Recombinant human collagen with good cross-linking effect

Abstract

The invention relates to a recombinant human collagen with a relatively good cross-linking effect. The amino acid sequence of the recombinant human collagen is a stable network structure is formed, and structural defects caused by animal source differences are also avoided.

Description

Technical Field

[0001] The present invention relates to the field of collagen, and in particular to a recombinant human collagen with good cross-linking effect. Background Art

[0002] Collagen is one of the most abundant proteins in the human body. It forms a support network within the cell structure, maintaining the stability and strength of body tissues. With the growing market for recombinant collagen products in food, functional health supplements, skincare, and medical devices, particularly medical dressings, there is a growing demand for high-quality and high-volume collagen.

[0003] Currently, mainstream collagen is generally extracted from animal tissues such as cows and pigs. However, collagen from different sources and types has different amino acid compositions, sequences and spatial structures, which leads to incomplete collagen structure or lack of certain key sites, affecting the cross-linking effect.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute prior art known to ordinary technicians in the field. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention discloses a recombinant human collagen with better cross-linking effect.

[0006] The technical solutions adopted in the present invention are as follows: A recombinant human collagen with good cross-linking effect, the amino acid sequence of the recombinant human collagen with good cross-linking effect is shown as SEQ ID NO 1.

[0007] A cross-linking method for recombinant human collagen with good cross-linking effect comprises the following steps: Step S1, preparing a collagen solution; Step S2, adding NHS to the collagen solution and stirring; Step S3, adding EDC to the collagen solution and stirring; Step S4, allowing the collagen solution to stand; Step S5, obtaining a finished product.

[0008] Furthermore, in step S2, the stirring time ranges from 3 to 5 minutes.

[0009] Furthermore, in step S2, the collagen solution is slowly stirred or pipetted to mix at room temperature, and then allowed to stand to solidify.

[0010] Furthermore, in step S3, the stirring time ranges from 3 to 5 minutes.

[0011] Furthermore, in step S3, the collagen solution is slowly stirred or pipetted to mix at room temperature.

[0012] Furthermore, in step S4, the standing time ranges from 2 to 4 hours.

[0013] Furthermore, in step S4, in the mixed solution, the ratio of collagen, NHS, and EDC is in the range of 1:0.7-0.4:0.1-0.15.

[0014] The beneficial effects of the present invention are as follows: 1. Optimizing the amino acid sequence of collagen through recombination provides more active sites for cross-linking reactions, which is conducive to forming more covalent bonds to connect protein molecules, thereby enhancing the cross-linking effect of collagen, forming a stable network structure, and avoiding structural defects introduced by differences in animal sources.

[0015] 2. EDC and NHS have a synergistic effect in the cross-linking reaction. EDC first activates the carboxyl group, and NHS then reacts with the activated carboxyl group to generate a more stable active intermediate. The two cooperate with each other to enable the cross-linking reaction to proceed more orderly and efficiently. At a specific ratio, the synergistic effect of EDC and NHS can reach a better equilibrium state, so that the carboxyl groups on the collagen molecules can be moderately activated and converted into stable reaction intermediates. These intermediates can react with amino groups and other groups on the collagen molecules at an appropriate rate and degree to form a stable cross-linking structure, thereby improving the cross-linking effect.

[0016] 3. As cross-linking auxiliary agents, EDC and NHS need to be evenly dispersed in the collagen solution to fully contact and function with the collagen molecules. If the stirring time is too short, EDC and NHS may not be completely dissolved or evenly dispersed in the collagen solution, resulting in local concentrations that are too high or too low, affecting the uniformity of the cross-linking reaction. An appropriate stirring time is usually sufficient to achieve a good solubility and dispersion of EDC and NHS in the solution, ensuring that they can interact with the collagen molecules more evenly throughout the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the steps of the cross-linking method of recombinant human collagen.

[0018] Figure 2 This is the thromboelastogram of collagen in Example 1.

[0019] Figure 3 This is the thromboelastogram of collagen in control group 1. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention will be described below with reference to the accompanying drawings.

[0021] Example 1: The amino acid sequence of a recombinant human collagen with good cross-linking effect is shown in SEQ ID NO 1.

[0022] The specific amino acid sequence is: gppgpvgltgrpgpvglpghpglkgeegaegpqgprglqgphgppgrvgkmgrpgadgarglpgdtgpkgdrgfdglpglpgekgqrgdfghvgqpgppged gppgpvgltgrpgpvglpghpglkgeegaegpqgprglqgphgppgrvgkmgrpgadgarglpgdtgpkgdrgfdglpglpgekgqrgdfghvgqpgppged gpagpmgltgrpgpvgppgsgglkgepgdvgpqgprgvqgppgpagkpgrrgragsdgargmpgqtgpkgdrgfdglaglpgekghrgdpgpsgppgppgdd gpqgprgdkgetgergaagikghrgfpgnpgapgspgpagqqgaigspgpagprgpvgpsgppgkdgtsghpgpigppgprgnrgergsegspghpgqpgpp.

[0023] Example 2: A cross-linking method for recombinant human collagen with better cross-linking effect, such as Figure 1 As shown, the following steps are included: Step S1: preparing collagen solution.

[0024] Step S2: Add NHS to the collagen solution and stir for 3-5 minutes. Slowly stir or pipette to mix the collagen solution at room temperature, then let it stand and wait for the collagen solution to solidify. Step S3: Add EDC to the collagen solution and stir for 3 to 5 minutes. Slowly stir or pipette to mix the collagen solution at room temperature.

[0025] Step S4: Let the collagen solution stand for 2 to 4 hours. In the mixed solution, the ratio of collagen, NHS and EDC is 1:0.7 to 0.4:0.1 to 0.15. In this embodiment, the ratio of the three is 1:0.5:0.1. Step S5: Obtain the finished product.

[0026] Product cross-linking expression test: (1) Chassis cells Platform ID: Bio-52827.

[0027] Specifications: Glycerol / culture.

[0028] Genus name: Corynebacterium Glutamicum.

[0029] Strain name: Corynebacterium glutamicum.

[0030] Other numbers: ACCC16522=ATCC13032.

[0031] Culture medium number: 2.

[0032] Culture temperature: 30℃.

[0033] Cultivation time: 24-48 hours.

[0034] The treated sample of Example 1 was tested by thromboelastometry, and the results were as follows: Figure 2 shown.

[0035] Analysis of test results: The thromboelastometry curve of the sample in Example 1 has a larger opening, which further indicates that the fibrin cross-links to form a network. The larger the opening, the higher the hardness of the recombinant collagen cross-links and the higher the degree of cross-linking, which proves that the cross-linking effect of the recombinant collagen cross-links is better.

[0036] Control group 1: animal-derived raw protein, which was tested in the same manner as the sample in Example 1.

[0037] Analysis of test results: The thromboelastometry curve of the sample in control group 1 was a straight line with no opening, indicating that the blood coagulation process was not initiated, fibrin could not be cross-linked, and thus a fibrin network could not be formed.

[0038] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0039] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A recombinant human collagen with good cross-linking effect, characterized by: The amino acid sequence of the recombinant human collagen with better cross-linking effect is shown in SEQ ID NO 1.

2. A cross-linking method for recombinant human collagen with good cross-linking effect as claimed in claim 1, characterized in that: The following steps are involved: Step S1, preparing collagen solution; Step S2, adding NHS to the collagen solution and stirring; Step S3, adding EDC to the collagen solution and stirring; Step S4, allowing the collagen solution to stand; Step S5: Obtain the finished product.

3. The cross-linking method according to claim 2, wherein: In step S2, the stirring time ranges from 3 to 5 minutes.

4. The cross-linking method according to claim 3, wherein: In step S2, the collagen solution is slowly stirred or pipetted to mix at room temperature, and then allowed to stand to solidify.

5. The cross-linking method according to claim 2, wherein: In step S3, the stirring time ranges from 3 to 5 minutes.

6. The cross-linking method according to claim 5, wherein: In step S3, the collagen solution is slowly stirred or pipetted to mix at room temperature.

7. The cross-linking method according to claim 2, wherein: In step S4, the standing time ranges from 2 to 4 hours.

8. The cross-linking method according to claim 2, wherein: In step S4, in the mixed solution, the ratio of collagen, NHS, and EDC ranges from 1:0.7 to 0.4:0.1 to 0.15.

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