Type I humanized collagen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to I-type humanized collagen as well as a preparation method and application thereof. The amino acid sequence of the type I humanized collagen prepared by the invention is shown as a sequence SEQ ID NO. 1 (HTGAWGKTVIEYKTTKTSRLVMGFXGPKGAA, wherein X is hydroxyproline). The invention also discloses a preparation method of the type I humanized collagen. The I-type humanized collagen contains essential amino acids and semi-essential amino acids of a human body, promotes proliferation of human skin fibroblasts, up-regulates expression of elastin, fibronectin and collagen I genes, and has the effects of interacting with endothelial cells and osteoblasts, promoting creeping and growth of the osteoblasts, and promoting the growth of the osteoblasts. And the interaction between osteoblasts and collagen is promoted. Therefore, the I-type humanized collagen can be widely applied to orthopedic sports materials and has application prospects in the fields of medical cosmetology and sports medicine materials.

Description

[0001] This invention claims the priority of Chinese Patent Application CN202411259905.0 filed on September 10, 2024. The content recorded in the specification, the attached drawings of the specification, and the claims of the priority document are incorporated herein by reference in their entirety and are regarded as part of the original record of the specification of this invention. The applicant further declares that the applicant has the right to modify the specification and claims of this invention based on the priority document. Technical Field

[0002] This invention belongs to the field of biomedical technology, and specifically refers to a type I humanized collagen, its preparation method, and its application. Background Art

[0003] Collagen is the most abundant protein in the human body, accounting for 25% - 30% of the total protein content. As the most abundant protein in the extracellular matrix, collagen maintains the integrity of cell structure and various physiological functions. Collagen is the main structural component of all connective tissues and is also present in the interstitial tissues of almost all parenchymal organs. In the past decade, people's understanding of collagen has been continuously increasing, and the members of the collagen family have also increased rapidly. Through research, it has been found that more than 20 different types of collagen have been discovered in the human body, which can be roughly divided into the following categories: fibrillar collagen, basement membrane collagen, microfibrillar collagen, anchoring fibril, transmembrane collagen, and incompletely characterized collagen. Type I collagen is fibrillar collagen, which is widely present in the skin, bones, and tendons. It can strengthen the tissue strength and elasticity, provide sufficient nutrients for cells, and maintain normal physiological functions. The length of type I collagen is about 300 nm, and the diameter is about 1.4 nm - 1.5 nm.

[0004] Currently, collagen is mainly sourced from animal tissues. Naturally extracted collagen is a mixture composed of collagens with different molecular weights, insoluble in water, and has poor biocompatibility. In addition, due to its origin from animal tissues, there is a risk of cross-infection with animal-source diseases or human infectious diseases, and it cannot be compatible with the human body, which will lead to immune rejection and allergic symptoms. The traditional methods for producing collagen are to treat animal-derived tissues with acids, alkalis, and enzymatic hydrolysis to extract collagen derivatives. The collagen extracted by these methods has already lost its original biological activity and cannot be applied in the biomedical field to play its true function. Therefore, currently, collagen can only be used in cosmetics and health products and cannot play its original biological function at all.

[0005] In view of the disadvantages and deficiencies in the current sources of collagen and extraction methods, the best solution is to vigorously develop recombinant humanized collagen, especially type I humanized collagen. However, current domestic research only focuses on the Gly-Xaa-Yaa recombinant region of type I collagen, or a continuous amino acid sequence of type I collagen, or the synthesis methods of the a1 chain and a2 chain of type I collagen. There is still a lack of development in the proteins corresponding to certain specific amino acid sequences in type I collagen and their functions.

[0006] Based on this, the present invention conducts research and development on type I humanized collagen containing specific amino acid sequences, with the expectation of finding type I humanized collagen with high performance, non-toxicity, non-allergenicity, simple preparation method, and low cost, which can be widely applied in the fields of orthopedic materials, medical aesthetics, and sports medicine materials. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a type I humanized collagen with high performance, non-toxicity, non-allergenicity, simple preparation method, and low cost, and its preparation method and applications, which can be widely applied in the fields of orthopedic materials, medical aesthetics, and sports medicine materials.

[0008] To achieve the purpose of this invention, the following technical solutions are adopted:

[0009] In the first aspect, the present invention provides a type I humanized collagen, which comprises:

[0010] a) an amino acid sequence as shown in SEQ ID NO.1;

[0011] b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1, and retaining the biological function of the amino acid sequence shown in SEQ ID NO.1;

[0012] c) an amino acid sequence with 1 or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO.1, and retaining the biological function of the amino acid sequence of SEQ ID NO.1; or

[0013] d) an amino acid sequence encoded by a nucleotide sequence that hybridizes with the polynucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 under stringent conditions, and the amino acid sequence retains the biological function of the amino acid sequence of SEQ ID NO.1, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0014] As an alternative, in the above-mentioned type I humanized collagen, the biological function is to promote the proliferation of human skin fibroblasts, up-regulate the expression of elastin, fibronectin and collagen I genes, and it has the interaction with endothelial cells and osteoblasts, promotes the crawling and growth of osteoblasts, and promotes the interaction between osteoblasts and collagen.

[0015] In a second aspect, the present invention provides a polynucleotide encoding the type I humanized collagen described in the first aspect above.

[0016] As an alternative, those skilled in the art can expect that as long as the nucleotide sequence of the type I humanized collagen described in the first aspect above can be prepared by using conventional methods in the field of molecular biology, it is within the scope of the present invention.

[0017] In a third aspect, the present invention provides a recombinant plasmid comprising the polynucleotide described in the second aspect above.

[0018] In a fourth aspect, the present invention provides a transformant obtained by introducing the recombinant plasmid described in the third aspect above into a host.

[0019] In a fifth aspect, the present invention provides a method for preparing the type I humanized collagen described in the first aspect above, and the preparation method comprises the following steps:

[0020] Step 1: Prepare a crude polypeptide by using 2-chlorotrityl chloride resin through solid-phase synthesis, and the crude polypeptide comprises type I humanized collagen with an amino acid sequence as shown in SEQ ID NO.1;

[0021] Step 2: Purify the crude polypeptide by HPLC to obtain the type I humanized collagen.

[0022] As an alternative, in the above preparation method, in Step 1, it includes the following steps:

[0023] 1.1 Resin swelling: Weigh an appropriate amount of 2-chlorotrityl chloride resin with a substitution degree of 0.5 - 1.0 mmol / g, put the resin into a reaction tube, add an appropriate amount of DCM, and shake;

[0024] 1.2 Connect the first amino acid;

[0025] 1.3 Deprotection: Add an appropriate amount of 20% piperidine DMF solution by volume percentage, after a period of time, remove it, then add an appropriate amount of 20% piperidine DMF solution by volume percentage, and after another period of time;

[0026] 1.4 Detection: Withdraw 20% piperidine DMF solution by volume percentage. Take an appropriate amount of resin, wash it with ethanol 3 - 5 times, drain it, and the ninhydrin test is positive;

[0027] 1.5 Washing: Wash with DMF 2 - 5 times successively, wash with methanol 2 - 5 times, and wash with DMF 2 - 5 times;

[0028] 1.6 Condensation: The protected amino acid (Fmoc-Arg(Pbf)-OH) is 3 - 5 times in excess, and HBTU is 3 - 5 times in excess. Both are dissolved in as little DMF as possible, added to the reaction tube, and immediately add 10 times in excess of DIEA, and react for a period of time;

[0029] 1.7 Washing: Wash with DMF 1 - 3 times successively, wash with methanol 1 - 3 times, and wash with DMF 1 - 3 times. Repeat the operations in steps 1.2 to 1.7, and connect the amino acids in the amino acid sequence shown in SEQ ID NO.1 from right to left in turn;

[0030] 1.8 Detection: Wash with DMF 1 - 3 times successively, wash with DCM 1 - 3 times, and wash with DMF 1 - 3 times. The ninhydrin test is negative;

[0031] 1.9 Remove the lysine protecting group of the sequence: Use DMF containing one or two drops of hydrazine hydrate to remove the dde lysine protecting group of the sequence;

[0032] 1.10 Washing: Wash with methanol 2 - 5 times;

[0033] 1.11 Cut the polypeptide from the resin: Put the resin into a flask or centrifuge tube containing the cutting solution and oscillate it at a constant temperature;

[0034] 1.12 Blow dry and wash: Blow dry the cleavage solution (i.e., the mixture of the cutting solution and the resin polypeptide) as much as possible with nitrogen, chromatograph it with ether, wash it with ether again, and then volatilize it at room temperature to obtain the crude peptide.

[0035] As an optional method, in the above preparation method, in step 2, it includes the following steps:

[0036] 2.1 Take an appropriate amount of the crude peptide and put it into a container, and dissolve it clearly with an appropriate amount of 50% acetonitrile aqueous solution by volume percentage;

[0037] 2.2 Filter the dissolution solution with a 0.45μm filter membrane;

[0038] 2.3 Analysis: Analyze the crude peptide by analytical HPLC. The mobile phase is water and acetonitrile, the time is 30 min, gradient elution. First, equilibrate the HPLC column (Diamonsil 5μm C18 250×4.6mm) with the starting gradient for 5 min and then inject the sample. By volume percentage, the starting gradient is 95% water and 5% acetonitrile, and the ending ratio is 5% water and 95% acetonitrile.

[0039] 2.4 Preparation: Prepare the crude peptide solution for injection. First, equilibrate the preparative HPLC column (Diamonsil 5μm C18 250×4.6mm) for 10 min. In terms of volume percentage, the initial gradient is 95% water and 5% acetonitrile, and the final gradient is 25% - 50% water and 50% - 75% acetonitrile, with a gradient time of 40 min. Collect the sample coming out of the detector to obtain purified type I humanized collagen.

[0040] 2.5 Identification: Take a sample of the purified type I humanized collagen prepared in step 2.4 for purity identification.

[0041] In the sixth aspect, the present invention provides the application of the type I humanized collagen described in the first aspect above in the preparation of pelvic floor repair products, medical beauty products, bone repair products, and sports medicine products.

[0042] In the seventh aspect, the present invention provides the application of the type I humanized collagen described in the first aspect above in the preparation of products for skin tissue.

[0043] Optionally, the products for skin tissue are used to maintain or promote the proliferation of human skin fibroblasts.

[0044] Alternatively, the products for skin tissue are used to maintain or improve skin elasticity.

[0045] In the eighth aspect, the present invention provides the application of the type I humanized collagen described in the first aspect above in products for promoting tissue growth, and the products are used to maintain or up-regulate the expression of elastin, fibronectin, and type I collagen genes and / or proteins.

[0046] In the ninth aspect, the present invention provides the application of the type I humanized collagen described in the first aspect above in bone repair products and sports medicine products.

[0047] Optionally, the products have preventive and repair functions and are used to promote the crawling and growth of osteoblasts.

[0048] The beneficial effects of the present invention are as follows:

[0049] The amino acid sequence of the type I humanized collagen prepared by the present invention is shown in SEQ ID NO.1 (HTGAWGKTVIEYKTTKTSRLVMGFXGPKGAA, where X is hydroxyproline). It contains essential amino acids and semi-essential amino acids of the human body, and also contains six amino acids: glycine, proline, alanine, valine, leucine, and lysine. This sequence contains essential amino acids of the human body in the shortest sequence and has the most water-soluble amino acids, ensuring the hydrophilicity of the protein, which is the first of its kind for recombinant humanized collagen. The above six amino acids have a positive effect on the components of the extracellular matrix (ECM), especially elastin, fibronectin, collagen I, and collagen VI;

[0050] (2) The type I humanized collagen described in the present invention provides a new synthesis method for a specific amino acid sequence of type I collagen. By using a chemical synthesis method, specific proline is accurately hydroxylated to achieve 100% consistency with the amino acid sequence of human type I collagen, filling the gap in the market where humanized collagen has no hydroxyproline or a low proline hydroxylation rate, becoming the first case of recombinant humanized collagen and leading the domestic market. In addition, the type I humanized collagen prepared by the present invention has a higher degree of proline hydroxylation, enabling the collagen to have the functions of antioxidant, regulating cell metabolism, growth, and development;

[0051] (3) The type I humanized collagen prepared by the present invention can promote the proliferation of human skin fibroblasts, up-regulate the expression of elastin, fibronectin, and type I collagen genes, facilitate wound healing, scar repair, maintain skin moisture, keep skin tension, and smooth fine lines, and is widely used in the medical beauty field. The protein synthesized by this invention can interact with endothelial cells and osteoblasts, promote the crawling and growth of osteoblasts, and can be widely used in orthopedic sports materials, having application prospects in the fields of medical beauty and sports medicine materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0053] Figure 1 : Mass spectrometry diagram of the type I humanized collagen described in the present invention;

[0054] Figure 2 : Experimental result diagrams of the type I humanized collagen described in the present invention promoting the proliferation of mouse fibroblasts and osteoblasts;

[0055] Figure 3 : Experimental result diagrams of the type I humanized collagen described in the present invention enhancing skin elasticity;

[0056] Figure 4 : The type I humanized collagen of the present invention improves the expression results of collagen synthesis-related genes in human skin fibroblasts. Detailed implementation manners

[0057] The following will explain the solution of the present invention in combination with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] Example 1: Preparation and characterization of type I humanized collagen

[0059] 1. Preparation of recombinant type I collagen

[0060] Step 1: Use 2-chlorotrityl chloride resin to prepare crude polypeptide by solid-phase synthesis method. The crude polypeptide contains type I humanized collagen (HTGAWGKTVIEYKTTKTSRLVMGFXGPKGAA, where X is hydroxyproline) with the amino acid sequence shown in SEQ ID NO.1. The specific steps are as follows:

[0061] 1.1 Resin swelling: Weigh 5 g of 2-chlorotrityl chloride resin with a substitution degree of 0.5 - 1.0 mmol / g, put the resin into a reaction tube, add an appropriate amount of DCM (15 mL / g), and shake for 30 min;

[0062] 1.2 Connect the first amino acid;

[0063] 1.3 Deprotection: Add 15 - 30 mL of 20% piperidine DMF solution (15 mL / g) for 5 min, remove it, then add 15 mL of 20% piperidine DMF solution (15 mL / g) and continue for 15 min;

[0064] 1.4 Detection: Draw out the 20% piperidine DMF solution, take a dozen resin particles, wash them 3 - 5 times with ethanol, drain, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105°C - 110°C for 5 min, and a dark blue color change indicates a positive reaction;

[0065] 1.5 Washing: Wash successively with DMF (10 mL / g) for 2 - 5 times, methanol (10 mL / g) for 2 - 5 times, and DMF (10 mL / g) for 2 - 5 times;

[0066] 1.6 Condensation: Use 3 - 5 times the excess of protected amino acid (Fmoc-Arg(Pbf)-OH) and 3 times the excess of HBTU, both dissolved in as little DMF as possible, add them to the reaction tube, immediately add 10 times the excess of DIEA, and react for 40 min;

[0067] 1.7 Washing: Wash successively with DMF (10 mL / g) for 1 - 3 times, methanol (10 mL / g) for 2 times, and DMF (10 mL / g) for 2 times. Repeat the operations in steps 1.2 to 1.7, and connect the amino acids in the amino acid sequence shown in SEQ ID NO.1 from right to left in turn;

[0068] 1.8 Detection: Wash successively with DMF (10 mL / g) for 2 times, DCM (10 mL / g) for 2 times, and DMF (10 mL / g) for 2 times, dry by suction for 10 min, and the ninhydrin test is negative;

[0069] 1.9 Removal of the protecting group of lysine in the sequence: Use DMF containing one or two drops of hydrazine hydrate to remove the protecting group dde of lysine in the sequence. Add 3 times the excess of hexadecanoic acid and 3 times the excess of HBTU, both dissolved in as little DMF as possible, add them to the reaction tube, immediately add 10 times the excess of DIEA, and react for 40 min;

[0070] 1.10 Washing: Wash with methanol (10 mL / g) for 3 times;

[0071] 1.11 Cleavage of the polypeptide from the resin: Prepare the cleavage solution (10 mL / g): by volume percentage, TFA 94.5%; water 2.5%; EDT 2.5%; TIS 0.5%. Load the resin into a flask or centrifuge tube, and the ratio of resin to cleavage solution is 10 g / mL, shake at a constant temperature for 120 min;

[0072] 1.12 Drying and washing: Blow dry the cleavage solution (i.e., the mixture of cleavage solution and resin polypeptide) as much as possible with nitrogen, chromatograph it with ether, wash it with ether 6 times, and then volatilize it at room temperature to obtain the crude peptide.

[0073] Step 2: Purify the crude polypeptide by HPLC to obtain the type I humanized collagen, and the specific steps are as follows:

[0074] 2.1 Take 200 - 500 mg of the crude peptide and put it into a container, dissolve it with 2 - 15 mL of 50% aqueous acetonitrile solution by volume percentage, and ultrasonic treatment can be carried out for 2 - 10 min;

[0075] 2.2 Filter the dissolution solution with a 0.45 μm filter membrane;

[0076] 2.3 Analysis: Take 3 - 6 μL of the crude peptide for analysis by analytical HPLC. The mobile phase is water and acetonitrile, for 30 min with gradient elution. First, equilibrate the HPLC column (Diamonsil 5 μm C18 250×4.6 mm) with the initial gradient for 5 min and then inject the sample. In terms of volume percentage, the initial gradient is 95% water and 5% acetonitrile, and the final ratio is 5% water and 95% acetonitrile.

[0077] 2.4 Preparation: Prepare the dissolved crude peptide for injection. First, equilibrate the preparative HPLC column (Diamonsil 5 μm C18 250×4.6 mm) for 10 min. In terms of volume percentage, the initial gradient is 95% water and 5% acetonitrile, and the final gradient is 25% - 50% water and 50% - 75% acetonitrile, with a gradient time of 40 min. Collect the sample coming out of the detector to obtain purified type I humanized collagen.

[0078] 2.5 Identification: Take a sample of the purified type I humanized collagen prepared in step 2.4 for mass spectrometry analysis.

[0079] 2. Characterization of Type I Humanized Collagen

[0080] Use a mass spectrometer to analyze the molecular weight of the purified type I humanized collagen. The mass spectrometry results are as Figure 1 shown, and the measured molecular weight of the target protein is 3306.88.

[0081] Example 2: Experimental Results of Type I Humanized Collagen Promoting the Proliferation of Fibroblasts and Osteoblasts

[0082] (1) Mouse fibroblasts (L - 929) and osteoblasts (MC3T3 - E1 Subclone24) were purchased from pricella company. Both types of cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin - streptomycin. The cell culture conditions were 37°C, 5% CO 2 , with a humidity greater than 90%. After the cells were passaged 3 times, a cell proliferation experiment was conducted.

[0083] (2) Seed the cells at 2×10 3The cells were seeded at an inoculation density of

[0084] (3) The medium was aspirated, and 20 μL of MTT solution (concentration 5 mg / mL) and 100 μL of basal medium were added to each well, and the cells were further cultured in the cell incubator for 4 hours. After the culture, 150 μL of DMSO was added to each well and incubated at 37 °C for 10 minutes. Finally, the absorbance (OD value) at a wavelength of 570 nm was measured using a microplate reader, and the relative viability of the cells in the experimental group and the positive control group was calculated according to the following formula:

[0085] Cell viability (%) = (OD 实验组 - OD 空白组 ) / (OD 空白对照组 - OD 空白组 ) × 100. The experimental results are shown in Table 1.

[0086] Table 1: Relative viability of cells in the positive control group and the experimental group

[0087]

[0088] The results were statistically analyzed using GraphPad Prism software. The t-test was used to compare the mean differences between the positive control group and the blank group, and between the experimental group and the blank group. When P < 0.05, it was considered that there was a statistically significant difference between the two groups of data. As Figure 2 shown, the results of the fibroblast proliferation experiment showed that compared with the blank control group, the cell viability in the positive control group increased by 51.6%, and the cell viability in the experimental group increased by 61.9%; the results of the osteoblast proliferation experiment showed that compared with the blank control group, the cell viability in the positive control group increased by 23.9%, and the cell viability in the experimental group increased by 27.24%, and both had significant statistical differences (***P < 0.001).

[0089] The above results indicate that the type I humanized collagen of the present invention can significantly promote the proliferation of fibroblasts and osteoblasts.

[0090] Example 3: Experimental Results of Type I Humanized Collagen in Enhancing Skin Elasticity

[0091] Six-week-old BALB / c nude mice with a body weight of (19±2) g were selected for the experiment and purchased from Spf (Beijing) Biotechnology Co., Ltd. All mice were housed in separate cages and had free access to food and water. They were randomly divided into three groups, with 10 animals in each group: Group 1 was injected with phosphate-buffered saline (PBS) as the blank control group; Group 2 was injected with HA (sodium hyaluronate) filler (purchased from Shandong Zhongshan Biotechnology Co., Ltd.) as the positive control group (or positive group); Group 3 was injected with the target protein (Type I humanized collagen of the present invention, the amino acid sequence is shown in SEQ ID NO.1) as the experimental group. All mice were exposed to ultraviolet light 3 times a week; a skin elasticity tester Cutometer (MPA580, Courage Khazaka, Germany) was used to measure tissue elasticity before injection and on the 1st, 3rd, 7th, and 14th days after injection. A 2-mm probe test hole was used, and the back skin was continuously stretched for 2 seconds under a constant negative pressure of 50 mbar, and then after the negative pressure ended, the skin rebound time of 2 seconds was maintained.

[0092] Skin elasticity parameters were calculated based on the experimental results. After the negative pressure was applied to the skin, the elastic amount of the skin at 0.1 second was called the elastic amount of the stretching part, denoted by Ue; after the negative pressure ended, the elastic amount of the skin rebound at 0.1 second was called the elastic amount of the rebound part, denoted by Ur. The better the skin elasticity, the closer the value of Ur / Ue is to 1.

[0093] The experimental results are as Figure 3 shown. The statistical analysis of the skin elasticity detection results was performed using Graphpad Prism software. The t-test was used to compare the mean differences between the positive control group and the blank group, and between the experimental group and the blank group, respectively. ***P<0.001, indicating that there was a significant statistical difference between the two groups of data. There was no significant statistical difference in the skin elasticity of the control group mice before and after injection; compared with the control group, the skin tissue elasticity of the experimental group and the positive control group mice was significantly improved on the 1st day after injection, and on the 3rd, 7th, and 14th days, it was observed that the skin elasticity of the experimental group and the positive drug control group mice was still significantly higher than that of the control group injected with phosphate-buffered solution (***P<0.001), indicating that the Type I humanized collagen described in the present invention has the effect of improving skin elasticity.

[0094] Example 4: Type I Humanized Collagen Increases the Expression of Collagen Synthesis-related Genes in Human Skin Fibroblasts

[0095] (1) Human skin fibroblasts (HFF-1) (purchased from Sunncel Company) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture conditions were 37°C, 5% CO 2 , and greater than 90% humidity. After the cells were passaged 3 times, the fibroblasts were seeded into 6-well plates, incubated for 24 h, the medium was changed and samples were added, and the expression levels of genes related to collagen synthesis were detected after continuous culture for 24 h.

[0096] (2) For the detection of genes related to collagen synthesis, a blank control group, a positive control group (5 mg / mL commercially available recombinant humanized type III collagen product, molecular weight 54 KD), and a sample group (5 mg / mL type I humanized collagen of the present invention) were set up, with 3 replicates in each group. Except for the blank control group, all were irradiated with UVA, and the irradiation dose was 30 J / cm 2 . After the irradiation was completed, they were placed in an incubator (37°C, 5% CO 2 ) and continued to be cultured for 24 h. Each well was washed twice with 2 mL of PBS, 1 mL of RNA extraction reagent (RNAisoPlus) was added, and the cells were lysed and the samples were collected. According to the instructions of the reverse transcription kit, RNA extraction, reverse transcription, and quantitative PCR were performed to detect the expression of genes related to collagen synthesis (COL1A1, COL3, SMAD 3).

[0097] Quantitative PCR instrument: ABI 7500 Sequence Detection System. The reaction program was: 95°C for 5 min; 95°C for 15 s, 60°C for 32 s for reading the plate, a total of 40 cycles, the melting curve was analyzed, and its threshold cycle number (Ct value) was recorded. The Ct value of the internal reference GAPDH was used as the basis to correct the difference of the target gene, and the relative gene expression analysis was performed using the 2 -ΔΔCT method for calculation. The primer sequence information used for PCR is shown in Table 2 below.

[0098] Table 2: Primer sequences and product sizes used for PCR

[0099]

[0100] Graphpad Prism software was used to perform statistical analysis on the results of gene expression levels. The t-test was used to compare the mean differences in the expression levels of the same genes between the positive control group and the blank group, and between the experimental group and the blank group, respectively. ***P < 0.001 was considered to have a significant statistical difference between the two groups of data.

[0101] The results of the expression levels of genes related to collagen synthesis are as Figure 4As shown, compared with the blank control group, the expression levels of collagen synthesis-related genes COL1, COL3, and SMAD 3 in the positive control group and the experimental group were all significantly up-regulated (***P<0.001), and the up-regulation effect in the experimental group was better than that in the positive control group. The above results indicate that the type I humanized collagen described in the present invention has the effect of increasing the expression of collagen synthesis-related genes in human skin fibroblasts.

[0102] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A humanized type I collagen, characterized in that: The humanized type I collagen comprises: a) the amino acid sequence shown in SEQ ID NO.1; b) an amino acid sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO.1, and which retains the biological function of the amino acid sequence shown in SEQ ID NO.1; c) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO.1, and which retains the biological function of the amino acid sequence of SEQ ID NO.1; d) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 under stringent conditions, and the amino acid sequence retains the biological function of the amino acid sequence of SEQ ID NO.1, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

2. The humanized type I collagen according to claim 1, characterized in that: The biological function is to promote the proliferation of human skin fibroblasts, up-regulate the expression of elastin, fibronectin and collagen I genes, interact with endothelial cells and osteoblasts, promote the crawling and growth of osteoblasts, and promote the interaction between osteoblasts and collagen.

3. A polynucleotide, characterized in that: The polynucleotide encodes the humanized type I collagen according to claim 1.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the polynucleotide according to claim 3.

5. A transformant, characterized in that: The transformant is obtained by introducing the recombinant plasmid according to claim 4 into a host.

6. The method for preparing type I humanized collagen according to claim 1 or claim 2, characterized in that: The preparation method comprises the following steps: Step 1: using 2-chlorotrityl chloride resin to prepare a crude polypeptide by a solid phase synthesis method, wherein the crude polypeptide comprises type I humanized collagen with an amino acid sequence as shown in SEQ ID NO.1; Step 2: Purify the crude polypeptide by HPLC to obtain the type I humanized collagen.

7. The preparation method according to claim 6, characterized in that: In step 1, the following steps are included: 1.1 Resin swelling: Weigh an appropriate amount of 2-chlorotrityl chloride resin with a degree of substitution of 0.5-1.0 mmol / g, put the resin into a reaction tube, add an appropriate amount of DCM, and shake; 1.2 connects to the first amino acid; 1.3 Deprotection: add an appropriate amount of 20% piperidine DMF solution by volume, remove it after a period of time, and then add an appropriate amount of 20% piperidine DMF solution by volume, and then add it for a period of time; 1.4 Detection: Draw off the 20% piperidine DMF solution by volume, take an appropriate amount of resin, wash it with ethanol 3 to 5 times, drain it, and the ninhydrin test is positive; 1.5 Washing: Wash with DMF 2 to 5 times, methanol 2 to 5 times, and DMF 2 to 5 times; 1.6 Condensation: Protected amino acid Fmoc-Arg(Pbf)-OH 3-5 times excess, HBTU 3-5 times excess, both dissolved in as little DMF as possible, added to the reaction tube, immediately added DIEA 10 times excess, react for a while; 1.7 Washing: Wash with DMF 1 to 3 times, methanol 1 to 3 times, and DMF 1 to 3 times, repeat steps 1.2 to 1.7, and connect the amino acids in the amino acid sequence shown in SEQ ID NO.1 from right to left; 1.8 Detection: Wash with DMF 1 to 3 times, DCM 1 to 3 times, and DMF 1 to 3 times in sequence. Ninhydrin test is negative; 1.9 Removal of sequence lysine protecting group: Use DMF containing one or two drops of hydrazine hydrate to remove the sequence lysine protecting group dde; 1.10 Washing: Wash with methanol 2 to 5 times; 1.11 Cut the peptide from the resin: Place the resin into a flask or centrifuge tube containing cutting solution and shake at constant temperature; 1.12 Blow dry and wash: Blow dry the lysate with nitrogen as much as possible, chromatograph it with ether, wash it with ether, and then evaporate it at room temperature to obtain the crude peptide.

8. The preparation method according to claim 6, characterized in that: In step 2, the following steps are included: 2.1 Take an appropriate amount of crude peptide and put it into a container, and dissolve it with an appropriate amount of 50% acetonitrile aqueous solution by volume; 2.2 Filter the solution with a 0.45 μm filter membrane; 2.3 Analysis: The crude peptide was analyzed by analytical HPLC with a mobile phase of water and acetonitrile for 30 min with gradient elution. The HPLC column was equilibrated with a starting gradient for 5 min before injection. The starting gradient was 95% water, 5% acetonitrile, and the ending ratio was 5% water, 95% acetonitrile in terms of volume percentage. 2.4 Preparation: Prepare the dissolved crude peptide for injection. First, balance the preparative HPLC column for 10 minutes. The starting gradient is 95% water and 5% acetonitrile by volume, and the ending gradient is 25% to 50% water and 50% to 75% acetonitrile. The gradient time is 40 minutes. Collect the sample from the detector to obtain the purified humanized type I collagen. 2.5 Identification: Take a sample of the purified humanized type I collagen prepared in step 2.4 for purity identification.

9. Use of the humanized type I collagen according to claim 1 or claim 2 in the preparation of pelvic floor repair products, medical cosmetic products, bone repair products and sports medicine products.

10. Use of the humanized type I collagen according to claim 1 or claim 2 in the preparation of skin tissue products.

11. The use of humanized type I collagen according to claim 1 or claim 2 in a product for promoting tissue growth, characterized in that: The preparation is used to maintain or upregulate the expression of elastin, fibronectin and collagen type I genes and / or proteins.

12. Use of the recombinant humanized type I collagen according to claim 1 or claim 2 in bone repair products and sports medicine products, characterized in that: The product has preventive and repairing functions and is used for promoting the crawling and growth of osteoblasts.

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