Recombinant I-type collagen as well as preparation method and application thereof

Abstract

The invention discloses a recombinant I-type collagen, a preparation method and application, and belongs to the technical field of binding tissue peptides, the recombinant I-type collagen is formed by sequentially connecting the following modules in series from the N terminal to the C terminal: a signal peptide, a truncated I-type collagen and a His tag; the nucleotide sequence of the truncated I type collagen is as shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; the nucleotide sequence of the truncated I type collagen is as shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; according to the invention, a mammalian expression system and a 293T cell line are used for expressing recombinant I-type collagen, so that collagen with natural biological activity is obtained, and the proliferation efficiency and repair capacity of the collagen for promoting HSF cells can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue peptide combination, relates to recombinant type I collagen, and also relates to a preparation method and application of the recombinant type I collagen. Background Art

[0002] Type I collagen is a protein present in various parts of the human body, including the skin, bones, tendons, cartilage, cornea, and connective tissues. It is the most abundant collagen in the human body, accounting for approximately 90% of the total collagen. Type I collagen provides structure, strength, and support to various parts of the body, including the skin, bones, tendons, and ligaments. It can promote the growth of new tissues and the healing of wounds.

[0003] Type I collagen is a natural skin moisturizer. It helps to retain moisture and improve skin hydration, making it a popular ingredient in moisturizers and other skin care products. Type I collagen has the ability to stimulate the growth of new skin cells and promote tissue regeneration. It can help improve skin elasticity and reduce the appearance of fine lines and wrinkles. Type I collagen is a useful ingredient in the cosmetic industry due to its moisturizing, regenerating, film-forming, and biocompatible properties. It is commonly used in moisturizers, anti-aging creams, and other skin care products to improve skin hydration, texture, and elasticity.

[0004] Currently, there are mainly four major systems for the production of recombinant proteins: prokaryotic expression systems (the most commonly used Escherichia coli protein expression), eukaryotic expression systems (such as yeast), mammalian cell protein expression systems (commonly used cells CHO, HEK293), and insect cell protein expression systems. Each expression system has its own advantages and disadvantages. Using the Escherichia coli expression system can obtain the expression product in a relatively short time, and the required cost is relatively low. The Escherichia coli system has also been proven to be the most popular host for the expression of recombinant drug proteins. However, the target protein is often expressed in the form of inclusion bodies, and the product purification is difficult. In addition, the post-translational processing and modification system of the prokaryotic expression system is not perfect, and the biological activity of the expression product is relatively low. The expression levels of the yeast and insect cell protein expression systems are high and the costs are low, but the post-translational processing and modification systems are not exactly the same as those of mammals. The proteins produced by the mammalian cell expression system are closer to the natural state, but the expression level is low and the operation is cumbersome.

[0005] Chinese Patent CN118620065A discloses recombinant humanized type I collagen, its expression vector and genetically engineered bacteria. Chinese Patent CN118852409A discloses a type I recombinant collagen, vector, host cell and their applications. Both patents screen the obtained recombinant humanized type I collagen, construct a vector and transform it into Pichia pastoris cells, and screen for high-copy recombinants to obtain a Pichia pastoris genetically engineered bacterium capable of expressing recombinant humanized type I collagen, thereby obtaining type I collagen for use in preparing cosmetics, biomaterials, medical devices, etc.

[0006] Chinese Patent CN118994370A discloses a recombinant type I collagen Pro.C1, its preparation method and applications. This recombinant type I collagen Pro.C1 can be expressed by prokaryotic fermentation, has no cytotoxicity, high biological activity, and has the effects of promoting cell proliferation, promoting collagen regeneration, and firm support. It has good water solubility and stable quality, and can be widely used in skin care products, skin repair dressings, medical beauty and other fields.

[0007] Chinese Patent CN118791595A discloses a type I humanized collagen, its preparation method and applications. The humanized collagen is obtained by chemically synthesizing polypeptides and purifying the polypeptides. This type I humanized collagen contains essential and semi-essential amino acids of the human body, can promote the proliferation of human skin fibroblasts, up-regulate the expression of type I collagen, fibronectin and collagen I genes, and has interactions with endothelial cells and osteoblasts, promotes the crawling and growth of osteoblasts, and promotes the interaction between osteoblasts and collagen.

[0008] However, when preparing or producing recombinant humanized type I collagen in the above patents, most choose Escherichia coli or yeast, and a few directly synthesize it. Although the Escherichia coli expression system and yeast expression system can obtain expression products in a relatively short time and the required cost is relatively low, the target proteins obtained by these two expression systems are usually expressed in the form of inclusion bodies, and the expression system lacks a post-translational modification mechanism or the post-translational processing and modification system is imperfect, making it difficult to form type I collagen close to the natural state, and the biological activity of the protein expression product is relatively low. What is generally directly synthesized is the polypeptide of humanized type I collagen, which has a short amino acid sequence and does not have the corresponding protein tertiary structure and corresponding modifications. There is no report in the prior art on expressing recombinant humanized type I collagen using a mammalian cell expression system. Summary of the Invention

[0009] Aiming at the deficiencies of the existing technology, the present invention provides a recombinant type I collagen, its preparation method and applications. Using a mammalian expression system, the 293T cell line is used to express recombinant type I collagen to obtain collagen with natural biological activity.

[0010] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A recombinant type I collagen, wherein the recombinant type I collagen is sequentially connected in series from the N-terminus to the C-terminus by the following modules: a signal peptide, a truncated type I collagen, and a His tag; The nucleotide sequence of the truncated type I collagen is shown as any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6; The nucleotide sequence of the signal peptide is shown as SEQ ID NO.1; The nucleotide sequence of the His tag is shown as SEQ ID NO.2.

[0011] The preparation method is to connect the nucleotide sequence of the recombinant type I collagen to a vector to obtain a recombinant expression vector, and then transfect 293T cells to express the recombinant type I collagen.

[0012] The application of the recombinant type I collagen in the preparation of medical devices.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: By truncating and codon-optimizing type I collagen, the present invention improves the expression level of type I collagen, and can improve the proliferation efficiency and repair ability of collagen on HSF cells, and screens out ColaI-3 with the best effect. Description of the Drawings

[0014] Figure 1 It is the structural diagram of the recombinant type I collagen in Example 1; Figure 2 It is the transfection rate in different groups of cells in Example 2; Figure 3 It is the content of the recombinant type I collagen in the supernatant of 293T cells in Example 2; Figure 4 It is the Western blot identification result of the recombinant type I collagen collected in different groups in Example 3; Figure 5 It is the result diagram of the ability of the recombinant type I collagen to promote the repair of HSF cells in Example 5. Detailed Embodiments

[0015] In order to have a clearer understanding of the technical features, objectives, and effects of the present invention, the specific embodiments of the present invention are now described.

[0016] Example 1 Design of the Structure of Recombinant Type I Collagen The recombinant type I collagen is sequentially connected in series from the N-terminus to the C-terminus by the following modules: Signal peptide, truncated type I collagen (abbreviated as Col-I), His tag; The signal peptide is used to direct and promote protein secretion, and its nucleotide sequence is shown as SEQ ID NO.1 in the sequence listing; The His tag is used for protein identification and purification, and its nucleotide sequence is shown as SEQ ID NO.2 in the sequence listing; The structural diagram of the recombinant type I collagen is as shown in Figure 1 shown.

[0017] Four truncated type I collagen amino acid sequences with different lengths were selected from the full-length type I collagen amino acid sequence (NCBI reference sequence: AB209597.1), specifically: (Col-I)-1: 165-1221; (Col-I)-2: 197-883; (Col-I)-3: 716-869; (Col-I)-4: 666-769, and their nucleotide sequences were codon-optimized.

[0018] The optimized nucleotide sequences are respectively: ① The nucleotide sequence of (Col-I)-1 (SEQ ID NO.3); ② The nucleotide sequence of (Col-I)-2 (SEQ ID NO.4); ③ The nucleotide sequence of (Col-I)-3 (SEQ ID NO.5); ④ The nucleotide sequence of (Col-I)-4 (SEQ ID NO.6); The nucleotide sequences of the above 4 truncated type I collagens were respectively recombined according to the structure of the recombinant type I collagen described above to construct the nucleotide sequences encoding (Col-I)-1, the nucleotide sequence encoding (Col-I)-2, the nucleotide sequence encoding (Col-I)-3, and the nucleotide sequence encoding (Col-I)-4.

[0019] The nucleotide sequences encoding the recombinant type I collagen were entrusted to Shandong Hongnuo Biotechnology Co., Ltd. for synthesis, and they were respectively ligated to the pTT5 vector, and the obtained recombinant vectors were named pTT5-(Col-I)-1, pTT5-(Col-I)-2, pTT5-(Col-I)-3, and pTT5-(Col-I)-4.

[0020] The above vectors were transformed, shaken in bacteria and plasmids were extracted according to conventional experimental methods, and the concentrations of various plasmids were adjusted to 1.0 μg / μL to obtain 4 kinds of pTT5 recombinant expression vectors containing Col-I.

[0021] In addition, replace the nucleotide sequences of the above 4 truncated type I collagens with the corresponding nucleotide sequences in AB209597.1. The nucleotide sequences before optimization of (Col-I)-1 (SEQ ID NO.7), (Col-I)-2 (SEQ ID NO.8), (Col-I)-3 (SEQ ID NO.9), and (Col-I)-4 (SEQ ID NO.10) were respectively constructed according to the structure of the recombinant type I collagen described in Example 1, synthesized by Shandong Hongnuo Biotechnology Co., Ltd., and ligated to the pTT5 vector. The obtained recombinant vectors were named pTT5-(Col-I)-1-before optimization, pTT5-(Col-I)-2-before optimization, pTT5-(Col-I)-3-before optimization, and pTT5-(Col-I)-4-before optimization respectively.

[0022] Transform, shake the bacteria, and extract the plasmids of the above vectors according to the conventional experimental methods. The concentrations of various plasmids were adjusted to 1.0 μg / μL to obtain 4 kinds of pTT5 recombinant expression vectors containing Col-I before optimization.

[0023] Example 2 Transfection of 293T cells with the recombinant expression vector and detection of the content of recombinant type I collagen 1. Culture of 293T cells (1) Resuscitate the cells: Quickly shake and thaw 1 tube of 293T cells (with a volume of 1 mL) cryopreserved in liquid nitrogen in a 40°C water bath. Transfer the thawed cells to a 50 mL centrifuge tube, add 10 mL of DMEM medium and mix well. Centrifuge at 400 g for 5 min, discard the supernatant, add 5 mL of complete DMEM medium and resuspend evenly. Then add all the cell suspension into a culture flask, add complete DMEM medium to 10 mL, and culture in an incubator at 37°C and 5% CO2; The complete DMEM medium is DMEM high-glucose medium containing 10% Vol FBS (purchased from Gibco).

[0024] (2) Cell passage: When the cell density reaches more than 80%, subculture can be carried out. After the first passage, the cells are cultured until the cell state is good and the viability is more than 90%. Dilute the cells in the logarithmic growth phase to 1×10 6 cells / mL, inoculate into a six-well plate (add 1 mL of cell suspension to each well), add 1 mL of complete DMEM medium to each well, and culture in an incubator at 37°C and 5% CO2. Transfection can be carried out when the cell density reaches a confluence rate of 50%.

[0025] 2. Transfection of 293T cells with the recombinant expression vector Before transfection, change the culture medium to fresh high-glucose DMEM medium. Prepare the DNA solution according to the conventional method, that is, mix 246 μL of high-glucose DMEM medium with 4 μL of pTT5 recombinant expression vector (4 μg); Lipofiter solution: mix 238 μL of high-glucose DMEM medium with 12 μL of Lipofiter. Then, after thoroughly mixing the above DNA solution and Lipofiter solution, incubate at room temperature for 20 min to obtain the Lipofiter-DNA culture solution. For the prepared 293T cells, remove the supernatant, and add 500 μL of the Lipofiter-DNA mixture to one well of a six-well plate, and culture in an incubator at 37 °C and 5% CO2 for 6 h. Remove the culture solution containing Lipofiter-DNA, and add 2 mL of fresh complete DMEM medium to each well to continue culturing for transfection.

[0026] After culturing and transfecting for 48 hours, use His flow antibody to measure the transfection rate in each group of cells (such as Figure 2 ). Take a part of the cell culture solution, centrifuge at 3000 rpm for 5 min, collect the supernatant, and detect the content of recombinant type I collagen in the supernatant by a type I collagen ELISA kit (ml057630; purchased from Enzyme-linked Biotechnology).

[0027] The results are shown in Table 1 and Figure 3 as follows. It can be seen that the 293T cells transfected with each recombinant expression vector of the present invention can secrete recombinant type I collagen, but there are significant differences in the content of recombinant type I collagen in the supernatant, which indicates that the sequence of truncated type I collagen affects the ability of the recombinant expression vector to express recombinant type I collagen. After the nucleotide sequence of the truncated type I collagen of the present invention is codon-optimized, the constructed recombinant expression vector has a stronger ability to express recombinant type I collagen, and the content of recombinant type I collagen expressed by the encoded (Col-I)-3 is the highest.

[0028] Table 1 Content of recombinant type I collagen in 293T cell supernatant

[0029] Example 3 Identification, purification and concentration of recombinant type I collagen Expand the system of different recombinant expression vectors to transfect 293T cells, collect the supernatant to obtain recombinant type I collagen, and purify the collected supernatant according to the Ni-NTA agarose gel affinity chromatography purification method to obtain a purified recombinant type I collagen solution, and identify the purified protein with His antibody.

[0030] (Col-I)-1 and (Col-I)-1-recombinant type I collagen before optimization had a size of approximately 117 KD; (Col-I)-2 and (Col-I)-2-recombinant type I collagen before optimization had a size of approximately 88 KD; (Col-I)-3 and (Col-I)-3-recombinant type I collagen before optimization had a size of approximately 20 KD; (Col-I)-4 and (Col-I)-4-recombinant type I collagen before optimization had a size of approximately 14.6 KD; Identified using His antibody, the results were as Figure 4 shown, the actual size of the recombinant type I collagen was consistent with the predicted size of the recombinant type I collagen.

[0031] The purified recombinant type I collagen solutions with optimized 4 nucleotide sequences and the recombinant type I collagen solutions with unoptimized 4 nucleotide sequences were respectively diluted with PBS (purchased from Solarbio, product number: P1020) to 1 mg / mL for standby; they were respectively named: (Col-I)-1, (Col-I)-2, (Col-I)-3, (Col-I)-4, (Col-I)-1-before optimization, (Col-I)-2-before optimization, (Col-I)-3-before optimization, (Col-I)-4-before optimization.

[0032] Example 4 Detection of the ability of recombinant type I collagen to promote the proliferation of HSF cells 1. Take human fibroblasts (HSF cells) in the logarithmic growth phase, dilute them with a special medium for fibroblasts to obtain a cell suspension, and inoculate the cell suspension into a 48-well culture plate at a density of 8×10 4 cells / well, and culture them in an incubator at 37 °C and 5% CO2 for 24 h.

[0033] The special medium for fibroblasts was purchased from Shanghai Xinyu Biotechnology Co., Ltd., and its components were: DMEM / F12 + 10% FBS + 1% p / s + 0.005 mg / mL insulin + 5 ng / mL BfGF + 1 μg / mL hydrocortisone + 50 μg / mL ascorbic acid (vitamin C) + 7.5 mM L-Gln.

[0034] 2. The experiment was divided into a normal group, a sample group, and a control group; the original culture medium was aspirated and discarded. In the normal group, fresh fibroblast-specific medium was added at a volume of 300 μL / well. In the sample group, various fibroblast-specific media containing recombinant type I collagen (0.5 mg / mL) were added at a volume of 300 μL / well. In the control group, fibroblast-specific medium containing commercially available type I collagen (NCBI reference sequence: BC036531.2) at 0.5 mg / mL was added at a volume of 300 μL / well. There were 3 replicate wells in each group, and the cells were cultured under the conditions of 37 °C and 5% CO2; after 24 h, the culture medium was aspirated and discarded, and fibroblast-specific medium containing 0.5 mg MTT was added at a volume of 300 μL / well. After culturing for 2 h, the culture medium was removed, 100 μL of DMSO solution was added, and the absorbance value of each well was measured at 490 nm, and the relative cell viability was calculated. The results are shown in Table 2.

[0035] Relative cell viability = sample group / normal group × 100%; As shown in Table 2, the ability of the recombinant type I collagen in the present invention to promote cell proliferation is higher than that of the commercially available type I collagen. The ability of the recombinant type I collagen with optimized nucleotide sequence to promote cell proliferation is better than that of the recombinant type I collagen before nucleotide sequence optimization. Among them, (Col-I)-3 showed the best effect.

[0036] Table 2 Viability of recombinant type I collagen in promoting HSF cells

[0037] Example 5 Detection of the ability of recombinant type I collagen to promote HSF cell repair HSF cells in the logarithmic growth phase were seeded into a 6-well plate at a density of 1×10 6 cells / well and incubated overnight in an incubator (37 °C, 5% CO2). When the confluence reached more than 80%, a scratch experiment was performed. The cell repair ability was measured by the cell scratch method, and the cells were washed 3 times with PBS after scratching. The experiment was divided into a normal group, a sample group, and a control group; in the normal group, 2 mL of fresh fibroblast-specific medium was added, in the sample group, fibroblast-specific medium containing various recombinant type I collagens (0.5 mg / mL) was added, and in the control group, fibroblast-specific medium containing 0.5 mg / mL commercially available type I collagen (NCBI reference sequence: BC036531.2) was added at a volume of 300 μL / well.

[0038] There were 3 replicate wells in each group, and the cells were further cultured in an incubator (37 °C, 5% CO2) for 24 h. Photos were taken and the pictures were analyzed to calculate the repair efficiency of HSF cells.

[0039] The results are as Figure 5As shown in Table 3, it can be seen that the repair rate of the recombinant type I collagen of the present invention for human fibroblasts is higher than that of the commercially available type I collagen. The recombinant type I collagen with optimized nucleotide sequence in the present invention shows more excellent ability to promote cell repair compared with the recombinant type I collagen before nucleotide sequence optimization. Among them, (Col-I)-3 shows the best effect.

[0040] Table 3 Repair rate of recombinant type I collagen promoting HSF cells

Claims

1. A recombinant type I collagen, characterized in that, The recombinant type I collagen is sequentially concatenated from the N-terminus to the C-terminus by the following modules: a signal peptide, a truncated type I collagen, and a His tag; The nucleotide sequence of the truncated type I collagen is shown as any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.

6.

2. The recombinant type I collagen according to claim 1, wherein The nucleotide sequence of the signal peptide is shown as SEQ ID NO.1; the nucleotide sequence of the His tag is shown as SEQ ID NO.

2.

3. The preparation method of the recombinant type I collagen according to claim 2, characterized in that, The preparation method is to ligate the nucleotide sequence of the recombinant type I collagen to a vector to obtain a recombinant expression vector, and then transfect 293T cells to express the recombinant type I collagen.

4. Use of the recombinant type I collagen according to claim 1 in the preparation of medical devices.

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