CN114369156B

CN114369156B - Injection containing stabilized macromolecular type I recombinant collagen

Info

Publication number: CN114369156B
Application number: CN202210100917.3A
Authority: CN
Inventors: 范代娣; 王小军; 罗培培; 严建亚; 刘琳
Original assignee: Shaanxi Giant Biogene Technology Co ltd
Current assignee: Shaanxi Giant Biogene Technology Co ltd
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2023-04-25
Anticipated expiration: 2042-01-27
Also published as: CN114369156A; WO2023143394A1

Abstract

The invention relates to an injection containing stable macromolecular type I recombinant collagen. The collagen injection comprises macromolecular type I recombinant collagen, water for injection and auxiliary materials, wherein the macromolecular type I recombinant collagen is formed by repeating a plurality of times by taking a short amino acid sequence (G A P G A P G S Q G A P G L Q) from natural human type I collagen as a repeating unit, and the repeating times are 75-110 times. The collagen injection disclosed by the invention can be subjected to damp-heat sterilization under the condition that the macromolecular type I recombinant collagen is not crosslinked by using a crosslinking agent, so that the toxicity of the crosslinking agent residue in the collagen injection product to a human body can be effectively avoided.

Description

Injection containing stabilized macromolecular type I recombinant collagen

Technical Field

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

Background

Collagen is a biological high molecular protein, is the main component in animal connective tissue, and is the functional protein with the greatest content and the greatest distribution in the mammal body, and accounts for 25% -30% of the total protein. Collagen has close relation with formation, maturation, intercellular information transmission, joint lubrication, wound healing, calcification, blood coagulation, aging and the like, is one of the most critical raw materials in the biotechnology industry, and has wide application in medical materials, cosmetics and food industry.

Natural human collagen is limited in source, and the natural collagen used in industry at present is mainly extracted from animal skin or bones by an acid, alkali or enzyme method, and the main source of the natural collagen is animal connective tissue. However, collagen extracted from animal tissues is at risk of diseases of animal origin and the like, and large-scale production causes great stress on animal feeding at the supply side.

With the wide application of genetic engineering technology, the bottleneck problem of large-scale preparation of human collagen is successfully solved by adopting proper engineering strains (such as escherichia coli, pichia pastoris and the like) to exogenously express the human collagen. However, when human collagen is expressed by using an engineering strain such as a pichia pastoris gene engineering strain, the human collagen is degraded during fermentation, purification and preservation, which increases the production cost and affects the performance of the human collagen produced by the method.

It is presumed that the reason for degradation of human collagen is that the amino acid sequence of human collagen contains many sites which are susceptible to hydrolysis. Therefore, the skilled artisan constructs recombinant collagen by selecting a short amino acid sequence from natural human collagen for repetition in an effort to avoid sites where hydrolysis is likely to occur, thereby improving the stability of collagen while maintaining the superior properties of natural human collagen. However, recombinant collagen constructed by repeating short amino acid sequences derived from natural human collagen has relatively monotonic amino acid composition and distribution, and in theory, this causes a large charge load on the surface, and the whole is not easy to reach a stable equilibrium state, so that hydrolysis and denaturation are easy in an aqueous solution, and the shorter the short amino acid sequence repeating unit, the more the number of times of repetition, the more unstable the recombinant collagen molecule in the aqueous solution tends to be.

It is possible to attempt to obtain recombinant collagen more resistant to degradation by mutating a short amino acid sequence derived from natural collagen and taking this as a repeating unit. However, the recombinant collagen obtained by mutation modification has reduced homology with natural human collagen, and may have an immunogenicity problem, so that it is not suitable for use in biological materials requiring long-term contact with human body.

On the other hand, tissue engineering materials such as subcutaneous fillers and the like are an important application direction of human collagen, and as tissue engineering materials, collagen is required to have good mechanical strength and stability in aqueous solutions (to be stored in aqueous solutions for a long period of 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 repetition of short amino acid sequences from natural human collagen.

From the practical standpoint, the molecular weight of collagen suitable for replacing natural human collagen as a tissue engineering material is generally considered to be more than 100kD by the skilled person. However, as described above, natural human collagen is limited in source, animal-derived collagen is at risk of transmitting diseases, genetically engineered human collagen is easily degraded during fermentation, purification and preservation, recombinant collagen constructed repeatedly by short amino acid sequences derived from natural human collagen is unstable in aqueous solution, and recombinant collagen obtained by mutation modification has an immunogenicity problem, so how to obtain collagen suitable for replacing natural human collagen as a tissue engineering material is a limiting problem in the art.

In addition, since the 70 s of the 20 th century, injectable bovine collagen was studied and passed FDA certification in 1981 for soft tissue filling, which restores the volume and elasticity of soft tissue by injection or filling to achieve cosmetic effects. The collagen has high safety and degradability, and can be used for restoring lips, repairing facial wrinkles and other soft tissue contour loss treatments.

Up to now, collagen injection has been widely used in the field of medical cosmetology. Based on the safety requirements of the product, the product needs to reach a sterile level, so various forms of collagen injections must be sterilized after encapsulation to kill the (pathogenic) microorganisms present therein. Aiming at the characteristics of collagen macromolecules and variability, from the viewpoints of sterilization effect and product performance after sterilization meeting requirements, the wet heat sterilization is more suitable for the sterilization of the products, and the sterilization of pathogenic microorganisms is most effective, but collagen is thermolabile, the temperature condition of wet heat sterilization (usually 121 ℃) can cause the thermal degradation of the collagen, the whole structure is separated and broken, the viscous liquid is changed into a water sample, and the biological activity and mechanical property are lost.

Crosslinking is known to be advantageous for collagen against thermal degradation, but for collagen injection products, the crosslinking agents used for crosslinking (e.g., glutaraldehyde, etc.) eventually enter with collagen and exist in the human body for a long period of time, and these crosslinking agents may be toxic to the human body. Accordingly, it is always desirable for those skilled in the art to find recombinant collagen that can withstand damp heat sterilization without cross-linking.

Disclosure of Invention

The inventors have conducted intensive studies in order to solve the above-mentioned technical problems existing in the prior art. The inventor firstly conducts technical literature investigation on recombinant collagen repeatedly constructed through short amino acid sequences from natural human collagen, selects some short amino acid sequences from natural human type I collagen (tissue engineering materials with the most wide application) in the prior art, then respectively uses the short amino acid sequences as repeated units to construct type I recombinant collagen with different molecular weights, and examines the stability of the type I recombinant collagen in aqueous solution for long-term storage so as to obtain large molecular weight (more than 100 kD) recombinant collagen which can be stably stored in aqueous solution for long time and can meet the mechanical strength requirement as the tissue engineering materials.

As a result of the above-described studies, the inventors have unexpectedly found that a type I recombinant collagen obtained by repeating 75 to 110 times a pentadecapeptide (G A P G A P G S Q G A P G L Q) derived from natural human type I collagen has an exceptionally excellent stability. The concrete steps are as follows: (1) Although the length of its repeat unit is the shortest of all recombinant collagens tested by the inventors, its stability in aqueous solution is the best; it is generally believed that the shorter the repeating unit, the more monotonic the amino acid composition and distribution, and the greater the surface charge loading of the recombinant collagen thus constructed, the less likely it is to reach a stable equilibrium state and thus more prone to hydrolysis. (2) It is even more stable than the type I recombinant collagen obtained by repeating the pentadecapeptide 52 times or 62 times or 72 times, and it is generally considered that the larger the number of repetitions, the larger the molecular weight, the larger the surface charge load of the recombinant collagen, the less likely it is to reach a stable equilibrium state, and thus the more easily hydrolyzed.

Based on the above findings, the inventors have completed the present invention. Namely, the invention comprises the following steps:

1. a macromolecular type I recombinant collagen is formed by repeating a plurality of times by taking a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is shown as SEQ ID No.1 (G A P G A P G S Q G A P G L Q) and the number of times of repetition is 75-110.

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

3. The macromolecular type I recombinant collagen according to item 1 having a molecular weight of 100kD or more.

4. The macromolecular type I recombinant collagen according to item 1, which is further provided with a tag that makes it easy to purify.

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. The 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 subcutaneous fillers, artificial bones, artificial skin, orally absorbable biological membranes, bone implants, vascular stents, intercellular scaffolds, and collagen sponges.

8. The use of the macromolecular type I recombinant collagen according to any one of items 1 to 5 as subcutaneous filler, artificial bone, artificial skin, orally absorbable biofilm, bone implant, vascular scaffold, cell matrix scaffold or collagen sponge.

9. An aqueous collagen solution comprising the macromolecular type I recombinant collagen according to any one of claims 1 to 5.

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

The inventors have made intensive studies on the reason why the macromolecular type I recombinant collagen of the present invention has an exceptionally excellent stability. Preliminary research results indicate that this may be due to:

the recombinant collagen which uses a certain amino acid sequence as a repeating segment to repeat has the stability closely related to the surface charge, the surface charge is related to the amino acid composition and the space structure of the protein, and a certain space structure is just formed after a certain number of times of repetition is achieved, so that the surface load is in a balanced or near-balanced state, and therefore, the abnormal stable state can be shown. The inventor just finds that the 15 amino acid repeated sequence collagen is in the load balance range, so the macromolecular type I recombinant collagen has extremely excellent stability.

In addition, the inventors have found that the macromolecular type I recombinant collagen of the present invention has particularly excellent heat resistance, can withstand damp heat sterilization (e.g., 121 ℃ for 20 min) without thermal degradation even without crosslinking, and exhibits good properties suitable for use as a collagen injection.

Thus, the present invention also includes:

2-1. The collagen injection comprises macromolecular type I recombinant collagen, water for injection and auxiliary materials, wherein the macromolecular type I recombinant collagen is formed by repeatedly taking a short amino acid sequence from natural type I collagen as a repeating unit, and the number of times of repetition is 75-110 as shown in SEQ ID No.1 (G A P G A P G S Q G A P G L Q).

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

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

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

The collagen injection according to claim 2-4, wherein the large molecule type I recombinant collagen has a His tag, a Flag tag or a c-Myc tag which makes it easy to purify.

2-6 the collagen injection according to claim 2-1, wherein the auxiliary material is one or more 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.

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

2-8 the collagen injection according to claim 2-1 for use in alleviating skin aging and correcting skin wrinkles.

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

the macromolecular type I recombinant collagen is formed by repeating a short amino acid sequence from natural human type I collagen as a repeating unit for a plurality of times, wherein the short amino acid sequence is shown as SEQ ID No.1 (G A P G A P G S Q G A P G L Q), and the repeating times are 75-110 times;

the collagen injection is subjected to damp-heat sterilization.

Use according to claims 2-10, wherein the number of repetitions is 80-105, preferably 82-102.

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

Use according to claims 2-12, wherein the macromolecular type I recombinant collagen is further provided with a tag which makes it easy to purify.

Use according to claims 2-13, wherein the macromolecular type I recombinant collagen carries a tag that makes it easy to purify a His tag, a Flag tag or a c-Myc tag.

Use according to claims 2-14, wherein the conditions of the wet heat sterilization are 110-130 ℃ for 20-60 min.

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

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

Use according to claims 2-17, wherein the collagen injection does not comprise a cross-linker component. In the present specification, the crosslinker component refers to a substance derived from a crosslinker, and includes a crosslinker molecule itself, and also includes a molecule formed by a chemical reaction of a crosslinker.

Use according to claims 2-18, wherein the collagen injection is used for alleviating skin ageing and correcting skin wrinkles.

2-19 the method for preparing 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 carrying out damp-heat sterilization on the mixed solution.

The process according to claim 2 to 19, wherein the conditions for wet heat sterilization are 110 to 130℃for 20 to 60 minutes.

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

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

Drawings

FIG. 1 is a SDS-PAGE electrophoresis of a portion of the recombinant collagen type I prepared in example 1. Control proteins are exemplified by Nos. 4, 6, 7, 10.

Fig. 2 is an HPLC plot of a portion of the test specimen prepared in example 2 after 12 months of placement. Control proteins are exemplified by Nos. 4, 7, 10, 13.

FIG. 3 is an infrared spectrum of type I recombinant collagen of Nos. 1 and 6.

FIG. 4 is a Raman spectrum of type I recombinant collagen of Nos. 1 and 6.

FIG. 5 is an SDS-PAGE gel of a wet heat sterilized collagen injection.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the invention.

General description: all enzymes used in the specific embodiments were purchased from TaKaRa, plasmid DNA extraction kit and DNA gel recovery kit were purchased from Beijing Soy Bao, and the gene recombination kit (Reorganization Kits) was purchased from Tiangen organism, and the specific operations were performed exactly as described in the kit.

Example 1 preparation of various recombinant collagens type I Using Yeast expression System

1. Construction of Yeast expression Strain

Yeast expression strains respectively expressing type I recombinant collagens No.1 to 18 shown in Table 1 were constructed. The specific operation is as follows: synthesizing a corresponding target gene by a total gene synthesis mode after optimization according to pichia pastoris codon preference, adding SnaB I and Not I enzyme cutting sites at two ends of the gene respectively, carrying out double enzyme cutting on the target gene by SnaBI and Not I enzymes, connecting the target gene with pPIC9k which is also subjected to enzyme cutting by SnaB I and Not I enzymes under the action of T4 ligase, transferring the target gene into Top10 competent cells after overnight connection at 16 ℃, coating an ampicillin resistance plate, picking a positive transformant, carrying out linearization by SacI after extracting plasmids, carrying out electric shock transfer into Pichia pastoris GS115 competent cells, and screening multicopy transformants by G418 resistance plates to obtain the expression strain of the type I recombinant collagen.

Table 1 type I recombinant collagen expressed by each Yeast expression Strain

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2. Inducible expression of a protein of interest

(1) Single colonies of yeast expression strains were picked and added to 5ml YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubated overnight at 30℃and 200rpm for activation;

(2) Inoculating to 100ml BMGY liquid culture medium at 1% inoculum size, culturing at 30deg.C and 200rpm to OD ₆₀₀ ＝6.0～9.0；

(3) Collecting thallus by centrifugation at 25deg.C for 6min under the action of 1500g centrifugal force, and suspending in 200ml BMMY liquid culture medium to give initial concentration of OD ₆₀₀ =1.0, cultured at 30 ℃,200 rpm;

(4) Adding methanol with the final concentration of 0.5-1.0% every 24h, and performing induced expression;

(5) Inducing for 72h, centrifuging the culture solution at 12000rpm for 2min, and collecting supernatant.

3. Preparation of type I recombinant collagen

And (3) performing ultrafiltration concentration on the supernatant prepared by fermentation through a 30kD ultrafiltration membrane, performing column separation through a CM ion exchange column, eluting with 35% NaCl solution, collecting eluent, desalting, concentrating, and freeze-drying to obtain the type I recombinant collagen. 0.1g of the lyophilized powder was dissolved in 100ml of physiological saline, and subjected to SDS-PAGE gel electrophoresis after sufficient dissolution, to confirm the molecular weight and the purity of the protein.

The result shows that the 18 constructed expression bacteria can successfully express target proteins, the electrophoretic purity of the proteins prepared by separation and purification is over 99 percent, and the electrophoretic result is shown in figure 1.

Example 2: stability experiment of various type I recombinant collagens in aqueous solution

A test Material

The materials used in the experiments were type I recombinant collagen Nos. 1 to-18 prepared in example 1.

B experiment method

The experimental material in A was subjected to ddH ₂ O is prepared into a protein solution with the protein concentration of 1mg/mL, the protein solution is filtered by a sterile filter with the concentration of 0.22 mu m in an ultra-clean workbench, and then is packaged into a sterile centrifuge tube for sealing, and is sampled at 25+/-2 ℃ for 0 month, 6 months and 12 months respectively, 3 tubes are sampled each time, the protein purity is detected (the protein purity is measured by high performance liquid chromatography), and the stability of the protein is judged according to the purity change.

C experiment results

The test results are shown in the following table:

table 2 results of 12 months stability test of recombinant collagen solution (purity,%)

It is generally believed that (1) recombinant collagen proteins having the same or similar molecular weights have shorter repeating units, monotonic amino acid composition and distribution, and higher surface charge loading, and are less likely to reach a stable equilibrium state, and thus are more likely to hydrolyze; (2) The more the number of repetitions, the greater the molecular weight, the greater the surface charge loading of the recombinant collagen, and the less likely the recombinant collagen reaches a stable equilibrium state, and thus is more susceptible to hydrolysis.

However, as can be seen from Table 2, the recombinant collagen type I (Nos. 1 to 3) of the present invention shows an exceptionally excellent stability in aqueous solutions, which can be left in aqueous solutions for 12 months, and the purity can be still as high as 97% or more (see FIGS. 2A to G).

Example 3: type I recombinant collagen Nos. 1-3 have abnormal aqueous stability of the cause initial detection

1) Infrared spectrometry

After preparing the recombinant collagens No.1 and No.6 in Table 1 into solutions respectively, carrying out infrared spectrum measurement, carrying out Fourier deconvolution on spectrum data by Bruker OPUS7.2, and intercepting 1700-1600 cm ^-1 And (3) carrying out second derivative peak-dividing fitting treatment on band spectrum data by using peak v4.12, plotting the treated data by using orgin to obtain secondary structure distribution, and calculating the relative content of a secondary structure. The results of comparing the secondary structures of the two proteins are shown in Table 3 and FIG. 3.

TABLE 3 secondary Structure of recombinant collagen No.1 and No.6 measured by IR Spectroscopy

	β-sheet/％	random/％	α-helix/％	β-turn/％	R^2
						No.1	45.95	27.34	10.56	16.14	0.9998
No.6	34.01	34.74	13.79	17.45	0.9995

2) Raman spectroscopy

After preparing the recombinant collagens No.1 and No.6 in Table 1 into solutions, respectively, raman spectrometry was performed, the spectrum data was smoothed by using a ThermoFisher Omnic9.2, and 1700-1600 cm was cut ^-1 And (3) carrying out second derivative peak-dividing fitting treatment on band spectrum data by using peak v4.12, plotting the treated data by using orgin to obtain secondary structure distribution, and calculating the relative content of a secondary structure. The results of comparing the secondary structures of the two proteins are shown in Table 4 and FIG. 4.

Table 4 secondary structures of recombinant collagen Nos. 1 and 6 as measured by Raman Spectroscopy

	β-sheet/％	random/％	α-helix/％	β-turn/％	R^2
						No.1	37.96	35.78	13.08	13.18	0.9983
No.6	27.35	26.02	17.04	29.58	0.9966

As can be seen from the measurement results of example 3, recombinant collagens having the same repeating units but different repeating times have a large difference in secondary structure, which also suggests a difference in tertiary structure between them. It is presumed that the spatial structure of the type I recombinant collagens of Nos. 1 to 3 makes the surface charges more balanced, thereby exhibiting good stability in aqueous solutions.

Example 4 thermal degradation resistance test 1 of collagen injection containing recombinant collagen type I according to the present invention

Experiment group 1: based on the volume of 1L of the components, 8g/LI type recombinant collagen (No. 2) and 5g/L crosslinked sodium hyaluronate are dissolved in a proper amount of sodium chloride injection, and stirred at normal temperature until the components are dissolved uniformly. The pH was adjusted to 7.0.+ -. 1.0 with an appropriate amount of phosphate/physiological saline buffer and the osmotic pressure was adjusted to 300.+ -.100 mOsmol/L. Filtering with a filter membrane, filling into a prefilled syringe, and sterilizing at 121deg.C for 20min under moist heat.

Experiment group 2: based on the volume of 1L of the components, 8g/LI type recombinant collagen (No. 1) and 5g/L crosslinked sodium hyaluronate are dissolved in a proper amount of sodium chloride injection, and stirred at normal temperature until the components are dissolved uniformly. The pH was adjusted to 7.0.+ -. 1.0 with an appropriate amount of phosphate/physiological saline buffer and the osmotic pressure was adjusted to 300.+ -.100 mOsmol/L. Filtering with a filter membrane, filling into a prefilled syringe, and sterilizing at 121deg.C for 20min under moist heat.

Experiment group 3: based on the volume of 1L of the components, 8g/LI type recombinant collagen (No. 3) and 5g/L crosslinked sodium hyaluronate are dissolved in a proper amount of sodium chloride injection, and stirred at normal temperature until the components are dissolved uniformly. The pH was adjusted to 7.0.+ -. 1.0 with an appropriate amount of phosphate/physiological saline buffer and the osmotic pressure was adjusted to 300.+ -.100 mOsmol/L. Filtering with a filter membrane, filling into a prefilled syringe, and sterilizing at 121deg.C for 20min under moist heat.

Control group 1: based on the volume of 1L of the components, 8g/LI type recombinant collagen (No. 4) and 5g/L crosslinked sodium hyaluronate are dissolved in a proper amount of sodium chloride injection, and stirred at normal temperature until the components are dissolved uniformly. The pH was adjusted to 7.0.+ -. 1.0 with an appropriate amount of phosphate/physiological saline buffer and the osmotic pressure was adjusted to 300.+ -.100 mOsmol/L. Filtering with a filter membrane, filling into a prefilled syringe, and sterilizing at 121deg.C for 20min under moist heat.

Control group 2: based on the volume of 1L of the components, 8g/LI type recombinant collagen (No. 7) and 5g/L crosslinked sodium hyaluronate are dissolved in a proper amount of sodium chloride injection, and stirred at normal temperature until the components are dissolved uniformly. The pH was adjusted to 7.0.+ -. 1.0 with an appropriate amount of phosphate/physiological saline buffer and the osmotic pressure was adjusted to 300.+ -.100 mOsmol/L. Filtering with a filter membrane, filling into a prefilled syringe, and sterilizing at 121deg.C for 20min under moist heat.

Preparing a sample solution of which the ratio is 1:20 by using the collagen injection subjected to damp-heat sterilization and purified water, then carrying out SDS-PAGE gel electrophoresis, and confirming the molecular weight and degradation condition, thereby representing the degree of thermal degradation of the type I recombinant collagen, and the electrophoresis result is shown in figure 5. As can be seen from fig. 5, after the wet heat sterilization, the type I recombinant collagen in the collagen injection of the experimental group was not substantially thermally degraded, while the type I recombinant collagen in the collagen injection of the control group was about 50% thermally degraded.

Sequence listing

<110> Shaanxi giant biotechnology Co.Ltd

<120> injection comprising stabilized macromolecular type I recombinant collagen

<130> TPF02060

<141> 2022-01-27

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Homo sapiens

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Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln

1 5 10 15

Claims

1. The collagen injection comprises macromolecular type I recombinant collagen, water for injection and auxiliary materials, wherein the macromolecular type I recombinant collagen is formed by repeatedly taking a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is shown as SEQ ID No.1 (G A P G A P G S Q G A P G L Q), and the number of times of repetition is 82;

the collagen injection is subjected to damp-heat sterilization;

the collagen injection does not contain a cross-linking agent component.

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

3. The collagen injection according to claim 1, which is used for relieving skin aging and correcting skin wrinkles.

4. The application of the macromolecular type I recombinant collagen in preparing the collagen injection comprises the steps of repeatedly taking a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is shown as SEQ ID No.1 (GAP G AP G S Q G AP G L Q), and the number of times of repetition is 82;

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

the collagen injection is subjected to damp-heat sterilization;

the collagen injection does not contain a cross-linking agent component.

5. The use according to claim 4, wherein the conditions of the wet heat sterilization are 110 to 130 ℃ for 20 to 60min.

6. The use according to claim 4, wherein the auxiliary material is one or more selected from sodium hyaluronate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, polylactic acid, glycine, alanine, proline, carnosine, potassium dihydrogen phosphate, dipotassium hydrogen phosphate.

7. The method for preparing the collagen injection as claimed in claim 1, which comprises the following steps: obtaining the mixed solution of the macromolecular type I recombinant collagen, water for injection and auxiliary materials, and carrying out damp-heat sterilization on the mixed solution.

8. The process according to claim 7, wherein the wet heat sterilization is carried out at a temperature of 110 to 130℃for 20 to 60 minutes.