Recombinant type I collagen, preparation method and application

Abstract

The present invention discloses a recombinant type I collagen, a preparation method and an application thereof, belonging to the technical field of tissue-binding peptides. The recombinant type I collagen comprises the following modules connected in series from the N-terminus to the C-terminus: a signal peptide, a truncated type I collagen, and a His tag; the nucleotide sequence of the truncated type I collagen is shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; the present invention utilizes a mammalian expression system and a 293T cell line to express the recombinant type I collagen, thereby obtaining collagen with natural biological activity and improving the proliferation efficiency and repair ability of HSF cells promoted by the collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue-binding peptides, 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 found throughout the body, including skin, bones, tendons, cartilage, cornea, and connective tissue. It is the most abundant collagen in the body, comprising approximately 90% of total collagen. Type I collagen provides structure, strength, and support to various parts of the body, including skin, bones, tendons, and ligaments. It promotes the growth of new tissue and aids wound healing.

[0003] Type I collagen is a natural skin moisturizer. It helps retain moisture and improve skin hydration, making it a popular ingredient in moisturizers and other skincare products. Type I collagen has the ability to stimulate new skin cell growth 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 cosmetics industry for its moisturizing, regenerative, film-forming, and biocompatible properties. It is commonly used in moisturizers, anti-aging creams, and other skincare products to improve skin hydration, texture, and elasticity.

[0004] Currently, there are four main systems for recombinant protein production: prokaryotic expression systems (most commonly using Escherichia coli for protein expression), eukaryotic expression systems (such as yeast), mammalian cell protein expression systems (commonly used cells such as CHO and HEK293), and insect cell protein expression systems. Each expression system has its own advantages and disadvantages. Using the E. coli expression system allows for the production of expression products in a relatively short time and at a relatively low cost, making the E. coli system the most popular host for recombinant pharmaceutical protein expression. However, the target protein is often expressed as inclusion bodies, making product purification difficult. Furthermore, the prokaryotic expression system has an imperfect post-translational processing and modification system, resulting in low biological activity of the expressed product. Yeast and insect cell protein expression systems offer high expression levels and low costs, but their post-translational processing and modification systems are not identical to those of mammals. Mammalian cell expression systems produce proteins closer to their native state, but at low expression levels and cumbersome procedures.

[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, a vector, a host cell and its application. Both patents construct a vector with the screened recombinant humanized type I collagen and transform it into Pichia pastoris cells, screen high-copy recombinants to obtain Pichia pastoris genetically engineered bacteria that can express recombinant humanized type I collagen, and thus obtain type I collagen for the preparation of cosmetics, biomaterials, medical devices, etc.

[0006] Chinese patent CN118994370A discloses a recombinant type I collagen protein Pro.C1, its preparation method, and application. The recombinant type I collagen protein Pro.C1 can be expressed by prokaryotic fermentation, is non-cytotoxic, has high biological activity, and has the effects of promoting cell proliferation, promoting collagen regeneration, and firming and supporting. 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 protein, its preparation method and application. The humanized collagen protein is obtained by chemically synthesizing a polypeptide and then purifying the polypeptide. The type I humanized collagen protein contains essential amino acids and semi-essential amino acids for the human body, can promote the proliferation of human skin fibroblasts, upregulate the expression of type I collagen, fibronectin and collagen I genes, interact with endothelial cells and osteoblasts, promote osteoblast crawling and growth, and promote the interaction between osteoblasts and collagen.

[0008] However, when preparing or producing recombinant humanized type I collagen, the above patents mostly use E. coli or yeast, and a few directly synthesize. Although the E. coli expression system and the yeast expression system can obtain expression products in a relatively short time and the cost required 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 nature, and the biological activity of the protein expression product is low. Direct synthesis is generally a polypeptide of humanized type I collagen, which has a short amino acid sequence and does not have the corresponding protein tertiary structure and corresponding modification. There is no report in the prior art on the use of mammalian cell expression systems to express recombinant humanized type I collagen. Summary of the Invention

[0009] In response to the shortcomings of the existing technology, the present invention provides a recombinant type I collagen, a preparation method and an application thereof, and utilizes a mammalian expression system to express the recombinant type I collagen in a 293T cell line to obtain collagen with natural biological activity.

[0010] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0011] A recombinant type I collagen, wherein the recombinant type I collagen comprises the following modules connected in series from the N-terminus to the C-terminus: a signal peptide, a truncated type I collagen, and a His tag;

[0012] The nucleotide sequence of the truncated type I collagen is shown in any one of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6;

[0013] The nucleotide sequence of the signal peptide is shown in SEQ ID NO.1;

[0014] The nucleotide sequence of the His tag is shown in SEQ ID NO.2.

[0015] The preparation method comprises connecting the nucleotide sequence of recombinant type I collagen to a vector to obtain a recombinant expression vector, and then transfecting 293T cells to express the recombinant type I collagen.

[0016] The use of the recombinant type I collagen in the preparation of medical devices.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention increases the expression level of type I collagen by truncation and codon optimization of type I collagen, and can improve the proliferation efficiency and repair ability of HSF cells promoted by collagen, and screens out ColaI-3 with the best effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a structural diagram of the recombinant type I collagen in Example 1;

[0020] Figure 2 is the transfection rate in different groups of cells in Example 2;

[0021] Figure 3 is the content of recombinant type I collagen in the supernatant of 293T cells in Example 2;

[0022] Figure 4 Western blot analysis of recombinant type I collagen collected from different groups in Example 3;

[0023] Figure 5 This is a graph showing the results of the ability of recombinant type I collagen to promote HSF cell repair in Example 5. DETAILED DESCRIPTION

[0024] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described.

[0025] Example 1 Design of recombinant type I collagen structure

[0026] The recombinant type I collagen is connected in series from the N-terminus to the C-terminus by the following modules:

[0027] signal peptide, truncated type I collagen (abbreviated as Col-I), His tag;

[0028] The signal peptide is used to guide and promote protein secretion, and its nucleotide sequence is shown in SEQ ID NO.1 in the sequence listing;

[0029] The His tag is used for protein identification and purification, and its nucleotide sequence is shown in SEQ ID NO.2 in the sequence listing;

[0030] The structure of the recombinant type I collagen is shown in FIG. Figure 1 shown.

[0031] Four truncated type I collagen amino acid sequences of 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.

[0032] The optimized nucleotide sequences are:

[0033] ①(Col-I)-1 nucleotide sequence (SEQ ID NO. 3);

[0034] ②(Col-I)-2 nucleotide sequence (SEQ ID NO.4);

[0035] ③(Col-I)-3 nucleotide sequence (SEQ ID NO.5);

[0036] ④(Col-I)-4 nucleotide sequence (SEQ ID NO.6);

[0037] The above four truncated nucleotide sequences of type I collagen were recombined according to the structure of the recombinant type I collagen described above to construct a nucleotide sequence encoding (Col-I)-1, a nucleotide sequence encoding (Col-I)-2, a nucleotide sequence encoding (Col-I)-3, and a nucleotide sequence encoding (Col-I)-4.

[0038] The above-mentioned nucleotide sequences encoding recombinant type I collagen were commissioned to Shandong Hongnuo Biotechnology Co., Ltd. for synthesis and ligated into pTT5 vectors respectively. The obtained recombinant vectors were named pTT5-(Col-I)-1, pTT5-(Col-I)-2, pTT5-(Col-I)-3, and pTT5-(Col-I)-4, respectively.

[0039] The above vectors were transformed, cultured and plasmids were extracted according to conventional experimental methods. The concentration of each plasmid was adjusted to 1.0 μg / μL to obtain four pTT5 recombinant expression vectors containing Col-I.

[0040] In addition, the nucleotide sequences of the above four truncated type I collagens were replaced with the corresponding nucleotide sequences in AB209597.1, and the nucleotide sequence of (Col-I)-1 before optimization (SEQ ID NO.7), the nucleotide sequence of (Col-I)-2 before optimization (SEQ ID NO.8), the nucleotide sequence of (Col-I)-3 before optimization (SEQ ID NO.9), and the nucleotide sequence of (Col-I)-4 before optimization (SEQ ID NO.10) were respectively constructed according to the structure of the recombinant type I collagen described in Example 1, and were commissioned to Shandong Hongnuo Biotechnology Co., Ltd. for synthesis, and ligated into 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.

[0041] The above vectors were transformed, cultured and plasmids were extracted according to conventional experimental methods. The concentration of each plasmid was adjusted to 1.0 μg / μL to obtain four pTT5 recombinant expression vectors containing Col-I before optimization.

[0042] Example 2 Transfection of 293T cells with recombinant expression vector and detection of recombinant type I collagen content

[0043] 1. Culture of 293T cells

[0044] (1) Cell recovery: One tube of 293T cells (1 mL) frozen in liquid nitrogen was quickly shaken and thawed in a 40°C water bath. The thawed cells were transferred to a 50 mL centrifuge tube, 10 mL of DMEM medium was added and mixed evenly, centrifuged at 400 g for 5 min, the supernatant was discarded, 5 mL of DMEM complete medium was added and blown evenly, and then all the cell suspension was added to a culture flask, DMEM complete medium was added to 10 mL, and cultured in a 37°C, 5% CO2 incubator; DMEM complete medium is DMEM high glucose medium containing 10% VolFBS (purchased from Gibco).

[0045] (2) Cell passage: When the cell density reaches more than 80%, the cell can be subcultured. After the first subculture, the cells are cultured until the cells are in good condition and the viability is more than 90%. The cells in the logarithmic growth phase are diluted to 1×10 6 Cells were seeded into six-well plates (1 mL of cell suspension was added to each well), and 1 mL of DMEM complete medium was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator. Transfection was performed when the cell density reached 50% confluence.

[0046] 2. Transfection of 293T cells with recombinant expression vector

[0047] Before transfection, replace the culture medium with fresh DMEM high-glucose medium. Prepare the DNA solution as standard: mix 246 μL of DMEM high-glucose medium with 4 μL of the pTT5 recombinant expression vector (4 μg). For the Lipofectamine solution, mix 238 μL of DMEM high-glucose medium with 12 μL of Lipofectamine. Then, thoroughly mix the DNA and Lipofectamine solutions and incubate at room temperature for 20 minutes to obtain the Lipofectamine-DNA culture medium. Remove the supernatant from the prepared 293T cells and add 500 μL of the Lipofectamine-DNA mixture to one well of a six-well plate. Incubate in a 37°C, 5% CO2 incubator for 6 hours. Remove the culture medium containing the Lipofectamine-DNA and add 2 mL of fresh DMEM complete medium to each well to continue transfection.

[0048] After 48 hours of culture and transfection, the transfection rate of each group of cells was determined using His flow cytometry antibody (e.g. Figure 2 A portion of the cell culture medium was centrifuged at 3000 rpm for 5 min, and the supernatant was collected. The content of recombinant type I collagen in the supernatant was detected using a type I collagen ELISA kit (ml057630; purchased from ELISA).

[0049] The results are shown in Table 1 and Figure 3As shown, 293T cells transfected with each recombinant expression vector of the present invention are all capable of secreting recombinant type I collagen, but the content of recombinant type I collagen in the supernatant varies significantly, indicating that the sequence of the truncated type I collagen affects the ability of the recombinant expression vector to express recombinant type I collagen. After codon optimization of the nucleotide sequence of the truncated type I collagen of the present invention, the recombinant expression vector constructed therefrom has a stronger ability to express recombinant type I collagen, among which the recombinant type I collagen encoding (Col-I)-3 has the highest content.

[0050] Table 1 Content of recombinant type I collagen in 293T cell supernatant

[0051]

[0052] Example 3 Identification, purification and concentration of recombinant type I collagen

[0053] Different recombinant expression vectors were amplified and transfected into 293T cells, and the supernatant was collected to obtain recombinant type I collagen. The collected supernatant was purified by Ni-NTA agarose gel affinity chromatography to obtain a purified recombinant type I collagen solution. The purified protein was identified using a His antibody.

[0054] The size of (Col-I)-1 and (Col-I)-1-recombinant type I collagen before optimization is about 117KD; the size of (Col-I)-2 and (Col-I)-2-recombinant type I collagen before optimization is about 88KD; the size of (Col-I)-3 and (Col-I)-3-recombinant type I collagen before optimization is about 20KD; the size of (Col-I)-4 and (Col-I)-4-recombinant type I collagen before optimization is about 14.6KD; using His antibody identification, the results are as follows Figure 4 As shown, the actual size of recombinant type I collagen is consistent with the predicted size of recombinant type I collagen.

[0055] The purified recombinant type I collagen solutions after nucleotide sequence optimization and the recombinant type I collagen solutions before nucleotide sequence optimization were diluted with PBS (purchased from Solebol, catalog number: P1020) to 1 mg / mL for later use; they were 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, and (Col-I)-4-before optimization.

[0056] Example 4 Detection of the ability of recombinant type I collagen to promote HSF cell proliferation

[0057] 1. Take human fibroblasts (HSF cells) in logarithmic growth phase and dilute them with fibroblast-specific culture medium to obtain a cell suspension. 4 The cell suspension was seeded into a 48-well culture plate at a density of 100 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0058] The fibroblast-specific culture medium was purchased from Shanghai Xinyu Biotechnology Co., Ltd., and its composition was: 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.

[0059] 2. The experiment was divided into a normal group, a sample group, and a control group. The original culture medium was aspirated and fresh fibroblast-specific culture medium was added to the normal group at 300 μL / well. The sample group was added with various fibroblast-specific culture media containing recombinant type I collagen (0.5 mg / mL) at 300 μL / well. The control group was added with fibroblast-specific culture media containing 0.5 mg / mL commercially available type I collagen (NCBI reference sequence: BC036531.2) at 300 μL / well. Each group was incubated at 37°C, 5% CO2. After 24 hours, the culture medium was aspirated and 300 μL / well of fibroblast-specific culture media containing 0.5 mg MTT was added. After 2 hours of incubation, the culture medium was removed and 100 μL of DMSO solution was added. The absorbance of each well was measured at 490 nm, and the relative cell viability was calculated. The results are shown in Table 2.

[0060] Relative cell viability = sample group / normal group × 100%;

[0061] As shown in Table 2, the recombinant type I collagen of the present invention exhibited a higher cell proliferation-promoting ability than commercially available type I collagen. The recombinant type I collagen after nucleotide sequence optimization exhibited a superior cell proliferation-promoting ability compared to the recombinant type I collagen before nucleotide sequence optimization. Among these, (Col-I)-3 exhibited the best effect.

[0062] Table 2 Recombinant type I collagen promotes the viability of HSF cells

[0063]

[0064] Example 5 Detection of the ability of recombinant type I collagen to promote HSF cell repair

[0065] HSF cells in the logarithmic growth phase were cultured at a rate of 1×10 6Cells were seeded into 6-well plates at a density of 100 μL / well and incubated overnight in an incubator (37°C, 5% CO2). A scratch wound assay was performed when the confluence reached 80% or higher. Cell repair capacity was assessed by a cell scratch assay, and the cells were washed three times with PBS. The experiment was divided into a normal group, a sample group, and a control group. The normal group received 2 mL of fresh fibroblast-specific culture medium, the sample group received fibroblast-specific culture medium containing various recombinant type I collagens (0.5 mg / mL), and the control group received 300 μL of fibroblast-specific culture medium containing 0.5 mg / mL of commercially available type I collagen (NCBI reference sequence: BC036531.2) per well.

[0066] Each group was divided into three replicate wells and cultured in an incubator (37°C, 5% CO2) for 24 hours. Photos were taken, and the images were analyzed to calculate the repair efficiency of HSF cells.

[0067] The results are as follows Figure 5 As shown in Table 3, the recombinant type I collagen of the present invention exhibits a higher repair rate for human fibroblasts than commercially available type I collagen. The recombinant type I collagen of the present invention, after nucleotide sequence optimization, exhibits superior cell repair-promoting abilities compared to the recombinant type I collagen before nucleotide sequence optimization. Among these, (Col-I)-3 exhibits the best effect.

[0068] Table 3 Repair rate of HSF cells promoted by recombinant type I collagen

[0069]

Claims

1. A recombinant type I collagen, characterized in that The recombinant type I collagen is 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 in 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, characterized in that The nucleotide sequence of the signal peptide is shown in SEQ ID NO.1; the nucleotide sequence of the His tag is shown in SEQ ID NO.

2.

3. The method for preparing recombinant type I collagen according to claim 2, characterized in that: The preparation method comprises connecting the nucleotide sequence of recombinant type I collagen to a vector to obtain a recombinant expression vector, and then transfecting 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.

Images