Recombinant humanized type III collagen and its encoding gene, protein preparation, preparation method of protein preparation, and their application, skin care products

Abstract

This invention, belonging to the fields of protein engineering and biotechnology, discloses a recombinant humanized type III collagen, its encoding gene, a protein preparation, a method for preparing the protein preparation, and applications thereof, as well as skin care products. The amino acid sequence of this recombinant humanized type III collagen is shown in SEQ ID NO. 1. This recombinant humanized type III collagen significantly enhances transdermal absorption efficiency while also significantly increasing the proliferation and migration of keratinocytes. This collagen 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 humanized type III collagen and its encoding gene, a protein preparation, a method for preparing the protein preparation, and applications thereof, and a skin care product. Background Art

[0002] Collagen is the primary protein in all connective tissues, including skin, bone, tendons, and cartilage. In the skin, for example, it forms large fiber bundles, each containing many individual collagen fibers, giving the skin its strong and flexible structure. Mature triple-helical collagen is abundant in the human body and is valuable for a range of industrial and medical applications. Collagen has been widely used in clinical applications, demonstrating safety and effectiveness in areas including soft tissue augmentation, wound and burn repair, orthopedics, and cardiovascular medicine.

[0003] Human type III collagen is a fibrillar collagen found in connective tissues such as the skin, internal organs, and the vascular system. It plays a key role in wound healing, collagen fibrillogenesis, and normal cardiovascular development.

[0004] Recombinant protein is a protein produced by applying genetic engineering technology. In recent years, recombinant protein has become an important part of the development of biopharmaceuticals and medical aesthetic proteins. The production of recombinant protein drugs mainly includes four major systems: prokaryotic protein expression, yeast protein expression, insect cell protein expression and mammalian cell protein expression. Recombinant collagen has advantages that natural collagen does not have, including processability, no viral risks and low rejection reactions, without changing the functional properties of natural collagen. It can also be mass-produced through genetic engineering, making it a research hotspot for the design and synthesis of collagen. Human recombinant type III collagen has strong water solubility and high biological activity, and has important application value in medicine, nutrition and other aspects. However, in designing short amino acid sequences as repeating units or splicing units,

[0005] Currently, there is no existing technology that provides effective guidance to ensure that the designed recombinant collagen can significantly enhance the proliferation and migration capabilities of keratinocytes while having excellent transdermal properties. Summary of the Invention

[0006] The purpose of the present invention is to overcome the problem in the prior art that recombinant humanized type III collagen cannot have good transdermal permeability and expression efficiency while also having high proliferation and migration capabilities of keratinocytes. The present invention provides a recombinant humanized type III collagen and its encoding gene, protein preparation, protein preparation preparation method and their applications, and skin care products. The recombinant humanized type III collagen has a small molecular weight and extremely high transdermal permeability, and has better migration and proliferation capabilities of keratinocytes, thereby improving the skin barrier function and wound healing effects.

[0007] In order to achieve the above-mentioned object, the present invention provides a recombinant humanized type III collagen in a first aspect. The recombinant humanized type III collagen has an amino acid sequence shown in SEQ ID NO. 1.

[0008] The second aspect of the present invention provides a gene encoding recombinant humanized type III collagen, wherein the gene has a nucleotide sequence encoding the recombinant humanized type III collagen as described above.

[0009] Preferably, the gene has the nucleotide sequence shown in SEQ ID NO. 2.

[0010] The third aspect of the present invention provides a protein preparation, which comprises the recombinant humanized type III collagen as described above.

[0011] A fourth aspect of the present invention provides a method for preparing a protein preparation, comprising: inoculating a recombinant strain containing the gene as described above into a fermentation medium for fermentation culture.

[0012] The fifth aspect of the present invention provides the use of at least one of the recombinant humanized type III collagen as described above, the gene as described above, the protein preparation as described above, and the recombinant humanized type III collagen preparation prepared by the method as described above in the preparation of a collagen product that enhances the proliferation and / or migration ability of keratinocytes.

[0013] Preferably, the collagen product is selected from at least one of an injection, a facial filler, a dressing, a cosmetic, and a tissue engineering material.

[0014] The sixth aspect of the present invention provides a skin care product, which contains at least one of the recombinant humanized type III collagen as described above, the gene as described above, the protein preparation as described above, and the recombinant humanized type III collagen preparation prepared by the method as described above.

[0015] Preferably, the content of recombinant humanized type III collagen in the skin care product is 0.0001-0.003% by weight.

[0016] 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 conducted in-depth mining and analysis of the biological information of the sequence, designing the hydrophilicity, charge distribution, and spatial structure of the amino acid sequence to achieve higher expression efficiency and high stability of the recombinant humanized type III collagen of the present invention while maintaining high biological activity. The amino acid sequence of the recombinant humanized type III collagen of the present invention is shown in SEQ ID NO. 1, which ensures the realization of the biological function of the recombinant humanized type III collagen, while giving the recombinant humanized type III collagen of the present invention extremely high skin permeability and expression efficiency, while greatly enhancing its ability to promote keratinocyte proliferation and migration.

[0017] The recombinant humanized type III collagen provided by the present invention has broad application prospects in the fields of medical cosmetic surgery, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The fermentation broth of recombinant type III humanized collagen in Example 1-3 SDS-PAGE Protein detection results, Line 1: S43 induced for 24 hours, Line 2, Line 4: Marker;

[0019] Figure 2 The purified recombinant humanized type III collagen in Example 1-4 SDS-PAGE Protein detection result diagram;

[0020] Figure 3 This is a diagram of the keratinocyte proliferation experiment using recombinant humanized type III collagen in Example 2;

[0021] Figure 4 This is a diagram of the keratinocyte migration experiment using recombinant humanized type III collagen in Example 3;

[0022] Figure 5 This is a diagram of the fibroblast adhesion experiment of the recombinant humanized type III collagen in Example 4;

[0023] Figure 6 This is a diagram of the fibroblast collagen metabolism experiment of the recombinant humanized type III collagen in Example 5. 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] In a first aspect, the present invention provides a recombinant humanized type III 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 recombinant humanized type III collagen, wherein the gene has a nucleotide sequence encoding the recombinant humanized type III collagen 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.

[0029] 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. The 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.

[0030] 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.

[0031] Table 1

[0032]

[0033] The present invention improves the expression efficiency of recombinant humanized type III collagen by adjusting the gene sequence. Specifically, by changing the synonymous codons in the gene to better align with the codon preference of Pichia pastoris, rare codons are eliminated, and translation efficiency is improved. The inventors have discovered that using this preferred embodiment can achieve higher expression levels of the recombinant humanized type III collagen provided by the present invention.

[0034] 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.

[0035] In addition, the relevant nucleotide sequences can also be synthesized using known artificial chemical synthesis methods.

[0036] The third aspect of the present invention provides a protein preparation, which contains the recombinant humanized type III collagen as described above.

[0037] In the present invention, the recombinant humanized type III 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.

[0038] A fourth aspect of the present invention provides a method for preparing a protein preparation, comprising: inoculating a recombinant strain containing the gene encoding the recombinant humanized type III collagen as described above into a fermentation medium for fermentation culture.

[0039] According to the present invention, 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.

[0040] According to the present invention, preferably, the encoding gene of the recombinant humanized type III collagen has the nucleotide sequence shown in SEQ ID NO.2. Further preferably, the expression strain of the recombinant strain is a yeast, more preferably a Pichia pastoris strain GS115. Exemplarily, the present invention uses the Pichia pastoris strain GS115 as a host strain, and transforms the recombinant vector pPIC9K-COL3-S43 containing the recombinant humanized type III collagen provided by the present invention into the GS115 host strain. The inventors have found that the use of the GS115 host strain has a higher ability to express recombinant humanized type III collagen and has higher stability.

[0041] 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 type III humanized collagen as a protein preparation. The separation and purification can adopt conventional protein separation methods in the art. That is to say, recombinant type III humanized 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 2 The inventors have found that the protein preparation prepared by the above method has higher purity.

[0042] The fifth aspect of the present invention provides the use of at least one of the aforementioned recombinant humanized type III collagen, the aforementioned gene, the aforementioned protein preparation, and the protein preparation prepared by the aforementioned method in the preparation of a collagen product that enhances keratinocyte proliferation and / or migration ability.

[0043] According to the present invention, preferably, the collagen product is selected from any one of an injection, a facial filler, a dressing, a cosmetic and a tissue engineering material.

[0044] The sixth aspect of the present invention provides a skin care product, which contains at least one of the recombinant humanized type III collagen as described above, the gene as described above, the protein preparation as described above, and the protein preparation prepared by the method as described above.

[0045] According to the present invention, the content of recombinant humanized type III collagen in the skin care product is preferably 0.0001-0.003% by weight. The inventors have found that adopting this preferred embodiment, the anti-aging skin care product prepared has excellent effects in promoting the proliferation and migration of keratinocytes, reducing skin sagging, reducing wrinkles, and maintaining skin elasticity.

[0046] The recombinant humanized type III collagen of the present invention achieves higher expression efficiency and high stability while maintaining high biological activity, can ensure that the recombinant humanized type III collagen has extremely high transdermal permeability and has significant ability to promote the proliferation and migration of keratinocytes. It has the characteristics of low risk of use and has broad application prospects in the fields of medical cosmetic surgery, cosmetics, etc.

[0047] The present invention will be described in detail below through examples.

[0048] In the following examples, Pichia pastoris pPIC9K was purchased from Cesuofi 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. The remaining reagents and raw materials were conventional commercial products.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] Example 1-1

[0054] This example illustrates the design of recombinant humanized type III collagen sequence

[0055] 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 to optimize its hydrophilicity, charge distribution, and spatial structure. This resulted in a recombinant humanized type III collagen protein with improved expression efficiency and stability while maintaining high biological activity. The resulting amino acid sequence is shown in SEQ ID NO. 1 as recombinant collagen (S43). This 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 ensures the biological function of the recombinant collagen, and it exhibits high biocompatibility and low risk of use.

[0056] Example 1-2

[0057] This example illustrates the construction of a strain expressing recombinant humanized collagen type III

[0058] 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-S43. 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-S43. The recombinant plasmid was linearized with Sac I and transformed into the GS115 host strain. After recovery, the bacterial suspension was plated on nutrient-deficient MD plates and incubated at 30°C for 3 days to obtain single colonies.

[0059] Examples 1-3

[0060] This example illustrates the induction expression of recombinant humanized collagen type III in recombinant strains.

[0061] 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 type III humanized collagen (S43) is expressed, and the molecular weight is less than 10 kDa, which is a small molecular weight. The results show that the recombinant type III humanized collagen prepared in this example has a higher transdermal absorption rate than conventional large-molecular recombinant type III humanized collagen.

[0062] Examples 1-4

[0063] This example illustrates the purification and preparation of recombinant humanized type III collagen

[0064] The fermentation broth containing recombinant humanized type III collagen (S43) 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, LexcapSP 6FF, to obtain a recombinant humanized type III collagen (S43) 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.

[0065] Example 2

[0066] This example illustrates the keratinocyte proliferation experiment of recombinant humanized type III collagen

[0067] On Day 0, HaCat cells (human immortalized keratinocytes) in the logarithmic growth phase were cultured at a rate of 1 E4 / well (1×10 4 cells / well), resuspended in 10 wt% FBS, and added to a 96-well plate.

[0068] On Day 1, the supernatant was removed and the protein sample (recombinant humanized type III collagen prepared in Examples 1-4) diluted with 0.1% by weight FBS was added. The cells were incubated at 37°C for 48 hours. After incubation, the supernatant was removed, the cells were washed once with PBS, and CCK8 solution (a reagent used for cell proliferation and toxicity testing) prepared in DMEM basal medium was added. The cells were incubated at 37°C for 1 hour. The experimental method for the control substances 02SC and BSA (bovine serum albumin) was consistent with the experimental method for recombinant humanized type III collagen described above. OD values ​​were obtained on a microplate reader and plotted using Graphpad.

[0069] The experimental results are as follows Figure 3 The results showed that recombinant humanized type III collagen (S43) with a dilution concentration of 0.0001-0.003 wt% was superior to the reference substances 02SC and BSA (bovine serum albumin) in promoting cell proliferation of keratinocytes. Therefore, S43 has better advantages and functions in promoting the thickening of the stratum corneum, improving the skin barrier function and maintaining stability.

[0070] Example 3

[0071] This example illustrates the keratinocyte migration experiment of recombinant humanized type III collagen

[0072] S1. First, draw two parallel straight lines across each well of an empty 24-well plate parallel to the edge of the plate. Then, resuspend HaCat cells (human immortalized keratinocytes) in 10 wt% FBS + DMEM complete medium and plate at a rate of 2 E5 / well (2 × 10 5 Cells were plated in a 24-well plate (100 cells / well). After the cells adhered overnight, a 200 μl pipette tip was used to streak the wells perpendicular to the streak made the previous day to create a scratch that would damage the cells.

[0073] S2. After all streaking is completed, remove the supernatant, wash the cells twice with PBS, and add the protein sample diluted with 0.1 wt% FBS + DMEM complete medium (recombinant type III humanized collagen S43 prepared in Example 1-4, diluted to a concentration of 0.01 wt%), mix thoroughly, and take a photo at 0 h. Then incubate at 37°C for 24 h and 48 h, and take photos respectively.

[0074] ImageJ was used to process the image healing area. The experimental results are shown in the figure below. Figure 4 As shown, the S43 protein has a good effect in promoting keratinocyte migration. Therefore, the recombinant humanized type III collagen provided by the present invention has good advantages in enhancing skin barrier function and promoting wound healing.

[0075] Example 4

[0076] This example illustrates the fibroblast adhesion experiment of recombinant humanized type III collagen

[0077] On Day 0, the recombinant humanized type III collagen sample prepared in Example 1-4 was diluted with PBS and coated in a 96-well plate at 4° C. overnight.

[0078] On Day 1, discard the protein solution and wash the plate twice with PBS. Discard the supernatant by shaking the plate. 4 HDF-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 for the recombinant humanized type III collagen samples. OD values ​​were obtained and plotted using GraphPad.

[0079] The experimental results are as follows Figure 5 The results showed that the recombinant humanized type III collagen (S43) prepared by the present invention significantly promoted cell adhesion compared to the control substance 02SC, both of which were superior to the control substance BSA (bovine serum albumin). Furthermore, when the S43 dilution concentration was 0.1% by weight, S43 had the strongest effect in promoting fibroblast adhesion, demonstrating excellent results in reducing skin sagging, wrinkles, and maintaining skin elasticity.

[0080] Example 5

[0081] This example illustrates the fibroblast collagen metabolism experiment of recombinant humanized type III collagen

[0082] HDF cells (human dermal fibroblasts) were resuspended in 10% FBS + DMEM medium and plated at 3 E5 / well (3 × 10 5 Cells were added to a 6-well plate (100 cells / well). After the cells attached, a protein sample (the recombinant humanized type III collagen prepared in Example 1-4, diluted to 0.01% by weight) diluted in 0.1% by weight FBS + DMEM medium was added. After thorough mixing, the plate was placed in a 37°C incubator. After 24 hours, RNA was extracted and reverse-transcribed to obtain cDNA, which was then subjected to qPCR. The control (02SC) was treated using the same experimental method as the recombinant humanized type III collagen sample.

[0083] The experimental results are as follows Figure 6 As shown in the results, the recombinant humanized type III collagen (S43) prepared by the present invention has a significant effect of promoting the production of COL-1 (type I collagen), COL-3 (type III collagen) and COL-4 (type IV collagen), and the effects are better than those of the control (02SC). Therefore, S43 has excellent advantages and effects in promoting skin elasticity and firmness, anti-wrinkle, wound healing, and maintaining tissue structure.

[0084] 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 humanized type III collagen, characterized in that: The amino acid sequence of the recombinant humanized type III collagen is shown in SEQ ID NO.

1.

2. A gene encoding recombinant humanized type III collagen, characterized in that: The gene is a nucleotide sequence encoding the recombinant humanized type III collagen described in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A protein preparation, characterized in that The protein preparation comprises the recombinant humanized type III collagen according to claim 1.

5. A method for preparing a protein preparation, characterized in that: The method for preparing the protein preparation comprises: inoculating a recombinant strain containing the gene according to claim 2 or 3 into a fermentation medium for fermentation culture.

6. Use of at least one of the recombinant humanized type III collagen according to claim 1, the protein preparation according to claim 4, and the protein preparation prepared by the method according to claim 5 in the preparation of a collagen product for wound healing.

7. The use according to claim 6, characterized in that The collagen product is selected from at least one of a facial filler, a dressing, and a tissue engineering material.

8. A skin care product, characterized in that: The skin care product contains at least one of the recombinant humanized type III collagen according to claim 1, the protein preparation according to claim 4, and the protein preparation prepared by the method according to claim 5.

9. The skin care product according to claim 8, characterized in that The content of recombinant humanized type III collagen in the skin care product is 0.0001-0.003% by weight.

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