Crosslinked recombinant collagen marker polypeptide and application thereof

Abstract

The invention discloses a cross-linked recombinant collagen marker polypeptide and application thereof, and belongs to the technical field of collagen detection. The amino acid sequence of the cross-linked recombinant collagen marker polypeptide is GESGPAGPAGAPGPAGSR. The cross-linked recombinant collagen marker polypeptide has the advantages that the cross-linked recombinant collagen marker polypeptide can be used for The cross-linked recombinant collagen marker polypeptide provided by the invention has strong specificity. The cross-linked recombinant collagen enzymatic hydrolysate is taken as a standard substance, a marker polypeptide is taken as a detection target, and the provided detection method is simple to operate and high in sensitivity, and can be used for quantitative analysis of the cross-linked recombinant collagen; the method can be used for quality control of a crosslinked recombinant collagen product and residual quantity analysis and degradation process evaluation of the crosslinked recombinant collagen product in animal bodies. The cross-linked recombinant collagen marker polypeptide provided by the invention has uniqueness, and a qualitative and quantitative detection method established by taking the marker polypeptide as a marker and a quantitative standard substance has important significance on quality control of a cross-linked recombinant collagen product.

Description

Technical Field

[0001] The present invention relates to the technical field of collagen detection, and in particular to a cross-linked recombinant collagen characteristic polypeptide and application thereof. Background Art

[0002] Cross-linked recombinant collagen refers to the product of combining collagen molecules by chemical or physical means. Cross-linking methods are mainly divided into physical cross-linking and chemical cross-linking. Physical cross-linking is to change the spatial arrangement and structure of collagen molecules through methods such as freeze-drying, ultraviolet light irradiation and heat treatment to form a cross-linked network, thereby enhancing its stability and biocompatibility. The advantage of this method is that it is simple to operate and does not require the introduction of chemical substances, but the disadvantage is that the degree of cross-linking is difficult to control. Chemical cross-linking is to introduce chemical cross-linking agents into collagen molecules to cause covalent bonding between collagen molecules, thereby enhancing the mechanical properties and anti-degradation ability of collagen. The advantage of chemical cross-linking is that the degree of cross-linking and the position of cross-linking can be precisely controlled, and the disadvantage is that toxic substances may be introduced or by-products may be produced.

[0003] Cross-linked recombinant collagen is widely used as an in vivo implant material due to its relatively long degradation cycle, and is applied in the fields of tissue repair and regeneration. After cross-linked recombinant collagen is implanted in the body, its degradation rate is the key to affecting the application effect. If the degradation rate is too fast, it may not play a role in mechanical support, and if the degradation is too slow, it may affect tissue repair and regeneration. Therefore, the dynamic evaluation of the in vivo degradation of recombinant collagen is an important means to predict and evaluate its expected clinical effect. Metals, ceramics, polymer materials and other materials that are difficult to degrade in the body can be evaluated by traditional methods, such as micro-computed tomography (micro-CT), mass loss method and HE staining. However, after cross-linked recombinant collagen is implanted in animals, it will merge with the animal's own tissues, gradually be degraded by proteases in the tissues and replaced by new tissues. The remaining materials will lose their original structure and shape, and it is impossible to separate them from the animal's own tissues. Therefore, traditional methods cannot analyze the residual amount of cross-linked recombinant collagen implanted in animals and evaluate its degradation process.

[0004] Traditional collagen detection methods mainly include: hydroxyproline method, Sirius red staining method, ELISA and other methods. The hydroxyproline quantitative detection method is based on the ratio between hydroxyproline and collagen, and converts the hydroxyproline content into collagen content. All collagens from animal sources contain hydroxyproline. This method can only quantify the total collagen content, and cannot distinguish between the collagen of the animal tissue itself and the recombinant collagen implanted into the body. Moreover, single-chain recombinant collagen does not contain hydroxyproline. Therefore, the hydroxyproline method cannot be used for quantitative analysis of recombinant collagen implanted into animals. The Sirius red staining method can only distinguish different types of collagen, but cannot distinguish collagen from different sources. The ELISA method is based on the binding reaction between antigen and antibody for determination, and recombinant collagen may not be full-length collagen, and some recombinant collagen is only multiple repetitions of an amino acid sequence, which can easily lead to false negative results. There are literature reports on the use of biotin labeling to detect recombinant collagen implanted in rats, but this method can only perform qualitative analysis and cannot achieve accurate quantitative analysis. Therefore, existing detection methods are unable to achieve qualitative and quantitative detection of cross-linked recombinant collagen and residual analysis of cross-linked recombinant collagen implanted in animals. Summary of the invention

[0005] The purpose of the present invention is to provide a cross-linked recombinant collagen characteristic polypeptide and its application to solve the problems existing in the above-mentioned prior art. The cross-linked recombinant collagen characteristic polypeptide provided by the present invention has strong specificity and uniqueness, and can be used for quality control of cross-linked recombinant collagen products and residue analysis and degradation process evaluation in animals.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a cross-linked recombinant collagen characteristic polypeptide, wherein the amino acid sequence of the cross-linked recombinant collagen characteristic polypeptide is GESGPAGPAGAPGPAGSR.

[0008] The present invention also provides the use of the cross-linked recombinant collagen characteristic polypeptide in any of the following items:

[0009] (1) Application in quality control of cross-linked recombinant collagen products;

[0010] (2) Application in the preparation of detection products for quality control of cross-linked recombinant collagen products;

[0011] (3) Application in the preparation of products for analyzing the in vivo residual content and / or degradation process of cross-linked recombinant collagen products;

[0012] (4) Application in the preparation of products for distinguishing cross-linked recombinant collagen from collagen of animal origin.

[0013] Optionally, the cross-linked recombinant collagen characteristic polypeptide is used as a standard to detect the cross-linked recombinant collagen product, to perform quality control on the cross-linked recombinant collagen product or to analyze the in vivo residue and / or degradation process.

[0014] Optionally, the cross-linked recombinant collagen characteristic polypeptide is used as a standard to detect the cross-linked recombinant collagen product, comprising the following steps:

[0015] After dissolving the cross-linked recombinant collagen characteristic polypeptide, a series of standard concentration working solutions are prepared;

[0016] The cross-linked recombinant collagen product to be tested is dissolved and trypsin is added for enzymatic hydrolysis. The enzymatic hydrolysis solution is centrifuged and the supernatant is taken as the test solution;

[0017] The standard working solution and the test solution are injected into a liquid chromatography-mass spectrometer for detection to determine whether the cross-linked recombinant collagen product to be tested contains the cross-linked recombinant collagen characteristic polypeptide or to quantitatively detect the content of recombinant collagen in the cross-linked recombinant collagen product to be tested.

[0018] Optionally, in the liquid chromatography-mass spectrometer, the liquid phase conditions are: chromatographic column: Zorbax C18 column; mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: water containing 0.1% formic acid and 60% acetonitrile; gradient elution: 0-2 min, 5%-15% B; 2-6 min, 15%-90% B; 6-8 min, 90% B; 8-8.1 min, 90%-5% B; 8.1-10 min, 5% B.

[0019] Optionally, in the liquid chromatography-mass spectrometer, mass spectrometry analysis uses electrospray ionization, and the ionization conditions are: m / z746.94→641.20, 994.80;

[0020] The mass spectrometry conditions were as follows: ion source spray voltage of 3.5 kV, capillary temperature of 320 °C, evaporation temperature of 350 °C, sheath gas flow rate of 19.8 mL / min, and auxiliary flow rate of 0.55 bar; positive ion mode scanning, SRM monitoring mode: m / z 746.94→641.20, m / z 732.84→994.80.

[0021] Optionally, if the retention time and daughter ions of the target ion in the cross-linked recombinant collagen product to be tested are consistent with the retention time and daughter ions of the cross-linked recombinant collagen characteristic polypeptide standard, it is determined that the cross-linked recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide.

[0022] Optionally, the content of recombinant collagen in the cross-linked recombinant collagen product to be tested is calculated based on the correspondence between the characteristic polypeptide of the cross-linked recombinant collagen and the molecular weight of the cross-linked recombinant collagen product.

[0023] The calculation formula is:

[0024]

[0025] Wherein, Mi is the content of cross-linked recombinant collagen in the sample to be tested, μg / mg; X is the concentration of characteristic polypeptide of cross-linked recombinant collagen in the sample to be tested, μg / mL; V is the final volume of the sample, mL; M1 is the molecular weight of cross-linked recombinant collagen, Da; M2 is the molecular weight of characteristic polypeptide of cross-linked recombinant collagen, Da; m is the mass of the sample to be tested, mg; n is the number of repetitions of characteristic polypeptide in cross-linked recombinant collagen.

[0026] Optionally, the cross-linked recombinant collagen product includes a recombinant collagen implant.

[0027] The present invention discloses the following technical effects:

[0028] The cross-linked recombinant collagen characteristic polypeptide provided by the present invention can be used to distinguish recombinant collagen from collagen from other animal sources. This method is not limited by the sample state and can be used to analyze the degradation process of cross-linked recombinant collagen implanted in animals, providing an accurate evaluation method for the in vivo degradation evaluation of cross-linked recombinant collagen.

[0029] The cross-linked recombinant collagen characteristic polypeptide provided by the present invention has strong specificity. The cross-linked recombinant collagen enzymatic hydrolysis product is used as a standard product and the characteristic polypeptide is used as a detection target. The detection method provided is simple to operate and has high sensitivity. It can quantitatively analyze the cross-linked recombinant collagen. The method can be used for quality control of cross-linked recombinant collagen products and residual analysis and degradation process evaluation in animals.

[0030] The cross-linked recombinant collagen characteristic polypeptide provided by the present invention is unique. The qualitative and quantitative detection methods established by using the characteristic polypeptide as a marker and a quantitative standard are of great significance for the quality control of cross-linked recombinant collagen products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1It is the primary mass spectrum of characteristic peptides in the hydrolysis products of cross-linked recombinant collagen;

[0033] Figure 2 The figure is the secondary mass spectrum of characteristic peptides in the hydrolysis product of cross-linked recombinant collagen;

[0034] Figure 3 The spectra are of cross-linked recombinant collagen characteristic polypeptide standard solutions of different concentrations; from top to bottom, the concentrations are 2μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, and 50μg / mL;

[0035] Figure 4 This is a spectrum of recombinant collagen implants;

[0036] Figure 5 This is a spectrum for repeatability inspection of the quantitative method of characteristic peptides of cross-linked recombinant collagen;

[0037] Figure 6 This is a spectrum for precision investigation of the quantitative method of cross-linked recombinant collagen characteristic peptides; wherein 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3 represent three replicates of three samples respectively;

[0038] Figure 7 This is the spectrum of characteristic peptides of recombinant collagen in the skin of rats in the control group;

[0039] Figure 8 Spectra of characteristic peptides in the skin of rats injected with recombinant collagen implants in vivo. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1 Product Quality Evaluation of Recombinant Collagen Implants

[0046] 1 Experimental materials and instruments

[0047] 1.1 Experimental reagents

[0048] Recombinant collagen implant (cross-linked recombinant collagen product) and trypsin (sequence pure) were purchased from Promega Corporation, USA; chromatographic grade acetonitrile was purchased from Merck, USA; chromatographic grade formic acid was purchased from Thermo Fisher, USA; protein marker was purchased from Thermo Fisher, USA; other reagents were commercially available of analytical grade.

[0049] 1.2 Experimental instruments

[0050] The analytical balance (AL104) was from Mettler Toledo; the centrifuge (PK165) was from Hunan Pingke Scientific Instrument Co., Ltd.; the constant temperature water bath shaker (SHA-BA) was from Changzhou Ronghua Instrument Manufacturing Co., Ltd.; the liquid chromatograph (U 3000) and triple quadrupole liquid chromatography-mass spectrometer (Quantum ACCESS MAX) were both from Thermo Fisher, USA; and the data processing software was Xcalibur 3.1.

[0051] 2 Experimental methods

[0052] 2.1 Solution preparation

[0053] 2.1.1 Preparation of standard samples

[0054] Accurately weigh 10 mg of cross-linked recombinant collagen characteristic polypeptide (sequence GESGPAGPAGAPGPAGSR) standard into a 10 mL volumetric flask, dissolve and dilute with 0.1 mol / L Tris-HCl (pH 8.0) as the characteristic polypeptide standard stock solution. Use 0.1 mol / L Tris-HCl (pH 8.0) to dilute the cross-linked recombinant collagen characteristic polypeptide stock solution into a series of standard working solutions with concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL.

[0055] 2.1.2 Treatment methods of recombinant collagen implants

[0056] Accurately weigh 100 mg of recombinant collagen implant and place it in a 15 mL centrifuge tube. Add 10 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution to fully dissolve it. Take 0.5 mL of the dissolved recombinant collagen implant solution and mix it with 20 μg of trypsin. Place it in a 37°C constant temperature water bath shaker to react for 18 h. Centrifuge (10,000 r / min, 10 min) and take the supernatant for analysis.

[0057] 2.2 Quantitative analysis

[0058] (1) Liquid chromatography method

[0059] Chromatographic column: Zorbax C18 column (2.1mm×150mm×5μm); mobile phase A: water (0.1% formic acid), mobile phase B: 60% acetonitrile aqueous solution (V / V, 0.1% formic acid); gradient elution: 0-2min (5%-15% B), 2-6min (15%-90% B), 6-8min (90% B), 8-8.1min (90%-5% B); 8.1-10min (5% B); flow rate 0.2mL / min; column temperature 30°C; sample volume 5.0μL.

[0060] (2) Mass spectrometry

[0061] Ion source spray voltage 3.5 kV, capillary temperature 320 ° C, evaporation temperature 350 ° C, sheath gas flow rate 19.8 mL / min, auxiliary flow rate 0.55 bar, positive ion mode scanning, SRM monitoring mode: m / z 746.94 (doubly charged) → 641.20 (TubeLens = 111, CE = 29), m / z 746.94 (doubly charged) → 994.80 (Tube Lens = 111, CE = 30).

[0062] 2.3 Data processing

[0063] The qualitative analysis was performed based on the retention time, and the fragment ion peak m / z 641.20 of the characteristic polypeptide was used as the quantitative ion. The characteristic polypeptide peak area was used as the ordinate and the concentration of the cross-linked recombinant collagen characteristic polypeptide was used as the abscissa to establish a quantitative standard curve to calculate the content of cross-linked recombinant collagen in the sample.

[0064] The recombinant collagen content in the implant product was calculated according to formula (1).

[0065]

[0066] Wherein: Mi is the content of cross-linked recombinant collagen in the sample, μg / mg; X is the measured concentration of characteristic polypeptide of cross-linked recombinant collagen, μg / mL; V is the final volume of the sample, mL; M1 is the molecular weight of cross-linked recombinant collagen, Da; M2 is the molecular weight of characteristic polypeptide of cross-linked recombinant collagen, Da; m is the mass of the sample, mg; n is the number of repetitions of characteristic polypeptide in cross-linked recombinant collagen.

[0067] 2.4 Investigation of the precision of the quantitative method for characteristic peptides of cross-linked recombinant collagen

[0068] According to the method in 2.1.2, three recombinant type III collagens of the same concentration (the concentration used in this example is 10 mg / mL) were prepared and enzymatically hydrolyzed. Each sample was repeated 3 times and quantitatively analyzed. The RSD value was calculated based on the concentration value of the quantitative detection to determine the precision of the method.

[0069] 2.5 Repeatability of the quantitative method for cross-linked recombinant collagen characteristic peptides

[0070] A cross-linked recombinant collagen characteristic polypeptide standard with a concentration of 0.1 mg / mL was injected 6 times, and the peak area RSD value was calculated to determine the repeatability of the method.

[0071] 3 Experimental results

[0072] 3.1 Methodological validation

[0073] The primary mass spectrum of characteristic peptides in the enzymatic hydrolysis product of recombinant collagen implant is shown in Figure 1 , the secondary mass spectrum is shown in Figure 2 The chromatograms of the characteristic peptide standard solutions of different concentrations (2μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL) are shown in Figure 3 The linear regression equation of the quantitative standard curve is Y = 1.76 × 10 6 +1.07×10 6 X, R 2=0.9952. The linear correlation coefficient is greater than 0.99, indicating that the peptide has a good linear relationship. In the methodological investigation, the RSD value of the repeatability experiment was 3.81% (see the repeatability spectrum for details). Figure 5 ), indicating that the method has good repeatability; the RSD value of the precision experiment is 1.58% (see the precision investigation spectrum Figure 6 ), indicating that the method has good precision; the recovery rates of different addition levels were between 95% and 112%, which met the requirements of the recovery rate and the detection results were accurate; the quantitative limit and detection limit of the characteristic peptide were 0.2 ng / mL and 0.03 ng / mL, respectively.

[0074] 3.2 Measurement results

[0075] If the retention time and daughter ions of the target ions in the product to be tested are consistent with the retention time and daughter ions of the cross-linked recombinant collagen characteristic polypeptide standard, it is determined that the product to be tested contains the cross-linked recombinant collagen characteristic polypeptide.

[0076] The characteristic peptides in three batches of recombinant collagen implants were detected (see Figure 4 ), and the recombinant collagen content was calculated according to formula (1). The results showed that the average content of recombinant collagen in the recombinant collagen implant was 8.25 mg / g.

[0077] Example 2 Evaluation of the in vivo degradation process of recombinant collagen implants injected intradermally in rats

[0078] 1. Implantation of recombinant collagen implants in vivo

[0079] SD rats (weight 200g±5g) were randomly divided into two groups, 6 rats in each group (3 males and 3 females). 0.4mL of Shutai 50 combined with xylazine hydrochloride was injected intraperitoneally into the rats, and the backs of the rats were depilated. The experimental group was subcutaneously injected with 1g of recombinant collagen implant, and the rats were killed by dislocation one week after the injection, and 1cm*1cm skin samples were taken. The control group was not injected with recombinant collagen implants, and the rats were killed by dislocation, and 1cm*1cm skin samples were taken.

[0080] 2 Pretreatment of rat skin tissue

[0081] Fresh rat skin was weighed and mixed with chloroform / methanol solution (V / V, 2:1) at a mass ratio of 1:5, placed at 4°C, magnetically stirred, reacted for 24 hours, and fat was removed. After the organic solvent evaporated completely, the skin was mixed with 0.05 mol / L acetic acid solution at a mass ratio of 1:2, placed at 4°C for 12 hours to allow the skin to swell fully. The skin tissue was fully homogenized, freeze-dried, and weighed.

[0082] Accurately weigh 1.5 mg of freeze-dried skin tissue, add 1.0 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution (containing 20 μg of trypsin), react at 37°C for 18 h, take out, centrifuge (10 000 r / min, 10 min), and take the supernatant for analysis.

[0083] 3 Experimental results

[0084] The spectra of characteristic peptides of cross-linked recombinant collagen in the skin samples of rats in the control group and the experimental group are shown in Figure 7 and Figure 8 The results showed that the peak of characteristic polypeptide of cross-linked recombinant collagen was not detected in the skin of rats in the control group, but the peak of characteristic polypeptide of cross-linked recombinant collagen was detected in the experimental group. The average content of cross-linked recombinant collagen in the skin of rats in the experimental group was 0.35 μg / mg.

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A cross-linked recombinant collagen characteristic polypeptide, characterized in that: The amino acid sequence of the cross-linked recombinant collagen characteristic polypeptide is GESGPAGPAGAPGPAGSR.

2. Use of the cross-linked recombinant collagen characteristic polypeptide according to claim 1 in any of the following: (1) Application in quality control of cross-linked recombinant collagen products; (2) Application in the preparation of detection products for quality control of cross-linked recombinant collagen products; (3) Application in the preparation of products for analyzing the in vivo residual content and / or degradation process of cross-linked recombinant collagen products; (4) Application in the preparation of products for distinguishing cross-linked recombinant collagen from collagen of animal origin.

3. The use according to claim 2, characterized in that The cross-linked recombinant collagen characteristic polypeptide is used as a standard to detect the cross-linked recombinant collagen product, and the quality of the cross-linked recombinant collagen product is controlled or the in vivo residue and / or degradation process is analyzed.

4. The use according to claim 3, characterized in that The cross-linked recombinant collagen characteristic polypeptide is used as a standard to detect the cross-linked recombinant collagen product, comprising the following steps: After dissolving the cross-linked recombinant collagen characteristic polypeptide, a series of standard concentration working solutions are prepared; The cross-linked recombinant collagen product to be tested is dissolved and trypsin is added for enzymatic hydrolysis. The enzymatic hydrolysis solution is centrifuged and the supernatant is taken as the test solution; The standard working solution and the test solution are injected into a liquid chromatography-mass spectrometer for detection to determine whether the cross-linked recombinant collagen product to be tested contains the cross-linked recombinant collagen characteristic polypeptide or to quantitatively detect the content of recombinant collagen in the cross-linked recombinant collagen product to be tested.

5. The use according to claim 4, characterized in that In the liquid chromatography-mass spectrometer, the liquid phase conditions are: chromatographic column: Zorbax C18 column; mobile phase A: aqueous solution containing 0.1% formic acid, mobile phase B: water containing 0.1% formic acid and 60% acetonitrile; gradient elution: 0-2 min, 5%-15% B; 2-6 min, 15%-90% B; 6-8 min, 90% B; 8-8.1 min, 90%-5% B; 8.1~10min, 5%B.

6. The use according to claim 4, characterized in that In the liquid chromatography-mass spectrometer, mass spectrometry analysis uses electrospray ionization, and the ionization conditions are: m / z 746.94→641.20, 994.80; The mass spectrometry conditions were as follows: ion source spray voltage of 3.5 kV, capillary temperature of 320 °C, evaporation temperature of 350 °C, sheath gas flow rate of 19.8 mL / min, and auxiliary flow rate of 0.55 bar; positive ion mode scanning, SRM monitoring mode: m / z 746.94→641.20, m / z 732.84→994.

80.

7. The use according to claim 4, characterized in that If the retention time and daughter ions of the target ion in the cross-linked recombinant collagen product to be tested are consistent with the retention time and daughter ions of the cross-linked recombinant collagen characteristic polypeptide standard, it is determined that the cross-linked recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide.

8. The use according to claim 4, characterized in that The content of recombinant collagen in the cross-linked recombinant collagen product to be tested is calculated based on the correspondence between the characteristic polypeptide of the cross-linked recombinant collagen and the molecular weight of the cross-linked recombinant collagen product.

9. The use according to any one of claims 2 to 8, characterized in that: The cross-linked recombinant collagen product includes a recombinant collagen implant.

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