Recombinant collagen and its encoding gene, recombinant vector, recombinant strain, preparation method and application thereof

Abstract

The present invention belongs to the fields of protein engineering and biotechnology and discloses a recombinant collagen protein, its encoding gene, recombinant vector, recombinant strain, preparation method, and applications thereof. The amino acid sequence of the recombinant collagen protein is shown in SEQ ID NO. 1. The recombinant collagen protein has high transdermal permeability and exhibits excellent fibroblast adhesion and collagen metabolism. This collagen protein has broad application prospects in medical cosmetic surgery, cosmetics, and other fields.

Description

Technical Field

[0001] The present invention relates to the fields of protein engineering and biotechnology, and in particular to a recombinant collagen protein and its encoding gene, a recombinant vector, a recombinant strain, a preparation method and applications thereof. Background Art

[0002] Collagen is a vital structural protein, one of the most abundant and widespread proteins in mammals and many other organisms. It plays a central role in maintaining the body's structural support, tissue stability, and elasticity. Collagen is widely present in tissues such as bones, joints, muscles, skin, blood vessels, and the cornea, where it is a primary component. With its unique triple-helix structure, collagen forms molecular cables that strengthen tendons and support the membranes of the skin and internal organs. In bones and teeth, collagen binds to mineral grains to form strong bone tissue. In addition to providing structural support, collagen also acts as a signaling molecule, regulating cell proliferation, differentiation, and migration, and is crucial for maintaining normal physiological function and overall health. Recombinant collagen, due to its low toxicity, low antigenicity, low immunogenicity, ability to guide cell regeneration, and excellent biocompatibility, has found widespread application in the biopharmaceutical, cosmetic, and food industries. In the aesthetic medical field, collagen is also a key ingredient used to improve skin condition and maintain its youthfulness and elasticity.

[0003] When collagen is used in cosmetics and other fields, from the perspective of enhancing its biological activity, it is expected that collagen will have good transdermal absorption performance. However, since collagen is a large molecular bioactive substance, its own transdermal absorption capacity is relatively weak. Therefore, in previous studies, how to promote the transdermal absorption of collagen has become an important research hotspot. In recent years, with the widespread application of genetic engineering technology, researchers have created various types of recombinant collagen. Usually, short amino acid sequences from natural human collagen can be selected for splicing and / or repeated construction to obtain new recombinant collagen, and this type of recombinant collagen theoretically has the advantages of low immunogenicity and high biological activity.

[0004] However, there is currently no existing technology that provides effective guidance for designing short amino acid sequences as repeating units or splicing units to ensure that the designed recombinant collagen not only maintains good biological activity and transdermal absorption ability, but also has better fibroblast adhesion and collagen metabolism ability of fibroblasts. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem in the prior art that recombinant collagen, while maintaining good biological activity and transdermal absorption capacity, cannot take into account the better adhesion of fibroblasts and the collagen metabolism capacity of fibroblasts. A recombinant collagen and its encoding gene, recombinant vector, recombinant strain, preparation method and use thereof are provided. The recombinant collagen has high activity, good transdermal effect, and takes into account the better adhesion of fibroblasts and the collagen metabolism capacity of fibroblasts.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides a recombinant collagen protein, wherein the recombinant collagen protein is a collagen protein having an amino acid sequence as shown in SEQ ID NO. 1.

[0007] The second aspect of the present invention provides a gene encoding a recombinant collagen protein, wherein the gene has a nucleotide sequence encoding the recombinant collagen protein as described above.

[0008] Preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2.

[0009] The third aspect of the present invention provides a recombinant vector comprising the gene as described above.

[0010] Preferably, the expression vector of the recombinant vector is a pPIC9K plasmid.

[0011] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0012] Preferably, the expression strain of the recombinant strain is yeast, more preferably Pichia pastoris strain GS115.

[0013] A fifth aspect of the present invention provides a method for preparing recombinant collagen, which comprises: inoculating the aforementioned recombinant strain into a fermentation medium for fermentation culture.

[0014] Preferably, the fermentation culture conditions include: temperature of 20-35° C., rotation speed of 150-240 rpm, time of 60-80 h, and inoculation amount of 1-15% by volume.

[0015] The sixth aspect of the present invention provides the use of at least one of the aforementioned recombinant collagen, the aforementioned gene, the aforementioned recombinant vector, the aforementioned recombinant strain, and the recombinant collagen produced by the aforementioned method in the preparation of collagen products.

[0016] Preferably, the collagen product is selected from at least one of a skin product, a product for enhancing fibroblast adhesion, and a product for enhancing fibroblast collagen metabolism.

[0017] Preferably, the collagen product is selected from any one of an injection, a facial filler, a dressing, a cosmetic and a tissue engineering material.

[0018] Through the above technical solution, the present invention obtains the natural human type III collagen α1 chain protein amino acid sequence ( GenBank : AGL34959.1), and deeply mined and analyzed the biological information of the sequence, and designed the hydrophilicity, charge distribution and protein spatial structure of the amino acid sequence, so that the recombinant collagen of the present invention can achieve better fibroblast adhesion and collagen metabolism ability of fibroblasts while maintaining high biological activity. The amino acid sequence of the recombinant collagen of the present invention is shown in SEQ ID NO. 1, which ensures the realization of the biological function of the recombinant collagen and has the characteristics of high biocompatibility, good transdermal permeability and low risk of use.

[0019] The recombinant collagen provided by the present invention has broad application prospects in the fields of medical cosmetic surgery, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The recombinant collagen fermentation broth in Example 1-3 SDS-PAGE Protein detection results, Line 1: S37 induced for 24 hours, Line 2, Line 4: Marker;

[0021] Figure 2 The recombinant collagen protein after purification in Example 1-4 SDS-PAGE Protein detection result diagram;

[0022] Figure 3 This is a diagram of the fibroblast adhesion experiment of the recombinant collagen in Example 2;

[0023] Figure 4 This is a diagram of the fibroblast collagen metabolism experiment of the recombinant collagen in Example 3. DETAILED DESCRIPTION

[0024] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0025] A first aspect of the present invention provides a recombinant collagen protein, wherein the recombinant collagen protein is a collagen protein having an amino acid sequence as shown in SEQ ID NO. 1.

[0026] The second aspect of the present invention provides a gene encoding a recombinant collagen protein, wherein the gene has a nucleotide sequence encoding the recombinant collagen protein as described above.

[0027] It is well known in the art that among the 20 different amino acids that make up proteins, Met (ATG) or Trp (TGG) are each coded by a single codon, and the other 18 amino acids are coded by 2-6 codons ( Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic code, there is often more than one codon that specifies an amino acid. Substitution of the third nucleotide in a triplet codon often does not change the amino acid composition, so the nucleotide sequences of genes encoding the same protein can be different.

[0028] According to the present invention, preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2. In the present invention, the gene may comprise only the sequence shown in SEQ ID NO. 2, or may be a DNA molecule comprising the sequence shown in SEQ ID NO. 2 as the coding region and additionally comprising other components. These additional components may include any components known in the art for artificially synthesizing gene sequences and expressing them via expression vectors, such as promoters, enhancers, Kozak sequences, and the like.

[0029] The amino acid sequence shown in SEQ ID NO. 1 and the nucleotide sequence shown in SEQ ID NO. 2 are shown in Table 1.

[0030] Table 1

[0031]

[0032] The present invention improves the expression efficiency of recombinant collagen by adjusting the gene sequence. Specifically, by changing the synonymous codons in the gene to better match the codon preference of Pichia pastoris, rare codons are eliminated, and translation efficiency is improved. The inventors have found that the above preferred embodiment can achieve higher expression levels of the recombinant collagen provided by the present invention.

[0033] The nucleotide sequences provided herein can generally be obtained using polymerase chain reaction (PCR) amplification, recombination, or synthetic methods. Once the nucleotide sequence is obtained, the amino acid sequence can be obtained in large quantities using recombination methods. The resulting nucleotide sequence is typically cloned into a vector, then transferred into genetically engineered bacteria, and then the nucleotide sequence is isolated from the propagated host cells using conventional methods. The nucleotide sequence shown in SEQ ID NO. 2 of the present invention was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd.

[0034] In addition, the relevant nucleotide sequences can also be synthesized using known artificial chemical synthesis methods.

[0035] A third aspect of the present invention provides a recombinant vector comprising the gene described above. In the present invention, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as commercially available plasmids, cosmids, phages, and retroviruses. The expression vector of the present invention is preferably the pPIC9K plasmid, resulting in the recombinant plasmid pPIC9K-COL3-S37.

[0036] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0037] The host cell can be a prokaryotic cell or a eukaryotic cell. According to the present invention, preferably, the expression strain of the recombinant strain is a yeast, more preferably a Pichia pastoris strain GS115.

[0038] In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation using the calcium chloride method or high-voltage electroporation transformation.

[0039] The present invention uses the Pichia pastoris strain GS115 as a host strain and transforms the recombinant vector pPIC9K-COL3-S37 containing the recombinant collagen provided by the present invention into the GS115 host strain. The inventors have found that using the GS115 host strain has a higher ability to express recombinant collagen and has higher stability.

[0040] A fifth aspect of the present invention provides a method for preparing recombinant collagen, which comprises: inoculating the aforementioned recombinant strain into a fermentation medium for fermentation culture.

[0041] In the present invention, there is no particular limitation on the fermentation conditions of the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Exemplarily, the fermentation process includes: first randomly picking a single colony of the recombinant strain and inoculating it into an activation medium (the activation medium preferably contains: 8-12 g / L yeast extract, 15-25 g / L tryptone, 15-25 g / L glucose, and 15-25 g / L agar powder), and shaking and culturing it at room temperature at a speed of 150-250 rpm for 8-12 h for activation culture; inoculating the activated cultured strain into a seed culture medium (the seed culture medium preferably contains: 15-25 g / L tryptone, 5-15 g / L yeast extract, 10-15 g / L YNB, 15-25 g / L glycerol, and 80-120 mM phosphate at pH 6.0-7.0) for seed culture, and shaking and culturing it at room temperature at a speed of 150-250 rpm for 8-12 h, and then inoculating it into a fermentation medium (the fermentation medium preferably contains: 15-25 g / L tryptone, 5-15 g / L yeast extract, The expression of the recombinant collagen was induced in 1% dextrose (10-15 g / L) of YNB (10-15 g / L), the resulting fermentation broth was centrifuged, and the supernatant was collected to obtain a liquid containing the recombinant collagen. The inventors have found that the above fermentation process is beneficial for increasing the expression of the recombinant collagen.

[0042] The recombinant collagen provided by the present invention has a low molecular weight and can more easily pass through the stratum corneum of the skin, thereby being absorbed by the skin and more easily penetrating the skin barrier, thereby achieving a better transdermal effect.

[0043] The present invention also provides a protein preparation, which comprises the recombinant collagen prepared by the above method.

[0044] In the present invention, the recombinant collagen can be made into corresponding protein preparations. Specifically, the protein preparation can exist in solid, semi-solid or liquid form. The protein preparation can contain auxiliary materials or additives for preparing protein preparations. Those skilled in the art can select them according to needs and will not be elaborated here.

[0045] In the present invention, the fermentation broth obtained by the above-mentioned preparation method can be directly used as a protein preparation, or the fermentation broth can be separated and purified to obtain recombinant collagen as a protein preparation. The separation and purification can adopt conventional protein separation methods in the art. That is to say, recombinant collagen can be used in the form of whole cells of a recombinant strain, or it can be used in the form of a crude protein preparation separated from the cells of a recombinant strain and not purified, or a purified protein preparation. The protein preparation in the present invention is purified and separated multiple times using a conventional cation exchange column lexcap SP 6FF in the art, and the detection effect of the purified protein is as follows: Figure 2The inventors have found that the protein preparation prepared by the above method has higher purity.

[0046] Based on the high stability, small molecular weight and high transdermal permeability of the recombinant collagen provided by the present invention, it has higher expression efficiency and good biocompatibility. In a sixth aspect, the present invention provides the use of at least one of the recombinant collagen as described above, the gene as described above, the recombinant vector as described above, the recombinant strain as described above, and the recombinant collagen produced by the method as described above in the preparation of a collagen product. Preferably, the collagen product is selected from at least one of a skin product, a product that enhances fibroblast adhesion, and a product that enhances fibroblast collagen metabolism.

[0047] In the present invention, the collagen product is selected from any one of an injection, a facial filler, a dressing, a cosmetic and a tissue engineering material.

[0048] The recombinant collagen of the present invention achieves higher expression efficiency and high stability while maintaining high biological activity, can ensure the realization of the biological function of the recombinant collagen, and has the characteristics of high biocompatibility, good transdermal permeability and low use risk. It has broad application prospects in the fields of medical cosmetic surgery, cosmetics, etc.

[0049] The present invention will be described in detail below through examples.

[0050] In the following examples, pPIC9K plasmid was purchased from Saisuofei Biotechnology Co., Ltd.; type III small molecule collagen 02SC (reference substance, hereinafter referred to as 02SC) was purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.; BSA (bovine serum albumin) was purchased from Solebao; HDF cells were purchased from Guangdong Boxi Biological; HaCat cells were purchased from the Chinese Academy of Sciences. The remaining reagents and raw materials were conventional commercial products.

[0051] YPD medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar powder, sterilized by high-pressure steam at 115°C for 30 min.

[0052] MD medium: glucose 20 g / L, YNB 13.14 g / L, biotin 0.0004 g / L, agar powder 20 g / L, sterilized by high-pressure steam at 115°C for 30 min.

[0053] BMMY medium: tryptone 20 g / L, yeast extract 10 g / L, YNB 13.4 g / L, phosphate 100 mM at pH 6.0, sterilized by high pressure steam at 115°C for 30 min, and added with the prepared 4X10 -5% biotin aqueous solution, 100 mM phosphate buffer (pH 6.0), 0.5% methanol.

[0054] BMGY medium: 20 g / L tryptone, 10 g / L yeast extract, 20 g / L glycerol, 13.4 g / L YNB (containing 10 g / L ammonium sulfate), sterilized by high-pressure steam at 115°C for 30 min, and added with the prepared 4×10 -5 % biotin aqueous solution, 100 mM phosphate buffer (pH 6.0).

[0055] Example 1-1

[0056] This example illustrates the design of recombinant collagen sequence

[0057] The amino acid sequence of native human type III collagen α1 chain (GenBank: AGL34959.1) was obtained from the NCBI database. In-depth biological information mining and analysis of the sequence was conducted, including analysis of the hydrophilicity and charge distribution of the amino acid sequence using the peptides library; prediction of the three-dimensional structure of the recombinant collagen using alphafold; and molecular dynamics simulations of the recombinant collagen using molecular dynamics software such as Amber to assess its structural stability. Ultimately, based on these analyses, the native human type III collagen α1 chain was redesigned. This resulted in a recombinant collagen (S37) with a higher expression efficiency and stability while maintaining high biological activity. This amino acid sequence contains the bioactive sites of the native human type III collagen α1 chain and does not contain any exogenous amino acids, such as tags. This recombinant collagen is humanized, ensuring the biological function of the recombinant collagen and exhibiting high biocompatibility and low risk of use.

[0058] Example 1-2

[0059] This example illustrates the construction of recombinant collagen expression strains

[0060] Based on the codon preference of Pichia pastoris, the amino acid sequence shown in SEQ ID NO. 1 in Example 1-1 was codon-optimized. The optimized nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2 and is named COL3-S37. The DNA sequence was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. and cloned into the Pichia pastoris expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-COL3-S37. The recombinant plasmid was linearized with Sac I and transformed into the GS115 host strain. After recovery, the bacterial culture was plated on nutrient-deficient MD plates and incubated at 30°C for 3 days to obtain single colonies.

[0061] Examples 1-3

[0062] This example illustrates the induction expression of recombinant collagen in recombinant strains

[0063] Randomly pick a single colony from the MD plate described in Example 1-2 and inoculate it into YPD medium for activation, shake culture at 30°C, 220 rpm overnight, then transfer it to BMGY medium at a 10% volume inoculation rate for seed culture, shake culture at 30°C, 220 rpm overnight, and transfer it to BMMY medium at a 10% volume inoculation rate for induction expression. 1% by weight methanol was added every 24 hours for induction. After 72 hours of induction culture, the culture was terminated. The fermentation broth was taken, the supernatant was collected by centrifugation, and the electrophoresis test results were as follows: Figure 1 As shown, the results show that recombinant collagen (S37) is expressed, and the molecular weight is less than 10 kDa, which is a small molecular weight, indicating that the recombinant collagen prepared in this example has a higher transdermal absorption rate than conventional large-molecular recombinant collagen.

[0064] Examples 1-4

[0065] This example illustrates the purification and preparation of recombinant collagen

[0066] The fermentation broth containing recombinant collagen (S37) prepared in Example 1-3 was centrifuged, and the supernatant was collected as the feed solution. The feed solution was clarified using a 0.1 μm membrane, and then concentrated using a 1 kDa membrane. The top wash was performed with buffer until the conductivity reached 2.4 mS / cm, and the pH was adjusted to 4.0 to obtain the loading solution. The loading solution was purified multiple times using a cation exchange column, Lexcap SP 6FF, to obtain a recombinant collagen (S37) sample. The electrophoresis results are shown in Figure 2. Figure 2 The gel showed only one clear protein band, indicating that the protein sample was highly pure, exceeding 90%. The purified sample was replaced with PBS buffer and the protein concentration was determined by the BCA method before being stored for future use.

[0067] Example 2

[0068] This example illustrates the fibroblast adhesion experiment of recombinant collagen

[0069] On Day 0, the recombinant collagen (S37) prepared in Example 1-4 was diluted with PBS and coated in a 96-well plate at 4°C overnight. On Day 1, the protein solution was discarded and the plate was washed twice with PBS. The supernatant was discarded by shaking the plate. 4HDF-1 cells were added to the plate (100 cells / well) and incubated at 37°C for 1 hour. Cell adhesion was observed and discarded by shaking the plate. The cells were washed twice with PBS. CCK8 solution (a reagent used for cell proliferation and toxicity assays) prepared in Dulbecco's medium (DMEM) was added at 100 μl / well (indicating 100 microliters of CCK8 solution was added to each well). The plates were incubated at 37°C for 1 hour and then read on a microplate reader. Controls (02SC) and bovine serum albumin (BSA) were assayed using the same protocol as the recombinant collagen samples. OD values ​​were obtained and plotted using GraphPad.

[0070] The experimental results are as follows Figure 3 The results showed that the recombinant collagen (S37) prepared by the present invention significantly promoted cell adhesion compared to the control (02SC), outperforming the control BSA (bovine serum albumin). Furthermore, when the S37 dilution concentration was 0.1% by weight, the cell adhesion-promoting effect was strongest, demonstrating excellent results in reducing skin sagging, wrinkles, and maintaining skin elasticity.

[0071] Example 3

[0072] This example illustrates the fibroblast collagen metabolism experiment of recombinant collagen

[0073] HDF cells (human dermal fibroblasts) were resuspended in 10% FBS + DMEM medium and plated at 3E5 / well (3×10 5 Cells (100 cells / well) were added to a 6-well plate. After the cells attached, a recombinant collagen (S37) sample diluted to 0.1 mg / mL in 0.1% FBS + DMEM medium was added. After thorough mixing, the cells were placed in a 37°C incubator. After 24 hours, RNA was extracted and reverse-transcribed to obtain cDNA, which was then used for qPCR. The control (02SC) was treated using the same experimental method as the recombinant collagen sample.

[0074] The experimental results are as follows Figure 4 As shown, the recombinant collagen (S37) prepared by the present invention has a significant effect of promoting the production of COL-1 (type I collagen) and COL-3 (type III collagen), and the effects are better than those of the control (02SC). Therefore, S37 has excellent advantages and effects in promoting skin elasticity and firming and anti-wrinkle efficacy.

[0075] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A recombinant collagen, characterized in that: The recombinant collagen is the collagen with an amino acid sequence as shown in SEQ ID NO.

1.

2. A gene encoding recombinant collagen, characterized in that: The gene has a nucleotide sequence encoding the recombinant collagen according to claim 1.

3. The gene according to claim 2, characterized in that The gene has a nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that The recombinant vector contains the gene according to claim 2 or 3.

5. A recombinant strain, characterized in that The recombinant strain contains the gene according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A method for preparing recombinant collagen, characterized in that: The preparation method comprises: inoculating the recombinant strain according to claim 5 into a fermentation medium for fermentation culture.

7. Use of at least one of the recombinant collagen according to claim 1 and the recombinant collagen prepared by the method according to claim 6 in the preparation of collagen products.

8. The use according to claim 7, characterized in that The collagen product is selected from at least one of an injection, a facial filler, a dressing, a cosmetic, and a tissue engineering material.

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