Recombinant collagen marker polypeptide and application thereof

Abstract

The invention discloses a recombinant collagen marker polypeptide and application thereof, and belongs to the field of collagen detection. The recombinant collagen marker polypeptide provided by the invention is used as a standard substance, the recombinant collagen in a recombinant collagen product can be qualitatively and quantitatively detected, and the recombinant collagen marker polypeptide has the advantages of strong specificity, high sensitivity, no limitation of sample state and the like when being used for detection. According to the application of the recombinant collagen marker polypeptide, the quality control of recombinant collagen products is realized, and the residual quantity and the degradation process of the recombinant collagen implanted into an animal body can be evaluated and analyzed. The method disclosed by the invention is of great significance in guaranteeing the quality of the recombinant collagen product and improving the production process.

Description

Technical Field

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

[0002] Recombinant collagen is prepared by introducing the target gene into specific host cells for gene expression and protein translation, followed by extraction and purification. At present, recombinant collagen is mainly divided into three categories: recombinant human collagen protein, recombinant human-like collagen protein, and recombinant collagen-like protein. Compared with tissue-extracted collagen, recombinant collagen is widely used in medical devices, tissue engineering medical materials, and cosmetics because of its characteristics of no virus risk, low immunogenicity, good water solubility, and stable quality.

[0003] At present, there are many kinds of recombinant collagen products on the market, with large differences in purity and uneven quality. Moreover, after recombinant collagen is implanted in the body as a degradable biomaterial, the degradation rate is the key to its 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 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, and the remaining materials will lose their original structure and shape. It is impossible to separate it from the animal's own tissues. Therefore, traditional methods cannot analyze the residual amount of 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. Hydroxyproline is contained in collagen of all animal sources and types. This method can only quantify the total collagen content, and cannot distinguish the collagen of the animal tissue itself from 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 recombinant collagen of a specific sequence and analysis of the residual amount of recombinant collagen implanted in animals. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant collagen characteristic polypeptide and its application to solve the problems existing in the above-mentioned prior art. The recombinant collagen specific polypeptide has strong specificity and is used as a standard to perform quantitative detection and analysis on the recombinant collagen in the recombinant collagen product. The detection method is simple to operate and has high sensitivity. It can be used for quality control of recombinant collagen products and analysis of their residues in animals and evaluation of their degradation process.

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

[0007] The present invention provides a recombinant collagen characteristic polypeptide, the amino acid sequence of the recombinant collagen characteristic polypeptide is GESGPAGPAGAPGPAGSK (SEQ ID NO.1).

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

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

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

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

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

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

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

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

[0016] After dissolving the recombinant collagen product to be tested, it is enzymatically hydrolyzed with trypsin, the enzymatic hydrolyzate 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 recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide or to quantitatively detect the content of recombinant collagen in the 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 is an aqueous solution containing 0.1% formic acid, B is an aqueous solution containing 0.1% formic acid and 60% acetonitrile; gradient elution: 0-3 min, 5%-25% B; 3-7 min, 25%-100% B; 7-8 min, 100% B; 8-8.1 min, 100%-5% B; 8.1-10 min, 5% B.

[0019] Optionally, in the liquid chromatography-mass spectrometer, electrospray ionization is used for mass spectrometry analysis, and the ionization conditions are: m / z 732.84→613.20, 909.46; the ion source spray voltage is 3.5 kV, the capillary temperature is 320°C, the evaporation temperature is 350°C, the sheath gas flow rate is 19.8 mL / min, and the auxiliary flow rate is 0.55 bar; positive ion mode scanning is adopted, SRM monitoring mode: m / z 732.84→613.20, the ion focusing lens voltage is 102, and the collision energy is 28; m / z 732.84→909.46, the ion focusing lens voltage is 102, and the collision energy is 26.

[0020] Optionally, if the retention time of the target ion in the recombinant collagen product to be tested is consistent with the retention time and daughter ion of the recombinant collagen characteristic polypeptide standard, it is determined that the recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide;

[0021] The content of recombinant collagen in the recombinant collagen product to be tested is calculated by the corresponding relationship between the characteristic polypeptide of the recombinant collagen and the molecular weight of the recombinant collagen product. The calculation formula is:

[0022]

[0023] Wherein, Mi is the content of recombinant collagen in the sample to be tested, μg / mg; X is the concentration of the characteristic polypeptide of 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 recombinant collagen, Da; M2 is the molecular weight of the characteristic polypeptide of recombinant collagen, Da; m is the mass of the sample to be tested, mg; n is the number of repetitions of the characteristic polypeptide in the recombinant collagen.

[0024] Optionally, the recombinant collagen product includes solutions, sponges, gels and freeze-dried fibers made from recombinant collagen.

[0025] The present invention discloses the following technical effects:

[0026] (1) The characteristic polypeptide of recombinant collagen provided by the present invention is unique, and the qualitative and quantitative detection methods established using the characteristic polypeptide as a marker are of great significance for the quality control of recombinant collagen products.

[0027] (2) The characteristic polypeptide of recombinant collagen provided by the present invention can be used to distinguish recombinant collagen from collagen of other animal origin. This method is not limited by the sample state and can be used to analyze the degradation process of recombinant collagen implanted in animals, providing an accurate evaluation method for the in vivo degradation evaluation of recombinant collagen. The present invention is of great significance for ensuring the quality of recombinant collagen products and improving the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 This is the primary mass spectrum of the characteristic peptides of recombinant collagen;

[0030] Figure 2 It is the secondary mass spectrometry of the characteristic peptides of recombinant collagen;

[0031] Figure 3The spectra of characteristic peptide standard solutions with different concentrations; A, B, C, D, E, and F in the figure are 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, and 50μg / mL, respectively;

[0032] Figure 4 This is the spectrum of the recombinant collagen freeze-dried fibers;

[0033] Figure 5 This is a spectrum for repeatability inspection of the quantitative method of recombinant collagen characteristic peptides;

[0034] Figure 6 This is the spectrum for precision investigation of the quantitative method of recombinant collagen characteristic peptides;

[0035] Figure 7 This is the spectrum of the characteristic peptides of recombinant collagen in the skin of rats in the control group; A, B, C, D, E, and F in the figure are 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, and 50μg / mL, respectively;

[0036] Figure 8 This is a spectrum of characteristic polypeptides in the skin of rats injected with recombinant collagen freeze-dried fibers in vivo; in the figure, A, B, C, D, E, and F are concentrations of 1μg / mL, 2μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, and 50μg / mL, respectively. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1 Quality evaluation of recombinant collagen freeze-dried fiber products

[0043] 1. Experimental materials and instruments

[0044] 1.1 Experimental Reagents

[0045] Recombinant collagen freeze-dried fibers were provided by Jiangsu Chuangjian Medical Technology Co., Ltd.; trypsin (analytical grade) was purchased from Promega, 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; SD rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; other reagents were all commercially available analytical grade.

[0046] 1.2 Experimental instruments

[0047] 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 (QuantumACCESS MAX) were both from Thermo Fisher, USA; and the data processing software was Xcalibur 3.1.

[0048] 2. Experimental methods

[0049] 2.1 Preparation of characteristic peptide standard solution

[0050] Weigh 10 mg of recombinant collagen characteristic polypeptide standard accurately, place in 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. Dissolve with 0.1 mol / L Tris-HCl (pH 8.0) to dilute the recombinant collagen characteristic polypeptide stock solution into a series of standard working solutions with concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL.

[0051] 2.2 Processing method of recombinant collagen freeze-dried fibers

[0052] Accurately weigh 5.0 mg of recombinant collagen freeze-dried fiber 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 freeze-dried fiber solution, mix it with 20 μg of trypsin, place it in a 37°C constant temperature water bath shaker, react for 18 hours, centrifuge (10000 r / min, 10 min), and take the supernatant for analysis.

[0053] 2.3 Quantitative analysis

[0054] 2.3.1 Liquid chromatography conditions

[0055] Chromatographic column: Zorbax C18 column (2.1mm×150mm×5μm); mobile phase: A is water (0.1% formic acid), B is 60% acetonitrile aqueous solution (V / V, 0.1% formic acid); gradient elution: 0-3min (5%-25% B), 3-7min (25%-100% B), 7-8min (100% B), 8-8.1min (100%-5% B); 8.1-10min (5% B); flow rate 0.3mL / min; column temperature 30°C; sample volume 5.0μL.

[0056] 2.3.2 Mass spectrometry conditions

[0057] 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 732.84 (doubly charged) → 613.20 (TubeLens = 102, CE = 28), m / z 732.84 (doubly charged) → 909.46 (Tube Lens = 102, CE = 26).

[0058] 2.3.3 Data processing

[0059] The recombinant collagen content in the freeze-dried fiber product was calculated according to formula (1).

[0060]

[0061] Wherein: Mi is the content of recombinant collagen in the sample, μg / mg; X is the measured concentration of the characteristic polypeptide of recombinant collagen, μg / mL; V is the final volume of the sample, mL; M1 is the molecular weight of the recombinant collagen, Da; M2 is the molecular weight of the characteristic polypeptide of the recombinant collagen, Da; m is the mass of the sample, mg; n is the number of repetitions of the characteristic polypeptide in the recombinant collagen.

[0062] 2.4 Investigation of the precision of the quantitative method for characteristic peptides of recombinant collagen

[0063] According to the method in 2.2, three samples of recombinant type III collagen with the same concentration were prepared and enzymatically hydrolyzed. Each sample was repeated three 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.

[0064] 2.5 Repeatability study of the quantitative method of recombinant collagen characteristic peptides

[0065] A recombinant type III collagen characteristic polypeptide standard with a concentration of 5 μg / mL was injected 6 times, and the peak area RSD value was calculated to determine the repeatability of the method.

[0066] 3. Experimental results

[0067] 3.1 Methodological validation

[0068] Chromatograms of characteristic peptide standard solutions of different concentrations are shown in Figure 3 (In the figure, A, B, C, D, E, and F are concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL, respectively). The primary mass spectrum and secondary mass spectrum of the polypeptide are shown in Figure 1-Figure 2 The linear regression equation is y = 3766418.2974x – 1533588.8804, R 2 =0.9984. The linear correlation coefficient is greater than 0.99, indicating that the peptide has a good linear relationship. Figure 5-Figure 6 As shown, the RSD value of the repeatability experiment was 0.8%, indicating that the method had good repeatability; the RSD value of the precision experiment was 3.83%, indicating that the method had good precision; the results of the addition recovery rates at different addition levels were all between 96% and 118%, which met the requirements for the addition recovery rate, and the detection results were accurate; the quantitative limit and detection limit of the characteristic peptide were 0.5 ng / mL and 0.05 ng / mL, respectively.

[0069] 3.2 Measurement results

[0070] The recombinant collagen characteristic polypeptide standard and the sample to be tested are respectively injected into the liquid chromatography-mass spectrometry instrument. If the retention time of the target ion is consistent with that of the recombinant collagen characteristic polypeptide standard, and its daughter ion is consistent with that of the standard, then the sample to be tested contains recombinant collagen components. Through the correspondence between the characteristic polypeptide and the molecular weight of recombinant collagen, the content of recombinant collagen in the sample can be calculated, thereby realizing the qualitative and quantitative analysis of recombinant collagen.

[0071] The characteristic peptides in three batches of recombinant collagen freeze-dried fibers 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 freeze-dried fibers was 0.98 mg / mg.

[0072] Example 2 Evaluation of the in vivo degradation process of recombinant collagen freeze-dried fibers injected intradermally in rats

[0073] The difference between this embodiment and embodiment 1 is only that:

[0074] 1. In vivo implantation of recombinant collagen freeze-dried fibers

[0075] 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 20μL of 8mg / mL recombinant collagen freeze-dried fibers, 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 freeze-dried fibers, and the rats were killed by dislocation, and 1cm*1cm skin samples were taken.

[0076] 2. Pretreatment of rat skin tissue

[0077] 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, stirred magnetically, 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.

[0078] 1.5 mg of freeze-dried skin tissue was accurately weighed, and 1.0 mL of 0.1 mol / L Tris-HCl (pH 8.0) solution (containing 20 μg of trypsin) was added. The mixture was reacted at 37°C for 18 h, and then removed and centrifuged (10000 r / min, 10 min). The supernatant was collected for analysis.

[0079] 3. Experimental results

[0080] The spectra of characteristic peptides of 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 no peak of characteristic polypeptide of recombinant collagen was detected in the skin of rats in the control group, while the peak of characteristic polypeptide of recombinant collagen was detected in the experimental group. The average content of recombinant collagen in the skin of rats in the experimental group was 1.99 μg / mg.

[0081] 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 recombinant collagen characteristic polypeptide, characterized in that: The amino acid sequence of the recombinant collagen characteristic polypeptide is GESGPAGPAGAPGPAGSK.

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

3. The use according to claim 2, characterized in that The recombinant collagen characteristic polypeptide is used as a standard to detect the recombinant collagen product, so as to perform quality control on the recombinant collagen product or analyze the in vivo residual amount and degradation process.

4. The use according to claim 3, characterized in that The detection of the recombinant collagen product using the recombinant collagen characteristic polypeptide as a standard comprises the following steps: After dissolving the recombinant collagen characteristic polypeptide, a series of standard concentration working solutions are prepared; After dissolving the recombinant collagen product to be tested, it is enzymatically hydrolyzed with trypsin, the enzymatic hydrolyzate 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 recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide or to quantitatively detect the content of recombinant collagen in the 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 is an aqueous solution containing 0.1% formic acid, and B is an aqueous solution containing 0.1% formic acid and 60% acetonitrile; gradient elution: 0-3 min, 5%-25% B; 3-7 min, 25%-100% B; 7-8 min, 100% B; 8-8.1 min, 100%-5% B; 8.1-10 min, 5% B.

6. The use according to claim 4, characterized in that In the liquid chromatography-mass spectrometer, electrospray ionization is used for mass spectrometry analysis, and the ionization conditions are: m / z 732.84→613.20, 909.46; the ion source spray voltage is 3.5 kV, the capillary temperature is 320°C, the evaporation temperature is 350°C, the sheath gas flow rate is 19.8 mL / min, and the auxiliary flow rate is 0.55 bar; positive ion mode scanning is adopted, and the SRM monitoring mode is: m / z 732.84→613.20, the ion focus lens voltage is 102, and the collision energy is 28; m / z732.84→909.46, the ion focus lens voltage is 102, and the collision energy is 26.

7. The use according to claim 4, characterized in that If the retention time of the target ion in the recombinant collagen product to be tested is consistent with the retention time and daughter ion of the recombinant collagen characteristic polypeptide standard, it is determined that the recombinant collagen product to be tested contains the recombinant collagen characteristic polypeptide; The content of the recombinant collagen in the recombinant collagen product to be tested is calculated based on the correspondence between the characteristic polypeptide of the recombinant collagen and the molecular weight of the recombinant collagen product.

8. The use according to any one of claims 2 to 7, characterized in that: The recombinant collagen product includes solution, sponge, gel and freeze-dried fiber made from recombinant collagen as raw material.

Images