A recombinant collagen with transdermal effect and its preparation method and application

Abstract

The present invention belongs to the technical field of protein engineering and genetic engineering, and specifically relates to a recombinant collagen with transdermal effect, and a preparation method and application thereof. Specifically, the present invention obtains a new recombinant collagen by design through screening, replacement, splicing and other methods, and its nucleotide sequence is optimized by Pichia pastoris codon preference, and then a transmembrane peptide is introduced at the amino end of the sequence, and a corresponding genetically engineered yeast strain is constructed, and recombinant collagen is successfully fermented and prepared. Experiments have proved that the recombinant collagen prepared by the above technical scheme has better free radical scavenging performance and transdermal performance than commercially available recombinant collagen, thereby effectively solving the current problem of collagen transdermal difficulty, and further improving its biological activity and efficacy, so it has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering and gene engineering, and specifically relates to a recombinant collagen with a transdermal effect and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It plays a key role in connecting and supporting bones, skin, and joints. Collagen can also interact with various macromolecules such as integrins, decorative proteins, fibronectin, heparin, and matrix metalloproteinases, and plays an important role in regulating tissue regeneration. So far, 29 types of collagen have been discovered. Among them, type III collagen is mainly found in connective tissues such as skin, tendons, ligaments, and joints, forming a reticular structure of the extracellular matrix. It is the most important type of collagen in the dermis, so it is widely used in beauty and skin care, medical devices, medical beauty and other products.

[0003] Type III collagen obtained from animal tissues has limited its application and development to a certain extent due to its low content, difficulty in purification, and animal virus contamination. Recombinant collagen is synthesized by genetic engineering technology, and has higher biological activity and biocompatibility, lower immunogenicity, and lower risk of missed pathogens. Therefore, its application has received strong attention. In addition, the stratum corneum of the skin, as a natural barrier of the human body, often limits the entry of large molecules into the dermis through the epidermis. The molecular weight of collagen currently used in the market is generally large, and it is difficult to cross the epidermal barrier and enter the superficial dermis to exert its biological effects. Cell-penetrating peptides are a type of short peptide that can carry large molecules into cells and skin. The new protein molecule formed by connecting cell-penetrating peptides to collagen through molecular biological methods may have good transdermal activity. Therefore, using genetic engineering and protein engineering technology to artificially design and synthesize recombinant collagen with an amino acid sequence similar to type III collagen and with good transdermal effect is expected to solve the above-mentioned problems. Based on this technology, the applicant has previously developed a recombinant transdermal collagen SPACE-Col7 (CN118359703A). However, the antioxidant and transdermal properties of the recombinant transdermal collagen still need to be improved. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a recombinant collagen with transdermal effect and its preparation method and application. Specifically, the recombinant collagen SPACE-Col4 produced by the present invention has better free radical scavenging performance and transdermal performance compared with commercially available recombinant collagen and previously reported SPACE-Col7 (CN118359703A). Based on the above research results, the present invention is completed.

[0005] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides a recombinant collagen, wherein the recombinant collagen has:

[0007] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0008] (a2) a protein derived from the amino acid sequence shown in SEQ ID NO.1 with the same function as that of the amino acid sequence shown in SEQ ID NO.1 after one or more amino acid residues are substituted and / or deleted and / or added;

[0009] (a3) Proteins encoded by other genes that have an amino acid sequence composition that is more than 90% similar to that of SEQ ID NO.1 and have the protein activity of SEQ ID NO.1.

[0010] The recombinant collagen is also modified with a cell-penetrating peptide, thereby effectively enhancing its transdermal performance. The cell-penetrating peptide can be modified at the carboxyl end (C-terminus) or amino end (N-terminus) of the recombinant collagen, preferably the amino end.

[0011] The second aspect of the present invention provides a polynucleotide capable of encoding the above-mentioned recombinant collagen.

[0012] The third aspect of the present invention provides a recombinant expression vector, which comprises the above-mentioned polynucleotide.

[0013] The fourth aspect of the present invention provides a host bacterium, wherein the host bacterium contains the above-mentioned recombinant expression vector or has the above-mentioned polynucleotide integrated into its chromosome or is capable of expressing the above-mentioned recombinant collagen.

[0014] The fifth aspect of the present invention provides a method for preparing the recombinant collagen, the method comprising: culturing the above-mentioned host bacteria to express the recombinant collagen; and isolating and purifying the recombinant collagen.

[0015] A sixth aspect of the present invention provides the use of the above-mentioned recombinant collagen in the preparation of cosmetics, medical beauty products or medicines.

[0016] A seventh aspect of the present invention provides a cosmetic comprising at least the above-mentioned recombinant collagen.

[0017] Beneficial technical effects of one or more of the above technical solutions:

[0018] The above technical scheme designs a new recombinant collagen protein through screening, replacement, splicing and other methods. The nucleotide sequence is optimized by Pichia pastoris codon preference, and then a transmembrane peptide is introduced at the amino end of the sequence. The corresponding genetically engineered yeast strain is constructed and the recombinant collagen protein is successfully prepared by fermentation.

[0019] Experiments have shown that the recombinant collagen prepared by the above technical solution has better free radical scavenging and transdermal properties than commercially available recombinant collagen, thereby effectively solving the current problem of collagen transdermal penetration and further improving its biological activity and efficacy, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of SDS-PAGE protein electrophoresis and Western Blot of the fermentation supernatant of the recombinant collagen SPACE-Col4 in Example 4 of the present invention are shown in FIG. A is an SDS-PAGE protein electrophoresis diagram, in which lane M represents a standard protein with a molecular weight of 180 kDa; lane 1 represents a control bacterial P. pastoris GS115 / pPIC9K fermentation supernatant; Lane 2 represents the recombinant bacteria P. pastoris A is the supernatant of GS115 / pPIC9K-SPACE-Col4 fermentation; B is the Western Blot result.

[0021] Figure 2 This is a graph of the antioxidant activity of different collagens according to Example 6 of the present invention; wherein, Comparative Example 1 is commercially available recombinant collagen; Comparative Example 2 is recombinant transdermal collagen SPACE-Col7; Col4 is recombinant collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention.

[0022] Figure 3 It is a comparison chart of the transdermal performance test results of different collagens in Example 7 of the present invention; wherein, the control group is commercially available recombinant collagen; Experimental Group 1 is the recombinant transdermal collagen SPACE-Col7; Experimental Group 2 is the heavy collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is the recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention.

[0023] Figure 4 This is a fluorescence microscope image of the fluorescence penetration test of different collagens in suckling pig skin according to Example 7 of the present invention; the control group is a comparative experimental group of commercially available recombinant collagen; Experimental Group 1 is the recombinant transdermal collagen SPACE-Col7; Experimental Group 2 is the heavy collagen Col4 prepared based on Example 5 of the present invention; SPACE-Col4 is the recombinant collagen SPACE-Col4 prepared based on Example 5 of the present invention. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0026] As mentioned above, the collagen currently used in the market generally has a large molecular weight and is difficult to cross the epidermal barrier and enter the superficial dermis to exert its biological effects.

[0027] In view of this, in a typical embodiment of the present invention, a recombinant collagen is provided, wherein the recombinant collagen has:

[0028] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0029] (a2) a protein derived from the amino acid sequence shown in SEQ ID NO.1 with the same function as that of the amino acid sequence shown in SEQ ID NO.1 after one or more amino acid residues are substituted and / or deleted and / or added;

[0030] (a3) Proteins encoded by other genes that have an amino acid sequence composition that is more than 90% similar to that of SEQ ID NO.1 and have the protein activity of SEQ ID NO.1.

[0031] In another specific embodiment of the present invention, the recombinant collagen is further modified with a transdermal peptide to effectively enhance its transdermal performance. The transdermal peptide can be modified at the carboxyl end (C-terminus) or amino end (N-terminus) of the recombinant collagen, preferably the amino end.

[0032] The cell-penetrating peptide may be the cell-penetrating peptide SPACE (amino acid sequence as shown in SEQ ID NO.3), the cell-penetrating peptide TAT (amino acid sequence as shown in SEQ ID NO.4) or the cell-penetrating peptide ANTP (amino acid sequence as shown in SEQ ID NO.5); further preferably, the cell-penetrating peptide is the cell-penetrating peptide SPACE.

[0033] In another specific embodiment of the present invention, a polynucleotide is provided, wherein the polynucleotide can encode the above-mentioned recombinant collagen;

[0034] The polynucleotide is selected from:

[0035] (b1) the nucleotide sequence as shown in any one of SEQ ID NO.2 and SEQ ID NO.6;

[0036] (b2) a nucleotide sequence that encodes a protein with the same amino acid sequence as the nucleotide sequence in (b1) but differs in sequence due to the degeneracy of the genetic code;

[0037] (b3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes the same functional protein;

[0038] (b4) A nucleotide sequence complementary to any one of (b1) to (b3).

[0039] In another specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the above-mentioned polynucleotide.

[0040] In the present invention, the recombinant expression vector is obtained by effectively connecting the above-mentioned polynucleotide to an expression vector, and the expression vector is a plasmid, specifically any plasmid vector in the pPIC series. Further, the expression vector is a pPIC9K plasmid.

[0041] In another specific embodiment of the present invention, a host bacterium is provided, wherein the host bacterium contains the above-mentioned recombinant expression vector or has the above-mentioned polynucleotide integrated into its chromosome or is capable of expressing the above-mentioned recombinant collagen.

[0042] The host bacteria may be eukaryotic fungi or prokaryotic fungi, such as bacteria, fungi, actinomycetes, etc. Further, the host bacteria is a yeast among fungi. The yeast may be Pichia pastoris (such as Pichia pastoris GS115, KM71 or SMD1168).

[0043] In another specific embodiment of the present invention, a method for preparing the recombinant collagen is provided, and the method comprises: culturing the above host bacteria to express the recombinant collagen; and isolating and purifying the recombinant collagen.

[0044] Furthermore, the separation and purification includes the steps of hollow fiber clarification, ultrafiltration membrane concentration and replacement, ion exchange chromatography, ultrafiltration membrane desalination and freeze-drying, which are not specifically limited here.

[0045] In another specific embodiment of the present invention, there is provided the use of the above-mentioned recombinant collagen in the preparation of cosmetics, medical beauty products or medicines.

[0046] The cosmetics can be applied to the skin to achieve anti-oxidation and skin barrier repair effects, so the cosmetics in the present invention can be skin care products. The cosmetic dosage form can be an aqueous solution, emulsion, ointment (cream), etc. At the same time, based on the above basic cosmetic categories, other cosmetic categories are further derived and prepared, such as facial cleansers, sunscreens, toners, face creams, eye creams, essence water (lotions), hand creams (ointments), body lotions and facial masks, etc.

[0047] In the present invention, the medical beauty products refer to products used in the field of medical beauty. Some commonly used medical beauty products include medical beauty masks, dressings, etc. The use of these products needs to be carried out by professional medical staff, or relevant guidance is required.

[0048] In the present invention, the drug may be a drug-device combination product, which refers to a product composed of a drug and a medical device and produced as a single entity. A specific embodiment of the drug-device combination product may be an external dressing containing the above-mentioned recombinant collagen.

[0049] In another specific embodiment of the present invention, a cosmetic is provided, wherein the cosmetic at least comprises the above-mentioned recombinant collagen.

[0050] Furthermore, the cosmetics are specifically skin care products.

[0051] Of course, the cosmetics may also contain any other raw material ingredients allowed to be added in the cosmetics field, including but not limited to suspending agents, emulsifiers, emollients, moisturizers, pH adjusters, thickeners, fragrances, etc. Those skilled in the art may choose to add according to actual conditions, and no specific limitation is made here.

[0052] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods for the specific conditions in the following examples are usually carried out under normal conditions.

[0053] The culture medium components used in the examples are as follows:

[0054] YPD medium: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose.

[0055] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, K 2 HPO 4 3 g / L, KH 2 PO 4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4×10 -4 g / L, glycerol 10 g / L.

[0056] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, K 2 HPO 4 3 g / L, KH 2 PO 4 11.8 g / L, YNB 3.4 g / L, ammonium sulfate 10 g / L, biotin 4×10 -4 g / L, methanol 10 mL / L.

[0057] Example 1 Sequence Design of Recombinant Collagen

[0058] In this example, the amino acid sequence of natural human type III collagen α1 chain protein (sequence number NP_000081) was analyzed, and based on the characteristics of the natural collagen amino acid sequence and the reported active sites, an amino acid sequence was finally obtained through screening, replacement, and splicing. The amino acid sequence is shown in SEQ ID NO.1, and the recombinant collagen Col4 was obtained.

[0059] Example 2 Construction of a gene expression system containing recombinant collagen

[0060] Based on the codon preference of Pichia pastoris, the sequence of recombinant collagen Col4 was optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. Subsequently, the nucleotide sequence of the codon-optimized recombinant collagen Col4 was entrusted to GenScript Biotech Co., Ltd. for full gene synthesis, and cloned into the Pichia pastoris expression vector pPIC9K between the EcoRI and NotI restriction sites to obtain the recombinant expression vector pPIC9K-Col4. DNA sequencing and comparison showed that the recombinant sequence was correct. The recombinant expression plasmid pPIC9K-Col4 was linearized with SalI fast-cutting enzyme and then electroporated into P. pastoris In the GS115 expression host cell, the obtained recombinant transformants were screened by geneticin G418 to obtain high-copy recombinant Pichia pastoris P. pastoris GS115 / pPIC9K-Col4.

[0061] Example 3 Shake flask fermentation of recombinant collagen engineered yeast strain

[0062] Genetically recombinant engineered bacteria P. pastoris GS115 / pPIC9K-Col4 was fermented in shake flasks. The specific fermentation steps are as follows: Pick a single clone and inoculate it in 40 mL of YPD medium, and culture it at 30°C, 200 rpm for 24 h. Transfer it to 40 mL of initial expression medium BMGY at a 10% inoculum volume, and culture it at 30°C, 200 rpm for 24 h. Collect the bacteria by centrifugation, wash the bacteria with physiological saline, and then replace it with 40 mL of induced expression medium BMMY, culture it at 30°C, 200 rpm, and add pure methanol to the culture medium every 24 h to a final concentration of 1.0% ( v / v ), and the expression was induced for 96 h.

[0063] Example 4 Construction of high-copy recombinant collagen strain and protein expression

[0064] In order to improve the transdermal effect of recombinant collagen, the transmembrane peptides SPACE (amino acid sequence as shown in SEQ ID NO.3), TAT (amino acid sequence as shown in SEQ ID NO.4) and ANTP (amino acid sequence as shown in SEQ ID NO.5) were optimized according to the codon preference of Pichia pastoris and then connected to the N-terminus of the Col4 sequence, respectively, to obtain three recombinant collagen proteins SPACE-Col4 (nucleotide sequence as shown in SEQ ID NO.6), TAT-Col4 (nucleotide sequence as shown in SEQ ID NO.7) and ANTP-Col4 (nucleotide sequence as shown in SEQ ID NO.8).

[0065] The coding genes of the three recombinant collagen proteins were cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vectors pPIC9K-SPACE-Col4, pPIC9K-TAT-Col4 and pPIC9K-ANTP-Col4. DNA sequencing showed that the sequences of the recombinant expression vectors were correct. The recombinant expression plasmids were linearized with SalI enzyme and then electroporated into P. pastoris In GS115 expression host cells, recombinant transformants were screened by geneticin G418 to obtain high-copy recombinant Pichia pastoris P. pastoris GS115 / pPIC9K-SPACE-Col4, P. pastoris GS115 / pPIC9K-TAT-Col4 and P. pastoris GS115 / pPIC9K-ANTP-Col4.

[0066] The supernatant of the fermentation of the recombinant engineering bacteria and the supernatant of the fermentation of the control bacteria were subjected to SDS-PAGE protein electrophoresis analysis. The electrophoresis analysis results were as follows: Figure 1 As shown in A. At around the theoretical protein molecular weight of 35 kDa, the recombinant engineering bacteria P. pastoris The supernatant of GS115 / pPIC9K-SPACE-Col4 fermentation (lane 2) had an additional protein band (indicated by the arrow), which was further detected by Western Blot. The results were as follows: Figure 1 As shown in B, the band at this position was determined to be the recombinant collagen SPACE-Col4.

[0067] Example 5 Preparation of recombinant collagen Col4 and SPACE-Col4

[0068] Recombinant bacteria P. pastoris GS115 / pPIC9K-Col4 and P. pastoris GS115 / pPIC9K-SPACE-Col4 was fermented in a shake flask, and the fermentation supernatant was purified by hollow fiber clarification, ultrafiltration membrane concentration and replacement, ion exchange chromatography, ultrafiltration membrane desalting and freeze drying (for specific purification steps, see CN118359703A) to obtain pure recombinant collagen. The purity of the protein purified by the above steps is greater than 96%, and the yield is greater than 55%.

[0069] Example 6 Antioxidant Experiment of Recombinant Collagen

[0070] Take 2.5 mL of the purified recombinant collagen solution with a concentration of 1 mg / mL in Example 5 and place it in a test tube, add 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol, shake the mixture vigorously for 10 seconds, and then react at room temperature for 30 minutes. After the reaction, measure the absorbance of the reaction mixture at 517 nm, and use distilled water instead of the sample solution as a blank control. Comparative experiments are performed with commercially available and self-developed recombinant collagen (Comparative Example 1, the recombinant collagen was purchased from Hebei Naco Biotechnology Co., Ltd., model 3CH-FD05; Comparative Example 2, the recombinant collagen is independently developed by the company, i.e., the recombinant transdermal collagen SPACE-Col7 disclosed in CN118359703A).

[0071] DPPH free radical scavenging activity = (OD blank - OD sample) / OD blank,

[0072] Among them, OD blank is the absorbance value of the blank control group, and OD sample is the absorbance value of the recombinant humanized collagen or commercially available recombinant collagen group.

[0073] The results are as follows Figure 2As shown, the recombinant collagen SPACE-Col4 of the present invention has obvious DPPH free radical scavenging ability compared with the blank control, and its DPPH free radical scavenging rate is higher than the DPPH free radical scavenging rate of commercially available recombinant collagen. Therefore, the recombinant collagen provided in this example has obvious antioxidant effect.

[0074] Example 7 Test of transdermal performance of recombinant collagen

[0075] This test uses piglet skin tissue to conduct a permeability experiment on the sample, and observes the accumulation of fluorescent labeled substances in the ex vivo skin tissue at different time points using a fluorescence microscope to evaluate the skin permeation behavior of the sample. The test is performed according to the test scheme in Table 1.

[0076] Table 1 Test plan

[0077]

[0078] Test results such as Figure 3 and Figure 4 As shown in the figure, the cumulative amount of fluorescent substances in the three experimental groups increased significantly with the extension of the test time, and the fluorescence intensity of the experimental groups was greater than that of the commercially available recombinant collagen in the control group. At the same time, the fluorescence intensity of the recombinant collagen SPACE-Col4 was significantly greater than that of the recombinant collagen Col4 and SPACE-Col7. The above test results show that compared with the commercially available recombinant collagen and recombinant collagen Col4 and SPACE-Col7, the recombinant collagen SPACE-Col4 with transdermal peptide has the best transdermal performance.

[0079] Matters not covered by the present invention are known technologies.

[0080] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

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

1.

2. A recombinant collagen, characterized in that: The recombinant collagen is further modified with a cell-penetrating peptide, which is modified at the amino end of the recombinant collagen; the cell-penetrating peptide is the cell-penetrating peptide SPACE, and its amino acid sequence is shown in SEQ ID NO.3; the nucleotide sequence encoding the recombinant collagen gene is shown in SEQ ID NO.

6.

3. A polynucleotide, characterized in that The polynucleotide encodes the recombinant collagen according to claim 1 or 2; the polynucleotide is selected from: (b1) the nucleotide sequence as shown in any one of SEQ ID NO.2 and SEQ ID NO.6; (b2) A nucleotide sequence that encodes a protein having the same amino acid sequence as the nucleotide sequence of (b1), but differs in sequence due to the degeneracy of the genetic code.

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

5. A host bacterium, characterized in that The host bacteria contains the recombinant expression vector of claim 4 or has the polynucleotide of claim 3 integrated into its chromosome, or expresses the recombinant collagen of any one of claims 1-2.

6. The host bacteria according to claim 5, characterized in that The host bacteria are bacteria, fungi and actinomycetes.

7. The host bacteria according to claim 6, characterized in that The fungus is yeast.

8. The host bacteria according to claim 7, characterized in that The yeast is Pichia pastoris.

9. A method for preparing recombinant collagen as claimed in claim 1 or 2, characterized in that: The preparation method comprises: culturing the host bacteria according to any one of claims 5 to 8 to express the recombinant collagen; and separating and purifying the recombinant collagen.

10. Use of the recombinant collagen according to claim 1 or 2 in the preparation of cosmetics or medical beauty products.

11. A cosmetic, characterized in that: The cosmetic comprises the recombinant collagen according to claim 1 or 2.

12. The cosmetic according to claim 11, characterized in that The cosmetics are skin care products.

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