid sequences, and then use these short amino acid sequences as repeating units to construct type I recombinant collagens with different molecular weights, and investigate the long-term storage stability of these type I recombinant collagens in aqueous solution In order to obtain a large molecular weight (above 100kD) recombinant collagen that can be stored stably in aqueous solution for a long time and can meet the mechanical strength requirements of tissue engineering materials.

As a result of the above research, the inventors unexpectedly found that the type I recombinant collagen obtained by repeating 75-110 times of a segment of pentadecadecanoid peptide (GAPGAPGSQGAPGLQ) from natural human type I collagen has exceptionally excellent stability. It is embodied in: (1) although the length of its repeating unit is the shortest among all recombinant collagens tested by the inventor, its stability in aqueous solution is the best; The more monotonous the distribution, the greater the surface charge load of the recombinant collagen thus constructed, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze. (2) It is even more stable than the type I recombinant collagen obtained by repeating the pentapenteptide 52 times or 62 times or 72 times, and it is generally believed that the more the number of repetitions, the greater the molecular weight, and the surface of the recombinant collagen The greater the charge load, the less likely it is to reach a stable equilibrium state, and thus more susceptible to hydrolysis.

Based on the above findings, the inventors have accomplished the present invention. That is, the present invention includes:

1. A macromolecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is as SEQ ID No.: 1( GAPGAPGSQGAPGLQ), the number of repetitions was 75 to 110 times.

2. The macromolecular type I recombinant collagen according to item 1, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

3. The macromolecular type I recombinant collagen according to item 1, which has a molecular weight of more than 100 kD.

4. The macromolecular type I recombinant collagen according to item 1, further bearing a tag for easy purification.

5. The macromolecular type I recombinant collagen according to item 4, wherein the tag is a His tag, a Flag tag or a c-Myc tag.

6. Use of the macromolecular type I recombinant collagen according to any one of items 1 to 5 as a tissue engineering material.

7. The use according to item 6, wherein the tissue engineering material is selected from the group consisting of dermal fillers, artificial bone, artificial skin, oral cavity absorbable biofilms, bone implants, vascular scaffolds, intercellular matrix scaffolds and collagen sponge.

8. The macromolecular type I recombinant collagen according to any one of items 1 to 5 is used as a subcutaneous filler, artificial bone, artificial skin, oral cavity absorbable biofilm, bone implant, vascular scaffold, and intercellular matrix scaffold Or the use of collagen sponge.

9. An aqueous collagen solution, comprising the macromolecular type I recombinant collagen described in any one of items 1-5.

10. The collagen aqueous solution according to item 9, which has been stored at room temperature for more than 3 months, preferably for more than 6 months, more preferably for more than 12 months; or has been stored at 4°C for more than 12 months, preferably 24 months More than one month, more preferably more than 36 months.

The inventors are conducting more in-depth research on the reason why the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability. Preliminary findings suggest that this may be due to:

The stability of recombinant collagen that repeats with a certain amino acid sequence as a repeat segment is closely related to the surface charge, and the surface charge is related to the composition of amino acids and the spatial structure of the protein. After reaching a certain number of repetitions, a certain The spatial structure makes the surface load in a balanced or near-balanced state, so it will show an abnormally stable state. The inventor just found that the 15 amino acid repeat sequence collagen is in the range of load balance, so the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability.

In addition, the inventors also found that the macromolecular type I recombinant collagen of the present invention has particularly excellent heat resistance, and it can even withstand moist heat sterilization (for example, 121 ° C, 20 min) without thermal degradation without cross-linking , showing good properties suitable for collagen injection.

Therefore, the present invention also includes:

2-1. A collagen injection, which comprises macromolecular type I recombinant collagen, water for injection and auxiliary materials, said macromolecular type I recombinant collagen consists of a short amino acid sequence from natural human type I collagen as a repeating unit It is formed by repeating multiple times, wherein the short amino acid sequence is shown in SEQ ID No.: 1 (GAPGAPGSQGAPGLQ), and the number of repetitions is 75 to 110 times.

2-2. The collagen injection according to claim 2-1, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

2-3. The collagen injection according to claim 2-1, wherein the molecular weight of the macromolecular type I recombinant collagen is above 100 kD.

2-4. The collagen injection according to claim 2-1, wherein the macromolecular type I recombinant collagen is also provided with a tag for easy purification.

2-5. The collagen injection according to claims 2-4, wherein the macromolecular type I recombinant collagen has a tag that makes it easy to purify is a His tag, a Flag tag or a c-Myc tag.

2-6. The collagen injection according to claim 2-1, wherein the auxiliary material is selected from sodium hyaluronate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polylactic acid, glycine, One or more of alanine, proline, carnosine, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

2-7. The collagen injection according to claim 2-1, which does not contain a cross-linking agent component. In this specification, the cross-linking agent component refers to the substance derived from the cross-linking agent, including the cross-linking agent molecule itself, and also includes the molecules formed after the chemical reaction of the cross-linking agent.

2-8. The collagen injection according to claim 2-1, which is used for alleviating skin aging and correcting skin folds.

2-9. A use of macromolecular type I recombinant collagen in the preparation of collagen injection, wherein,

The macromolecular type I recombinant collagen is composed of a short amino acid sequence from natural human type I collagen repeated multiple times as a repeating unit, and the short amino acid sequence is shown in SEQ ID No.: 1 (GAPGAPGSQGAPGLQ), repeated The number of times is 75 to 110 times;

The collagen injection has been sterilized by moist heat.

2-10. The use according to claims 2-9, wherein the number of repetitions is 80-105 times, preferably 82-102 times.

2-11. The use according to claims 2-9, wherein the molecular weight of the macromolecular type I recombinant collagen is above 100 kD.

2-12. The use according to claims 2-9, wherein the macromolecular type I recombinant collagen is also provided with a tag for easy purification.

2-13. The use according to claims 2-12, wherein the macromolecular type I recombinant collagen has a tag that makes it easy to purify is a His tag, a Flag tag or a c-Myc tag.

2-14. The use according to claims 2-9, wherein the conditions for the moist heat sterilization are 110-130° C. for 20-60 minutes.

2-15. The use according to claims 2-9, wherein the collagen injection comprises the macromolecular type I recombinant collagen, water for injection and auxiliary materials.

2-16. The use according to claim 2-15, wherein the auxiliary material is selected from sodium hyaluronate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polylactic acid, glycine, alanine , proline, carnosine, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or one or more of them.

2-17. The use according to claims 2-9, wherein the collagen injection does not contain a cross-linking agent component. In this specification, the cross-linking agent component refers to the substance derived from the cross-linking agent, including the cross-linking agent molecule itself, and also includes the molecules formed after the chemical reaction of the cross-linking agent.

2-18. The use according to claims 2-9, wherein the collagen injection is used for alleviating skin aging and correcting skin folds.

2-19. The preparation method of the collagen injection according to any one of claims 2-1 to 2-8, comprising: obtaining the mixed solution of the macromolecular type I recombinant collagen, water for injection and auxiliary materials, And carry out moist heat sterilization to described mixed solution.

2-20. The preparation method according to claims 2-19, wherein the conditions for the moist heat sterilization are 110-130° C. for 20-60 minutes.

The content of the macromolecular type I recombinant collagen in the collagen injection is not particularly limited, and may be 1 g/L-15 g/L, for example, 5 g/L-10 g/L.

The content of the excipients in the collagen injection is not particularly limited, and may be 1 g/L-20 g/L, for example, 5 g/L-15 g/L.

Description of drawings

FIG. 1 is an SDS-PAGE electrophoresis image of a portion of type I recombinant collagen prepared in Example 1. Take No.4, 6, 7, and 10 as examples for control proteins.

Fig. 2 is the HPLC picture of part of the test samples prepared in Example 2 after standing for 12 months. Take No.4, 7, 10, and 13 as examples for control proteins.

Fig. 3 is the infrared spectrogram of No.1 and No.6 type I recombinant collagen.

Fig. 4 is the Raman spectrum of No.1 and No.6 type I recombinant collagens.

Fig. 5 is the SDS-PAGE gel electrophoresis picture of the collagen injection solution after moist heat sterilization.

Detailed ways

The present invention will be described in detail through specific examples below. It should be pointed out that these descriptions are only exemplary descriptions and do not limit the scope of the present invention.

General description: the enzymes used in the specific embodiment are all purchased from TaKaRa Company, the plasmid DNA extraction kit and the DNA gel recovery kit are purchased from Beijing Suolaibao Company, and the gene recombination kits (Reorganization Kits) are purchased from For Tiangen Biology, the specific operation was carried out in accordance with the instructions of the kit.

Example 1. Preparation of Various Type I Recombinant Collagens Using Yeast Expression System

1. Construction of yeast expression strains

Yeast expression strains expressing No. 1-18 type I recombinant collagen shown in Table 1 were constructed. The specific operation is: after optimization according to the codon preference of Pichia pastoris, synthesize the corresponding target gene by whole gene synthesis, and add SnaBI and Not I restriction sites at both ends of the gene respectively, and use SnaBI and NotI enzymes to synthesize the corresponding target gene. The target gene was double-enzymatically digested, and then ligated with pPIC9k, which was also digested with SnaB I and Not I enzymes, under the action of T4 ligase. After overnight ligation at 16°C, it was transferred into Top10 competent cells and coated with ampicillin-resistant plates. Pick the positive transformant, extract the plasmid, linearize it with SacI, transfer it into Pichia pastoris GS115 competent cells by electric shock, and screen the multi-copy transformant with G418 resistance plate, which is the expression strain of type I recombinant collagen.

Table 1 Type I recombinant collagen expressed by each yeast expression strain

2. Induced expression of target protein

(1) Pick a single colony of the yeast expression strain and add it to 5ml YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), cultivate overnight at 30°C and 200rpm for activation;

(2) Inoculate 100 ml of BMGY liquid medium with 1% inoculum size, culture at 30°C and 200 rpm until OD ₆₀₀ =6.0-9.0;

(3) Under the action of 1500g centrifugal force, centrifuge at 25°C for 6 minutes to collect the bacterial cells, suspend them in 200ml BMMY liquid medium, make the initial concentration OD ₆₀₀ =1.0, and cultivate them at 30°C and 200rpm;

(4) Methanol was added every 24 hours, with a final concentration of 0.5-1.0%, to induce expression;

(5) After induction for 72 hours, the culture solution was taken and centrifuged at 12,000 rpm for 2 minutes, and the supernatant was taken.

3. Preparation of type I recombinant collagen

After the supernatant prepared by fermentation is concentrated by 30kD ultrafiltration membrane, it is separated by CM ion exchange column, eluted with 35% NaCl solution, and the eluate is collected, desalted and concentrated, and then freeze-dried to obtain type I Preparation of recombinant collagen. Take 0.1g of lyophilized powder and dissolve it in 100ml of normal saline, fully dissolve it and perform SDS-PAGE gel electrophoresis to confirm the molecular weight and protein electrophoresis purity.

The results showed that all the 18 expression strains constructed could successfully express the target protein, and the electrophoresis purity of the prepared protein after separation and purification was all above 99%. The electrophoresis results are shown in Figure 1 .

Embodiment 2: Stability experiment of various type I recombinant collagens in aqueous solution

AExperimental material

The materials used in the experiment were type I recombinant collagen No.1--18 prepared in Example 1.

BExperimental method

Prepare the experimental material in A with ddH ₂ O to form a protein solution with a protein concentration of 1 mg/mL, filter it with a 0.22 μm sterile filter in a clean bench, then divide it into sterile centrifuge tubes, seal them, and place them at 25°C Under the condition of ±2°C, samples were taken at 0 month, 6 months, and 12 months respectively, and 3 tubes were sampled each time, and the protein purity was detected (protein purity was determined by high performance liquid chromatography), and the stability of the protein was determined according to the change of purity .

C Experimental results

The test results are as follows:

Table 2 Reconstituted collagen solution 12 months stability test result (purity, %)

It is generally believed that (1) for recombinant collagen with the same or similar molecular weight, the shorter the repeating unit, the more monotonous the amino acid composition and distribution, the greater the surface charge load, the less likely it is to reach a stable equilibrium state, and thus easier to hydrolyze; (2) For recombinant collagen with the same repeating unit, the more repetitions, the greater the molecular weight, the greater the surface charge load of the recombinant collagen, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze.

However, as can be seen from Table 2, the type I recombinant collagen (No.1～3) of the present invention shows exceptionally excellent stability in aqueous solution, and it is placed in aqueous solution for 12 months, and the purity can still be as high as more than 97% (see Figure 2A-G).

Example 3: Preliminary research on the reasons why type I recombinant collagen No. 1-3 has abnormal stability in aqueous solution

1) Infrared Spectroscopy Determination

After the recombinant collagens No.1 and No.6 in Table 1 were formulated into solutions, infrared spectroscopy was measured, and the spectral data was deconvoluted by Fourier with Bruker OPUS7.2, and the 1700-1600cm ^-1 band spectrum was intercepted The data was processed by peak fitting of the second-order derivative with peakfit v4.12, and the processed data was plotted with orgin to obtain the distribution of the secondary structure, and the relative content of the secondary structure was calculated. The comparison results of the secondary structures of the two proteins are shown in Table 3 and Figure 3.

The secondary structure of the recombinant collagen of No.1 and No.6 determined by infrared spectroscopy in table 3

2) Raman Spectroscopy Determination

After the recombinant collagens No.1 and No.6 in Table 1 were prepared into solutions, the Raman spectrum was measured, and the spectral data was smoothed and corrected with ThermoFisher Omnic9.2, and the spectral data in the 1700-1600cm- ¹ band was intercepted , used peakfit v4.12 to perform peak fitting of the second order derivative, plotted the processed data with orgin to obtain the secondary structure distribution, and calculated the relative content of the secondary structure. The comparison results of the secondary structures of the two proteins are shown in Table 4 and Figure 4.

The secondary structure of No.1 and No.6 recombinant collagen determined by Raman spectroscopy in table 4

It can be seen from the measurement results of Example 3 that the recombinant collagens with the same repeating unit but different repeating numbers have large differences in secondary structures, which also implies that there are differences in the tertiary structures of the two. It can be speculated that the spatial structure of Type I recombinant collagen No. 1-3 makes the surface charge more balanced, thus showing good stability in aqueous solution.

Example 4 The heat-resistant degradation test of the collagen injection containing the type I recombinant collagen of the present invention

Test 1

Experimental group 1: Dissolve 8g/L type LI recombinant collagen (No.2) and 5g/L cross-linked sodium hyaluronate in an appropriate amount of sodium chloride injection, and stir at room temperature until the volume of the components is 1L. Dissolve evenly. Adjust the pH value to 7.0±1.0 and the osmotic pressure to 300±100mOsmol/L with an appropriate amount of phosphate/saline buffer. After being filtered through a filter membrane, it is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 20 minutes.

Experimental group 2: Based on the volume of the components being 1L, 8g/L type LI recombinant collagen (No.1) and 5g/L cross-linked sodium hyaluronate were dissolved in an appropriate amount of sodium chloride injection, and stirred at room temperature until Dissolve evenly. Adjust the pH value to 7.0±1.0 and the osmotic pressure to 300±100mOsmol/L with an appropriate amount of phosphate/saline buffer. After being filtered through a filter membrane, it is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 20 minutes.

Experimental group 3: Based on the volume of the components as 1L, 8g/L type LI recombinant collagen (No.3) and 5g/L cross-linked sodium hyaluronate were dissolved in an appropriate amount of sodium chloride injection, and stirred at room temperature until Dissolve evenly. Adjust the pH value to 7.0±1.0 and the osmotic pressure to 300±100mOsmol/L with an appropriate amount of phosphate/saline buffer. After being filtered through a filter membrane, it is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 20 minutes.

Control group 1: Based on the volume of the components as 1L, 8g/L type LI recombinant collagen (No.4) and 5g/L cross-linked sodium hyaluronate were dissolved in an appropriate amount of sodium chloride injection, and stirred at room temperature until Dissolve evenly. Adjust the pH value to 7.0±1.0 and the osmotic pressure to 300±100mOsmol/L with an appropriate amount of phosphate/saline buffer. After being filtered through a filter membrane, it is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 20 minutes.

Control group 2: Based on the volume of the components being 1L, 8g/L type LI recombinant collagen (No.7) and 5g/L cross-linked sodium hyaluronate were dissolved in an appropriate amount of sodium chloride injection, and stirred at room temperature until Dissolve evenly. with the right amount Phosphate/saline buffer was used to adjust the pH to 7.0±1.0 and the osmolarity to 300±100 mOsmol/L. After being filtered through a filter membrane, it is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 20 minutes.

The collagen injection after damp heat sterilization and purified water were prepared as a test solution at a ratio of 1:20, and then subjected to SDS-PAGE gel electrophoresis to confirm the molecular weight and degradation, so as to characterize the thermal degradation of type I recombinant collagen The degree of electrophoresis is shown in Figure 5. It can be seen from Figure 5 that after moist heat sterilization, the type I recombinant collagen in the collagen injection of the experimental group basically did not undergo thermal degradation, while about 50% of the type I recombinant collagen in the collagen injection of the control group occurred thermal degradation.

Claims

A macromolecular type I recombinant collagen, which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is such as SEQ ID No.: 1 (G A P G A P G S Q G A P G L Q), the number of repetitions is 75 to 110 times, preferably 80 to 105 times, more preferably 82 to 102 times.
A collagen injection, which includes macromolecular type I recombinant collagen, water for injection and auxiliary materials, and the macromolecular type I recombinant collagen is repeated multiple times by a short amino acid sequence from natural human type I collagen as a repeating unit And constitute, wherein, the short amino acid sequence as shown in SEQ ID No.: 1 (GA P G A P G S Q G A P GL Q), the number of repetitions is 75 to 110 times, preferably 80 to 105 times , more preferably 82 to 102 times.
The collagen injection according to claim 2, wherein, the auxiliary material is selected from sodium hyaluronate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polylactic acid, glycine, alanine, proline One or more of acid, carnosine, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.
The collagen injection according to claim 2, which does not contain a cross-linking agent component.
The collagen injection according to claim 2, which is used for alleviating skin aging and correcting skin folds.
A use of a macromolecular type I recombinant collagen in the preparation of collagen injections, wherein the macromolecular type I recombinant collagen consists of a short amino acid sequence from natural human type I collagen that is repeated multiple times as a repeating unit. Composition, the short amino acid sequence is shown in SEQ ID No.: 1 (GA P G A P G S Q G A P G L Q), the number of repetitions is 75 to 110 times, preferably 80 to 105 times, more preferably 82 to 102 times;

The collagen injection comprises the macromolecule type I recombinant collagen, water for injection and auxiliary materials;

The collagen injection has been sterilized by moist heat.
The use according to claim 6, wherein the conditions of the moist heat sterilization are 110-130° C. for 20-60 minutes.
The use according to claim 6, wherein the auxiliary material is selected from sodium hyaluronate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polylactic acid, glycine, alanine, proline, carnosine , potassium dihydrogen phosphate, and dipotassium hydrogen phosphate, or one or more of them.
The use according to claims 2-9, wherein the collagen injection does not contain a cross-linking agent component.
The method for preparing collagen injection according to claim 2, comprising: obtaining a mixed solution of the macromolecular type I recombinant collagen, water for injection and auxiliary materials, and performing moist heat sterilization on the mixed solution. The conditions for the moist heat sterilization are, for example, 110-130° C. for 20-60 minutes.