A gene encoding and its use for producing recombinant humanized collagen type III with high transdermal performance

Abstract

The application discloses a kind of coding genes, the coding gene can encode human source III type collagen alpha 1 chain functional fragment and transdermal peptide TD-1, wherein the coding gene of human source III type collagen alpha 1 chain functional fragment and the coding gene of transdermal peptide TD-1 are expressed in series.The application also discloses a kind of recombinant vectors and a kind of recombinant bacteria, the coding gene and the coding gene of human source 4-proline hydroxylase are contained in the above-mentioned recombinant bacteria.The application also discloses the application of the above-mentioned coding gene, recombinant vector, recombinant bacteria in the production of high transdermal performance recombinant humanization III type collagen.The recombinant humanization III type collagen prepared by the application has similar structure with human source III type collagen, has good biological activity and stability, and has good transdermal ability, has good antioxidant, anti-carbonylation, inhibits tyrosinase activity, inhibits melanin production, promotes keratinocyte migration and promotes the ability of wound repair.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an encoding gene and its application in generating recombinant humanized type III collagen with high transdermal permeability. Background Technology

[0002] Approximately 70%-80% of the extracellular matrix of skin cells is collagen, mainly type I and type III collagen. Type I collagen is primarily found in adult skin, tendons, and bone tissue, while type III collagen is mainly found in infant skin, vascular endothelium, and intestines. Both type I and type III collagen are closely related to the skin damage repair process and the quality of repair. Type III collagen can promote the proliferation of dermal fibroblasts, increase cell activity, and be absorbed by fibroblasts as raw materials for collagen synthesis. This stimulates cells to synthesize more collagen, filling and repairing damaged and aging skin, rebuilding the reticular structure, enhancing the extensibility of damaged skin, and restoring skin elasticity.

[0003] Currently, collagen is mainly derived from animals, and its extraction methods generally involve acid and alkali hydrolysis from animal connective tissues (pigskin, cowhide, donkey skin, fish skin, etc.). However, the resulting products have complex compositions, and acid and alkali treatment can easily cause collagen to lose its original physiological activity. Furthermore, there are risks associated with animal-derived diseases, and its application in humans can lead to foreign body or xenogeneic rejection reactions, limiting its widespread use in the field of medical tissue materials.

[0004] With the large-scale application of genetic engineering technology, recombinant collagen expression has successfully overcome the bottleneck of large-scale collagen production by using a modeled host expression method for exogenous protein expression. For example, human collagen can be expressed in *E. coli*, but because *E. coli* lacks the enzymes necessary for post-translational modifications such as glycosylation and hydroxylation, it cannot form the natural triple helix higher-order structure. The product obtained from expression in *E. coli* is unhydroxylated collagen peptide, which lacks high biological activity and has poor stability, making it unsuitable for long-term storage. It also inhibits the self-assembly of natural collagen molecules into collagen fibers. Furthermore, *E. coli* requires cell disruption to extract the protein, introducing *E. coli* cell fragments into the final target protein. Due to the potential immunogenicity of the *E. coli* system, and the presence of endotoxins and pyrogens within the system itself, there are potential safety hazards. In contrast, the *Pichia pastoris* eukaryotic expression system for heterologous protein expression has high biosafety and has gained widespread recognition, including from the US FDA. It is more suitable for the heterologous expression of proteins for medical and food applications.

[0005] Human natural type III collagen consists of three polypeptide chains twisted to the right into a triple helix. Primary structure analysis shows that long segments of its polypeptide chain are composed of repeated Gly-xy amino acid sequences. Here, x is usually proline, and y is usually hydroxyproline and hydroxylysine. These latter two amino acids are rarely found in other proteins. The hydroxyl group of hydroxyproline participates in interchain hydrogen bonding, promoting the formation of the triple helix structure and improving the stability and functionality of collagen. Hydroxyproline is usually formed from proline by the action of 4-proline hydroxylase. Therefore, co-expression of the 4-proline hydroxylase gene is necessary to produce collagen with a triple helix structure similar to that of human type III collagen. Furthermore, collagen is a large bioactive molecule that is not easily absorbed through the skin, especially its nourishing effect on the dermis is very limited, and its cosmetic effects need improvement. Small molecule collagen peptides have poor collagen activity; therefore, the market needs recombinant collagen with high collagen activity and high transdermal permeability. Summary of the Invention

[0006] Based on the technical problems existing in the background technology, this invention proposes an application of a coding gene and the generation of a recombinant humanized type III collagen with high transdermal performance. This invention links the amino acid sequence of the transdermal peptide TD-1 to the N-terminus of the amino acid sequence of the functional fragment of the α1 chain of human type III collagen, and co-expresses its coding gene with the coding gene of 4-proline hydroxylase in Pichia pastoris. The resulting recombinant humanized type III collagen exhibits excellent transdermal ability, significantly superior to that of animal-derived collagen, thus solving the problem of difficult transdermal absorption of large-molecule recombinant collagen; furthermore, its molecular... The small amount further improves transdermal performance. The recombinant humanized type III collagen of this invention has a triple helix structure, exhibiting similar stability to naturally occurring human type III collagen, demonstrating good stability and enabling self-assembly of collagen fibers. The recombinant humanized type III collagen obtained by this invention also possesses good antioxidant capacity, effectively scavenging free radicals and inhibiting both major pathways of protein carbonylation modification (ROS and RSC pathways). This has been further verified in skin cells, suggesting its potential for anti-skin aging. The recombinant humanized type III collagen obtained by this invention also exhibits some inhibitory effect on melanin production and promotes wound repair.

[0007] This invention proposes a coding gene that encodes a functional fragment of human type III collagen α1 chain and a transdermal peptide TD-1, wherein the coding gene for the functional fragment of human type III collagen α1 chain and the coding gene for the transdermal peptide TD-1 are expressed in tandem.

[0008] Preferably, the encoding gene can also encode a His tag, wherein the encoding gene for the His tag, the encoding gene for the functional fragment of human type III collagen α1 chain, and the encoding gene for transdermal peptide TD-1 are expressed in tandem.

[0009] The His tag, also known as a multihistidine tag, consists of 6 to 10 consecutive histidine residues. This tag is used in many recombinant proteins to aid in protein purification, enabling researchers to extract target proteins from thousands of proteins in cells or cell lysates.

[0010] The aforementioned human type III collagen α1 chain functional fragment is a functional fragment that can realize the helical structure of human type III collagen.

[0011] The gene encoding the His tag in the above-mentioned coding genes has a small His tag structure, which can efficiently purify recombinant humanized type III collagen with high transdermal performance without affecting protein activity.

[0012] Preferably, the amino acid sequence of the functional fragment of the human type III collagen α1 chain is any one of the following:

[0013] a. An amino acid sequence as shown in SEQ ID NO.1;

[0014] b. An amino acid sequence with equivalent function formed by replacing, deleting, or adding amino acids to the amino acid sequence shown in SEQ ID NO.1.

[0015] Preferably, the amino acid sequence of the transdermal peptide TD-1 is any one of the following:

[0016] c. The amino acid sequence as shown in SEQ ID NO.2;

[0017] d. An amino acid sequence with equivalent function formed by replacing, deleting or adding amino acids to the amino acid sequence shown in SEQ ID NO.2.

[0018] Preferably, the nucleotide sequence of the gene encoding the functional fragment of human type III collagen α1 chain is any one of the following:

[0019] e. Nucleotide sequence as shown in SEQ ID NO.3;

[0020] f. A nucleotide sequence that hybridizes with the nucleotide sequence defined in SEQ ID NO.3 and encodes a protein having the same function.

[0021] Preferably, the nucleotide sequence of the gene encoding the transdermal peptide TD-1 is any one of the following:

[0022] g. A nucleotide sequence as shown in SEQ ID NO.4;

[0023] h. A nucleotide sequence that hybridizes with the nucleotide sequence defined in SEQ ID NO.4 and encodes a polypeptide with the same function.

[0024] Preferably, the amino acid sequence of the His tag is any of the following:

[0025] a. An amino acid sequence as shown in SEQ ID NO. 5;

[0026] b. An amino acid sequence with equivalent function formed by replacing, deleting, or adding amino acids to the amino acid sequence shown in SEQ ID NO.5.

[0027] Preferably, the nucleotide sequence of the gene encoding the His tag is any one of the following:

[0028] c. Nucleotide sequence as shown in SEQ ID NO. 6;

[0029] d. A nucleotide sequence that hybridizes with the nucleotide sequence defined in SEQ ID NO. 6 and encodes a polypeptide with the same function.

[0030] In the gene cluster, the gene encoding the His tag, the gene encoding the functional fragment of human type III collagen α1 chain, and the gene encoding the transdermal peptide TD-1 are expressed in tandem. The three coding genes may contain sequences that do not affect the expression of the His tag, the functional fragment of human type III collagen α1 chain, and the transdermal peptide TD-1; or the three coding genes may be directly linked.

[0031] Preferably, the amino acid sequence of the transdermal peptide TD-1 is located at the N-terminus of the amino acid sequence of the functional fragment of the human type III collagen α1 chain.

[0032] Preferably, the amino acid sequence of the His tag is located at the C-terminus of the amino acid sequence of the functional fragment of the human type III collagen α1 chain.

[0033] When the gene encoding the functional fragment of human type III collagen α1 chain and the gene encoding the transdermal peptide TD-1 are expressed in tandem, the nucleotide sequence of the encoding gene can be the nucleotide sequence shown in SEQ ID NO.7.

[0034] When the gene encoding the His tag, the gene encoding the functional fragment of human type III collagen α1 chain, and the gene encoding the transdermal peptide TD-1 are expressed in tandem, the nucleotide sequence of the encoding gene can be the nucleotide sequence shown in SEQ ID NO.8.

[0035] The present invention also proposes a recombinant vector in which the above-mentioned coding gene is inserted.

[0036] Preferably, the recombinant vector is a recombinant expression vector.

[0037] The recombinant vector is preferably a recombinant constitutive expression plasmid, for example, the recombinant vector can be obtained by recombination with the constitutive expression plasmid pGAPZα.

[0038] The present invention also proposes a recombinant bacterium containing the above-mentioned coding gene and the coding gene for human 4-proline hydroxylase.

[0039] 4-Proline hydroxylase (P4H) is a tetramer composed of two α subunits and two β subunits. The coding genes for the aforementioned human 4-proline hydroxylase include the coding genes for the P4H α subunit and the coding genes for the P4H β subunit.

[0040] The inventors refer to the above-mentioned segment of the encoding gene of human 4-proline hydroxylase as P4H. The encoding gene of human 4-proline hydroxylase is a naturally occurring known sequence, and its nucleotide sequence is shown in SEQ ID NO.9.

[0041] Preferably, the recombinant strain is Pichia pastoris.

[0042] Preferably, the recombinant strain is Pichia pastoris PX21, which was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on October 19, 2023, with accession number CCTCC NO: M 20231948.

[0043] The present invention also proposes a method for constructing the above-mentioned recombinant bacteria, comprising the following steps: co-expressing the above-mentioned encoding gene and the encoding gene of human 4-proline hydroxylase in the starting strain to obtain a recombinant bacteria capable of generating recombinant humanized type III collagen with high transdermal performance;

[0044] Alternatively, the above recombinant vector and a recombinant vector containing the encoding gene for human 4-proline hydroxylase can be co-expressed in the starting strain to obtain a recombinant strain capable of generating recombinant humanized type III collagen with high transdermal permeability.

[0045] The starting strain was Pichia pastoris X33, purchased from Invitrogen.

[0046] This invention also proposes the application of the above-mentioned encoding gene, recombinant vector, and recombinant bacteria in the production of recombinant humanized type III collagen with high transdermal permeability.

[0047] The present invention also proposes a recombinant humanized type III collagen, wherein the encoding gene of the recombinant humanized type III collagen is as described above.

[0048] Preferably, the amino acid sequence of the recombinant humanized type III collagen is any one of the following:

[0049] a. An amino acid sequence as shown in SEQ ID NO. 10;

[0050] b. An amino acid sequence with equivalent function formed by replacing, deleting, or adding amino acids to the amino acid sequence shown in SEQ ID NO.10.

[0051] Preferably, the apparent molecular weight of the recombinant humanized type III collagen is 22-24 kDa.

[0052] The present invention also proposes a method for preparing the above-mentioned recombinant humanized type III collagen, comprising the following steps: protein separation and purification of the fermentation broth of the above-mentioned recombinant bacteria to obtain recombinant humanized type III collagen.

[0053] This invention also proposes the application of the above-mentioned recombinant humanized type III collagen in the preparation of cosmetics or pharmaceuticals.

[0054] Beneficial effects

[0055] This invention uses genetic engineering to obtain recombinant humanized type III collagen, avoiding the risks associated with extracting collagen from animal tissues. It utilizes 4-proline hydroxylase to achieve triple helix formation of the α1 chain functional fragment of human type III collagen in vitro, obtaining collagen with similar stability to naturally occurring human type III collagen. Its structure is more similar to human type III collagen, exhibiting better bioactivity and stability. No significant degradation was observed after storage at -80℃ for 8 weeks, facilitating the development and preservation of cosmetic and medical devices. Furthermore, it can achieve self-assembly of collagen fibers.

[0056] This invention utilizes Pichiapastoris X33 to construct a constitutive Pichiapastoris engineered strain expressing recombinant human type III collagen. The glyceraldehyde-3-phosphate dehydrogenase promoter in Pichiapastoris X33 eliminates the need for methanol during fermentation, avoiding the pollution and dangers caused by using large amounts of methanol in the production process, making it more suitable for large-scale production.

[0057] This invention links the amino acid sequence of transdermal peptide TD-1 to the N-terminus of the amino acid sequence of the functional fragment of human type III collagen α1 chain, thereby giving the prepared recombinant humanized type III collagen good transdermal ability. Its transdermal performance is significantly better than that of animal-derived collagen, solving the problem of difficult transdermal absorption of large molecular weight recombinant collagen. In addition, the recombinant humanized type III collagen of this invention has a small molecular weight, which can further promote transdermal absorption.

[0058] The recombinant humanized type III collagen obtained in this invention also has good antioxidant capacity and has a significant inhibitory effect on oxidative stress in zebrafish.

[0059] The recombinant humanized type III collagen obtained in this invention also has good anti-carbonylation ability, and has good inhibitory ability on the two main pathways of protein carbonylation modification (ROS pathway and RSC pathway), which has been further verified in skin cells, suggesting that it has certain anti-skin aging potential.

[0060] The recombinant humanized type III collagen obtained in this invention has the ability to inhibit tyrosinase activity and melanin production, and can reduce melanin synthesis in B16-F10 cells.

[0061] The recombinant humanized type III collagen obtained in this invention also has the activity of promoting keratinocyte migration and has the potential to promote wound repair. Attached Figure Description

[0062] Figure 1 The results of the Western-Blot analysis of recombinant Pichia pastoris in Example 2 are shown. In the figure, A is the Western Blot image of P4HB antibody incubation, B is the Western Blot image of His-tagged antibody incubation, Control is recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC, and #21, #25, and #37 are recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC / pGAPK-P4H.

[0063] Figure 2 The results are the sampling and testing results at each fermentation time point in Example 3. Here, a is the wet weight of the cells, and b is the Western Blot test result of collagen. The time points corresponding to b from left to right are 21h, 27h, 33h, 39h, 45h, and 50h.

[0064] Figure 3 The results of the detection of recombinant humanized type III collagen in Example 4 are shown. In this figure, C is the HPLC chromatogram of the protein, D is the SDS PAGE detection result, and rHC is the recombinant humanized type III collagen.

[0065] Figure 4 The image shows the ABTS clearance rate curve, where TD-HrHC is recombinant humanized type III collagen and Trolox is the positive control.

[0066] Figure 5The results of ROS scavenging in zebrafish by recombinant humanized type III collagen in Example 6 are shown. TD-hrHC is recombinant humanized type III collagen. The bars from left to right are the positive control group, the test group with 25 μg / ml TD-hrHC, the test group with 10 μg / ml TD-hrHC, and the test group with 5 μg / ml TD-hrHC.

[0067] Figure 6 The results show the in vitro anti-protein carbonylation ability of recombinant humanized type III collagen. In the figure, A represents the ROS model, B represents the RCS model, rHC represents recombinant humanized type III collagen, and GSH represents glutathione.

[0068] Figure 7 The study aimed to assess the anti-protein carbonylation ability of recombinant humanized type III collagen in HACAT cells, where rHC is recombinant humanized type III collagen and GSH is glutathione.

[0069] Figure 8 The figure shows the effect of recombinant humanized type III collagen and β-arbutin on melanin content in B16-F10 cells. Arbutin is β-arbutin, and TD-hrHC is recombinant humanized type III collagen. The bars from left to right are the positive control group, the experimental group with 250 μg / ml TD-hrHC, the experimental group with 125 μg / ml TD-hrHC, and the experimental group with 63 μg / ml TD-hrHC.

[0070] Figure 9 The curve shows the hyaluronidase inhibition rate of recombinant humanized type III collagen, where TD-HrHC is recombinant humanized type III collagen and VC is the positive control vitamin C.

[0071] Figure 10 The results are for cell migration rate detection. In the figure, a is an optical microscope image, b is the calculated cell migration rate, PBS is the negative control group, NC is the negative control group, and 5, 10, and 20 μg / ml are the experimental groups with different concentrations of recombinant humanized type III collagen.

[0072] Figure 11 This is a schematic diagram of a diffusion cell.

[0073] Figure 12 The results show the transdermal performance of recombinant humanized type III collagen. In this data, B represents the transdermal performance of recombinant humanized type III collagen at different time points, C represents the transdermal performance of each collagen at the same time point, and rHC represents recombinant humanized type III collagen. Detailed Implementation

[0074] The technical solution of the present invention will now be described in detail through specific embodiments.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used in the examples are commercially available unless otherwise specified.

[0076] Example 1

[0077] Optimize gene design and synthesis

[0078] The amino acid sequence of the functional fragment of the human type III collagen α1 chain (i.e., amino acid sequence 597 to 777 in the collagen domain region, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.3) was selected, and the amino acid residue sequence of the transdermal peptide TD-1 was linked to the N-terminus (amino acid sequence as shown in SEQ ID NO.2, nucleotide sequence as shown in SEQ ID NO.4). The amino acid sequence of the His tag was linked to the C-terminus of the TD-1 sequence (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6). The gene sequence was optimized according to the codon selection preference of Pichia pastoris to obtain the target gene sequence. Then, the optimized full gene sequence (synthesized by Genewiz) was obtained through whole-genome synthesis and designated as plasmid pUC-TD1-rHC-6his (abbreviated as pUC-rHC).

[0079] 4-Proline hydroxylase (P4H) is a tetramer composed of two α-subunits and two β-subunits. Based on the codon selection bias of Pichia pastoris, the coding sequences of the P4H α-subunit and P4H β-subunit were optimized. Then, P4H α and P4H β fragments were synthesized and self-assembled into P4H(α2β2). The optimized full-length gene sequence was then obtained through whole-genome synthesis (synthesized by Genewiz). The synthesized plasmid is designated pUC-P4H, containing the coding genes for both the P4H α-subunit and the P4H β-subunit.

[0080] Example 2

[0081] Construction of recombinant expression engineered bacteria

[0082] 1. Construction and screening of pGAPZα-TD1-rHC-6his expression vector

[0083] The plasmid pUC-rHC was double-digested, and the target fragment TD1-rHC-6his (nucleotide sequence shown in SEQ ID NO.8) was obtained by gel extraction and recovery.

[0084] The constitutive vector plasmid pGAPZα was double-digested with enzymes, and the pGAPZα expression vector fragment was obtained by gel extraction.

[0085] The target fragment TD1-rHC-6his and the pGAPZα expression vector fragment were ligated using a ligase. The ligation product was transformed into competent Escherichia coli Top10, and positive clones were screened on LB resistant plates containing Zeocin. The recombinant constitutive expression plasmid was successfully obtained and named pGAPZα-rHC.

[0086] 2. Construction and screening of pGAPK-P4H expression vector

[0087] Using pUC-P4H as a template, PCR amplification was performed to obtain the target fragment P4H (nucleotide sequence shown in SEQ ID NO.9);

[0088] Using the pGAPK vector as a template, linearized pGAPK was obtained by PCR amplification.

[0089] The target fragment P4H and linearized pGAPK were recombined and ligated. The recombinant ligation product was transformed into competent E. coli Top10. Positive clones were screened on LB resistant plates containing Kana, and the recombinant constitutive expression plasmid was successfully obtained and named pGAPK-P4H.

[0090] 3. Construction and screening of recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC / pGAPK-P4H

[0091] The recombinant expression plasmid pGAPZα-rHC was linearized and electroporated into Pichia pastoris X33 (purchased from Invitrogen) competent cells. Using Zeocin as an antibiotic marker, Dot-Blot and Western-Blot detection were performed with his tag antibody to screen for high-copy-positive recombinants, thus obtaining recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC.

[0092] The recombinant expression plasmid pGAPK-P4H was linearized and electroporated into recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC competent cells. Using G418 as an antibiotic marker, high-copy-positive recombinants were screened by Dot-Blot and Western-Blot detection with P4HB antibody to obtain recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC / pGAPK-P4H.

[0093] The results of Western blotting are as follows: Figure 1 As shown, Figure 1The results of the Western-Blot analysis of recombinant Pichia pastoris in Example 2 are shown. In the figure, A is the Western Blot image of P4HB antibody incubation, B is the Western Blot image of His-tagged antibody incubation, Control is recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC, and #21, #25, and #37 are recombinant Pichia pastoris engineered strain X33 / pGAPZα-rHC / pGAPK-P4H.

[0094] Depend on Figure 1 It can be seen that the three recombinant Pichia pastoris engineered strains #21, #25, and #37, X33 / pGAPZα-rHC / pGAPK-P4H, can simultaneously express the functional fragment of human type III collagen α1 chain and P4H. Strain #21 was selected as the target strain, freeze-dried and preserved, and designated as Pichia pastoris PX21. It was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on October 19, 2023, with accession number CCTCC NO: M 20231948.

[0095] Example 3

[0096] Fermentation expression of recombinant humanized type III collagen

[0097] Culture medium:

[0098] (1) YPD liquid culture medium: 10g yeast extract, 20g trypton, 20g glycerol, ultrapure water to a final volume of 1000ml, sterilized at 121℃ for 20min.

[0099] (2) YPD solid medium: Add 15g of agar to YPD liquid medium per 1000ml.

[0100] (3) BMGY medium: 10g yeast extract, 20g trypton, 40g glycerol to a final volume of 800ml, add 100ml 1M phosphate buffer, sterilize at 121℃ for 20min. After cooling, add 100ml filtered and sterilized 10×YNB.

[0101] Fermentation process:

[0102] The recombinant Pichia pastoris engineered strain #21 X33 / pGAPZα-rHC / pGAPK-P4H from Example 2 was streaked onto YPD solid medium plates and cultured at 30°C until single colonies grew. A single colony was picked and inoculated into 10 ml of YPD liquid medium and cultured overnight at 30°C with shaking at 220 rpm to obtain the primary seed culture. 1 ml of the primary seed culture was added to 200 ml of BMGY medium and cultured at 30°C with shaking at 220 rpm for 24 hours to obtain the secondary seed culture. All of the secondary seed culture was added to the fermenter, the operating software was opened, the corresponding fermenter was connected, and the parameter curve was set for fermentation. Samples were taken at various time points during the fermentation process for analysis. The results are as follows: Figure 2 As shown.

[0103] Figure 2 The results are the sampling and testing results at each fermentation time point in Example 3. Here, a is the wet weight of the cells, and b is the Western Blot test result of collagen. The time points corresponding to b from left to right are 21h, 27h, 33h, 39h, 45h, and 50h.

[0104] Depend on Figure 2 It can be seen that during the fermentation process, the wet weight of the cells increases and the protein content accumulates with the increase of fermentation time, while there is no obvious degradation of recombinant humanized type III collagen.

[0105] Example 4

[0106] Isolation and purification of recombinant humanized type III collagen

[0107] Buffer solution:

[0108] (1) 0.2M phosphate buffer (PB): 27.22g K2HPO4, 8g NaCl, dissolved in 800ml ultrapure water, pH adjusted to 7.4, and volume adjusted to 1000ml. This is solution A.

[0109] (2) 3M NaCl: 175.32g NaCl dissolved in 1000ml ultrapure water, this is solution B.

[0110] (3) Nickel column chromatography Binding Buffer: 100ml solution A, 100ml solution B, add ultrapure water to 800ml, adjust pH to 7.4, and make up to 1000ml.

[0111] (4) Nickel column chromatography Wash Buffer: 100ml solution A, 100ml solution B, 1.36g imidazole, add ultrapure water to 800ml, adjust pH to 7.4, and make up to 1000ml.

[0112] (5) Nickel column chromatography Elution Buffer: 100ml solution A, 100ml solution B, 13.6g imidazole, add ultrapure water to 800ml, adjust pH to 7.4, and make up to 1000ml.

[0113] (6) Molecular sieve chromatography buffer: Dissolve 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 in 800mL distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to make up to 1L to obtain 0.01M PBS buffer.

[0114] Separation and purification process:

[0115] (1) Obtaining the supernatant sample: Take the fermentation broth prepared in Example 3, centrifuge at 4℃ and 12000g for 30min, discard the precipitate, collect the supernatant, filter the supernatant through 0.22μm, 500kD and 100kD hollow fiber columns to obtain the flow-through liquid, adjust the pH to 7.4 with ammonia water as the supernatant sample;

[0116] (2) Nickel column affinity chromatography: Rinse the nickel column with 5-10 column volumes of ultrapure water to remove 20% ethanol (stop when the UV value remains unchanged or fluctuates around a certain value); add 10 column volumes of Binding Buffer to equilibrate the column (the Binding Buffer and the supernatant sample should be placed on ice to minimize protein degradation loss); add the supernatant sample to the column, control the flow rate at 200-300 rpm / min, and collect the flow-through; then add 2 column volumes of Wash Buffer to wash the column (to elute impurities) to obtain the eluent; then add 5-10 column volumes of Elution Buffer to elute the protein to obtain the eluent. Start collecting when the UV280 value rises slightly and stop collecting when the value decreases slowly to obtain the crude protein extract.

[0117] (3) Molecular sieve gel filtration chromatography: The crude protein extract was concentrated and loaded onto a HiPrep 26 / 60 Superdex 100 HR molecular sieve. The washing buffer was PBS, the flow rate was 1 ml / min, and UV280 monitoring was used to collect each elution peak. The protein concentration was determined by BCA, and the purity of the collected protein was determined by SDS-PAGE and high-performance liquid chromatography. The results are as follows: Figure 3 , Figure 3 The results of the detection of recombinant humanized type III collagen in Example 4 are shown in Figure 4. In Figure 4, C is the HPLC chromatogram of the purified protein, D is the SDSPAGE detection result, and rHC is the recombinant humanized type III collagen.

[0118] Depend on Figure 3It can be seen that the obtained recombinant humanized type III collagen has high stability. The purified recombinant humanized type III collagen has high protein purity, with an apparent molecular weight of approximately 22 kDa, consistent with the theoretical molecular weight. High-performance liquid chromatography (HPLC) analysis shows that the obtained protein purity reaches 93.997%. It can be used for large-scale production of recombinant humanized type III collagen, and the production method is rapid and simple.

[0119] The amino acid sequence of recombinant humanized type III collagen is shown in SEQ ID NO.10.

[0120] Example 5

[0121] Detection of the degree of hydroxylation of recombinant humanized type III collagen

[0122] The kit used for hydroxylation degree detection was the Boxbio Hydroxyproline Content Detection Kit (catalog number: AKAM017).

[0123] Three batches of fermentation broth were obtained according to the method of Example 3, and three batches of recombinant humanized type III collagen were obtained according to the method of Example 4. 1 ml of each sample was taken, 1 ml of hydrochloric acid was added, and the mixture was acidified at 110°C for 6 h; centrifuged at 12000 g at room temperature for 20 min, and the supernatant was collected; reagent one (concentrated hydrochloric acid) was added to adjust the pH to 6-8, and distilled water was added to a final volume of 4 ml to obtain the sample to be tested.

[0124] Then, prepare the solutions for the test tubes, standard tubes, and blank tubes according to Table 1, shake thoroughly to mix, and let stand at room temperature for 20 min. Then, add the reagents according to Table 2, develop the color at 60℃ for 20 min, and let stand at room temperature for 15 min. Then, pipette 200 μl of the solution from each tube into a 96-well plate and measure the absorbance at 560 nm. Use ultrapure water as the zero point and record them as A_test, A_standard, and A_blank, respectively. Calculate: ΔA_standard = A_standard - A_blank, ΔA_test = A_test - A_blank.

[0125] A standard curve was established based on the measured data from the standard tubes. A standard equation was derived by plotting the corresponding hydroxyproline concentration on the x-axis and the corresponding ΔA standard on the y-axis. The hydroxyproline content in the test samples was then calculated, and subsequently, the degree of hydroxylation of each batch of recombinant humanized type III collagen was calculated. The results are shown in Table 3.

[0126] Table 1 Sampling Information

[0127]

[0128] Table 2 Sampling Information

[0129] reagents Measurement tube (μl) Standard tube (μl) Blank tube (μl) Reagent 3 (DMAB solution) 60 60 60 Ultrapure water 120 120 180

[0130] Table 3 Detection Results

[0131] Fermentation batch Degree of hydroxylation 20221128 11.55％ 20221207 12.01％ 20221215 11.92％ Average content of protein hydroxyproline in three batches 11.89±0.27％

[0132] As shown in Table 3, the proportions of hydroxyproline in the recombinant humanized type III collagen obtained from the three batches of fermentation were 11.55%, 12.01%, and 11.92%, respectively, which are close to the hydroxyproline proportion of 13% in natural human type III collagen.

[0133] Example 6

[0134] Antioxidant activity test of recombinant humanized type III collagen

[0135] 1. Antioxidant Activity Assay of Recombinant Humanized Type III Collagen - ABTS Method

[0136] ABTS (2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) reacts with potassium persulfate to produce green ABTS. + Substances with antioxidant properties can scavenge ABTS. + ABTS + The maximum absorption occurs at 734 nm. By measuring the absorbance, the magnitude of the absorbance value can be used to quantitatively determine the free radical scavenging ability of the reactant. That is, the lower the absorbance value, the stronger the ability of the antioxidant to scavenge ABTS free radicals.

[0137] Take 50 μl each of recombinant humanized type III collagen at different concentrations and positive control Trolox (water-soluble vitamin E derivative), add 150 μl of ABTS working solution (0.74 mmol / L LABTS, 0.26 mmol / L K2S2O8), incubate at room temperature for 6 min, and measure absorbance at 734 nm. Calculate the ATBS scavenging effect of the samples and plot the scavenging rate curve. Results are as follows: Figure 4 As shown.

[0138] Figure 4 The image shows the ABTS clearance rate curve, where TD-HrHC is recombinant humanized type III collagen and Trolox is the positive control.

[0139] Depend on Figure 4 It can be seen that recombinant humanized type III collagen has a good free radical scavenging ability, which is concentration-dependent. It has potential anti-wrinkle and firming effects.

[0140] 2. Antioxidant Activity Test of Recombinant Humanized Type III Collagen - Zebrafish Oxidative Stress Model

[0141] Zebrafish are small tropical freshwater fish, named for the zebra-like longitudinal stripes on their sides. Their tissue structure, growth and development, digestive and circulatory systems share high similarities with humans. Furthermore, with the completion of the zebrafish genome sequencing project, it was found that the structure and function of their proteins and genes are 87% conserved with humans. They have advantages such as early development, when the embryo is transparent and visible, and clearer observation of cell lines using fluorescent staining or other markers. Oxidative stress (OS) refers to a state of imbalance between oxidation and antioxidation in the body, with a tendency towards oxidation, leading to inflammatory infiltration of neutrophils, increased protease secretion, and the production of large amounts of oxidation intermediates. Oxidative stress is a negative effect produced by free radicals in the body and is considered a major factor contributing to aging and wrinkles. Oxidative stress can be directly assessed by measuring reactive oxygen species (ROS). Menadione, as a superoxide generator, can enter cells, be reduced by a single electron to a semiquinone free radical, and then enter the respiratory chain to react with O2 to form ·O. 2- Therefore, exogenous addition of menadione can increase reactive oxygen species (ROS), and menadione can be used to induce oxidative stress in zebrafish. The fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA), also known as a reactive oxygen species (ROS) fluorescent probe, is a cell-penetrating fluorescent probe. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to generate DCFH. Since DCFH is impermeable to the cell membrane, the probe is easily retained inside the cell. Intracellular ROS can oxidize the non-fluorescent DCFH to generate fluorescent DCF. Detecting the fluorescence of DCF allows us to determine the level of intracellular ROS.

[0142] a. Randomly select healthy zebrafish embryos at 2 dpf and place 30 embryos in each well of a 6-well plate. Carefully remove excess water using a pipette. According to the grouping, quickly add the corresponding culture medium to maintain a total solution volume of 4 mL for each group (i.e., add system water to the negative control group, add menadione solution to the model control group, add Trolox solution and menadione solution to the positive control group, and add different concentrations of recombinant humanized type III collagen solution and menadione solution to the test group). Place the 6-well plate in a constant temperature and light incubator at 28℃±1℃ and in the dark for 22h±2h.

[0143] b. Take out the above 6-well plate, add DCFH-DA solution to each group to make the final concentration of DCFH-DA 10 μg / mL, and place it in a constant temperature light incubator at 28℃±1℃ in the dark for 1 h.

[0144] c. Remove the 6-well plates and observe and record the number of abnormal and dead fish embryos (egg coagulation, immobility, no heartbeat, white and opaque body, no response to mechanical stimulation, and failure to separate the tail within approximately 48 hours indicates death). Remove the dead fish embryos, discard the culture medium, and wash the plates three times with system water. Place one fish from each group into a black 96-well plate, add 100 μL of system water, and repeat the process 24 times per group. Detect fluorescence at an emission wavelength of 485 nm and an excitation wavelength of 535 nm. Calculate the reactive oxygen species (ROS) scavenging rate using the following formula:

[0145]

[0146] in,

[0147] I 受试物组 —The average fluorescence intensity of the test group.

[0148] I 模型对照组 —The average fluorescence intensity value of the model control group.

[0149] Test results as follows Figure 5 As shown. Figure 5 The results of ROS scavenging in zebrafish by recombinant humanized type III collagen in Example 6 are shown. TD-hrHC is recombinant humanized type III collagen. The bars from left to right are the positive control group, the test group with 25 μg / ml TD-hrHC, the test group with 10 μg / ml TD-hrHC, and the test group with 5 μg / ml TD-hrHC.

[0150] Depend on Figure 5 It can be seen that recombinant humanized type III collagen, at a concentration of 5 μg / ml, exhibited antioxidant capacity no less than that of the positive control Trolox; it also showed a dose-dependent effect on antioxidant capacity, with higher concentrations resulting in stronger antioxidant capacity. It has potential anti-wrinkle and firming effects.

[0151] Example 7

[0152] Anti-carbonylation activity assay of recombinant humanized type III collagen

[0153] The introduction of carbonyl groups into the side chains of protein amino acid residues is a major marker of protein oxidative damage. The process by which carbonyl groups, such as aldehydes, ketones, and lactams, are introduced into the side chains of protein amino acid residues is called "protein carbonylation." Protein carbonylation is generally considered an irreversible post-translational modification that can cause conformational changes in polypeptide chains and backbone breakage, leading to loss of protein function. Keratin proteins in the keratinocytes of the skin are the main targets of protein carbonylation. Human skin, especially facial skin, is frequently exposed to ultraviolet radiation, triggering reactive oxygen species (ROS) chain reactions and producing large amounts of MDA and HNE, which in turn lead to extensive carbonylation of proteins in the keratinocytes of the skin. The carbonylation of keratin has the following effects: First, it reduces the skin's ability to retain moisture. Protein carbonylation is negatively correlated with skin hydration, and carbonylated proteins can significantly reduce the amount of bound water in the stratum corneum. Second, carbonylated proteins reduce skin elasticity. Carbonylation of proteins in facial skin keratinocytes can severely affect the skin barrier function and lead to decreased skin elasticity, which is more severe in autumn and winter. Third, carbonylated proteins reduce skin radiance. Carbonylation of keratinocytes alters the fibrous structure of keratin, affecting light refraction, reducing skin transparency, and making the skin appear dull and lackluster.

[0154] Protein carbonylation in the skin mainly occurs through two pathways: the ROS (reactive oxygen species) pathway and the RSC (lipid peroxidation) pathway. Cell-free models of protein carbonylation were established by first simulating the ROS and RSC reactions of proteins using H2O2 and MDA / BSA, respectively, with glutathione (GSH) as a positive control. Using the carbonyl content of the model group as 100%, the anti-carbonylation capacity of various concentrations of recombinant humanized type III collagen and GSH was calculated.

[0155] 1. Test of the anti-protein carbonylation ability of recombinant humanized type III collagen - biochemical level (ROS pathway):

[0156] Configure the reaction systems for each group according to Table 4, and incubate at 37°C in the dark for 24 hours.

[0157] The degree of protein carbonylation was determined using the DNPH colorimetric method. The specific steps were as follows: 100 μl of each of the above reaction systems was transferred, and 400 μl of 10 mmol / L DNPH solution (using 2 mol / L HCl aqueous solution) was added to each system. The systems were placed in the dark and reacted for 1 h, vortexing every 10 min. Then, 500 μl of 200 g / L trichloroacetic acid was added to each system, and the mixture was gently mixed. The systems were allowed to stand at 4 °C for 5 min to allow the reaction to complete. Afterward, the systems were centrifuged at 12000 g for 15 min at 4 °C, and the supernatant was discarded. The precipitates were washed three times (using an equal volume of ethanol and acetic acid mixture; 1 ml each time; centrifuged at 12000 g for 15 min at 4 °C). The precipitates were then dissolved in 1.25 ml of 6 mol / L guanidine hydrochloride in a water bath at 37 °C for 15-30 min. After centrifugation at 12000 g for 15 min, the supernatants were collected, and the absorbance of the supernatants was measured at 370 nm.

[0158] Table 4 Reaction System

[0159]

[0160]

[0161] Note: Each group was brought to a final volume of 1 ml with PBS. Three replicates were prepared for each group, and each replicate had three sets of wells.

[0162] 2. Test of the anti-protein carbonylation ability of recombinant humanized type III collagen - biochemical level (RCS pathway):

[0163] Configure the reaction systems for each group according to Table 5, and incubate at 37°C in the dark for 24 hours.

[0164] The degree of protein carbonylation was determined using the DNPH colorimetric method, with the specific steps being the same as the DNPH colorimetric method in the ROS pathway.

[0165] Table 5 Reaction System

[0166] reagents Model group Positive control group Experimental groups 1-3 BSA 15mg / ml 15mg / ml 15mg / ml MDA 3mmol / L 3mmol / L 3mmol / L rHC / / 1 / 5 / 10μM GSH / 10μM /

[0167] Note: Each group was brought to a final volume of 1 ml with PBS. Three replicates were prepared for each group, and each replicate had three sets of wells.

[0168] Test results are as follows Figure 6 As shown, Figure 6 The results show the in vitro anti-protein carbonylation ability of recombinant humanized type III collagen. In the figure, A represents the ROS model, B represents the RCS model, rHC represents recombinant humanized type III collagen, and GSH represents glutathione.

[0169] Figure 6The carbonylation inhibition rate was calculated with the carbonyl content of the model group being 100% as the baseline. The carbonylation inhibition rate of each group was calculated, and the data for each group are the mean ± standard deviation.

[0170] Depend on Figure 6 It can be seen that in the ROS model, the inhibition rate of 1 μM recombinant humanized type III collagen was 33%, while the inhibition rates of 5 μM and 10 μM were 48% and 52%, respectively, which were much higher than those of the 10 μM positive control GSH. In the RSC model, the inhibition rate of 1 μM recombinant humanized type III collagen was 5%, while the inhibition rates of 5 μM and 10 μM were 13% and 16%, respectively, which were close to those of the 10 μM positive control GSH. Therefore, the anti-carbonylation ability of recombinant humanized type III collagen is no less than that of the positive control GSH, and this anti-carbonylation ability also shows a certain dose-dependent effect, with higher concentrations exhibiting stronger anti-carbonylation ability.

[0171] 3. Anti-carbonylation capacity test of recombinant humanized type III collagen - cellular level

[0172] High concentrations of glucose can induce carbonylation modification of a large number of proteins, leading to cellular senescence. To investigate whether recombinant humanized type III collagen possesses anti-carbonylation activity, a high concentration of glucose (15 mg / ml) was added to HACAT cell culture medium to induce high intracellular carbonylation levels. The specific steps were as follows:

[0173] First, we examined the effect of recombinant humanized type III collagen on the viability of HACAT cells, and then selected the concentration that had no effect on the viability of B16F10 cells for subsequent experiments.

[0174] Then, the cell model was constructed. The specific steps were as follows: culture dishes were prepared, and the initial seeding amount of HaCaT cells was 1×10⁻⁶. 5 Cells were cultured at a concentration of 10 cells / ml according to Table 6 for 72 hours. When the cells reached approximately 90% confluence, they were collected and the cell count was determined. PBS was then added to dilute the cells to ensure equal cell concentrations in each group. Equal volumes of cell suspension were transferred, centrifuged at 500g for 5 minutes, and the supernatant was removed. This process was repeated twice to remove the supernatant. 600 μl of RIPA lysis buffer was added to the precipitate, and lysis was performed for 15 minutes. The cells were then centrifuged at 12000g for 5 minutes, and the supernatant was collected. 67 μl of 0.1 g / ml streptomycin sulfate was added, and the mixture was allowed to stand for 10 minutes. The cells were then centrifuged at 12000g for 5 minutes, and the supernatant was collected. Protein concentration was determined using the BCA method, and PBS was used to adjust the protein concentration in each group to ensure equal concentrations. Finally, the degree of protein carbonylation was determined using the DNPH colorimetric method, following the same steps as the DNPH colorimetric method in the ROS pathway.

[0175] Table 6 Sampling Information

[0176] Grouping Adding samples negative control group HACAT cells cultured in DMEM medium Model group HACAT cells were cultured in DMEM medium containing 80 mM glucose. Positive control group The model group was treated with glutathione (GSH) to a final concentration of 10 μM. Experimental groups 1-3 The model groups were supplemented with recombinant humanized type III collagen at final concentrations of 10 μM, 5 μM, and 1 μM, respectively.

[0177] Test results as follows Figure 7 As shown, Figure 7 The study aimed to assess the anti-protein carbonylation ability of recombinant humanized type III collagen in HACAT cells, where rHC is recombinant humanized type III collagen and GSH is glutathione.

[0178] Figure 7 The carbonylation inhibition rate was calculated with the carbonyl content in the model group being 100% as the baseline. The carbonylation inhibition rate of each group was calculated, and the data for each group are the mean ± standard deviation.

[0179] Depend on Figure 7 It can be seen that recombinant humanized type III collagen exhibited an anti-carbonylation ability of no less than 10 μM MSH at a concentration of 1 μM; in addition, recombinant humanized type III collagen also showed a dose-dependent effect on anti-carbonylation, with the higher the concentration, the stronger the anti-carbonylation ability. It has potential anti-wrinkle and firming effects.

[0180] Example 8: Assay of the melanin-inhibiting activity of recombinant humanized type III collagen

[0181] a. Quantitative detection method for melanin inhibition

[0182] Mouse melanoma cells B16-F10 are a commonly used cell model for studying melanin production. Melanocytes can synthesize and secrete melanin, which is an amino acid derivative. When melanin reacts with sodium hydroxide, it produces a water-soluble compound. The melanin content is calculated by detecting the ultraviolet absorbance at 405 nm.

[0183] Melanocyte-stimulating hormone (α-MSH) primarily activates tyrosinase and promotes its synthesis, thereby promoting melanin production and darkening skin and hair color. This study evaluated the skin-lightening activity of mouse melanoma cells B16-F10 by calculating the difference in melanin secreted by these cells compared to those in the sample group.

[0184] The inhibitory effect of recombinant humanized type III collagen on melanin synthesis in B16-F10 cells was investigated. The specific steps are as follows:

[0185] a. First, the effect of recombinant humanized type III collagen on the proliferation of melanoma cells B16-F10 was detected, and the concentration that had no effect on the proliferation of B16-F10 cells was selected for subsequent experiments;

[0186] b. Take a 6-well plate and add 2 ml of B16-F10 cell suspension to each well to make the cell density 4 × 10⁻⁶. 4 Cells / well were collected and incubated in a 5% CO2 incubator for 24 hours. The supernatant was then discarded. According to the sample addition information in Table 7, 2 ml of solution was added to each well, which were designated as blank control group, model group, positive control group, and experimental group, respectively. The cells were then incubated in a 5% CO2 incubator at 37°C for 72 hours (based on the drug administration time). The supernatant was then discarded.

[0187] c. Add samples again according to Table 7, then add 2 ml of DMEM complete culture medium to each well, and continue to incubate at 37°C for 48-72 h in an incubator containing 5% CO2 until the cell fusion rate reaches more than 90% under a microscope;

[0188] d. Then discard the culture supernatant, wash each well twice with 1 ml of sterile PBS buffer; discard the PBS buffer, add 200 μl of trypsin solution to each well for 3 min; add 1 ml of PBS buffer to each well and pipette the digested cells, and aspirate the cell suspension into a 1.5 ml EP tube, centrifuge at 300 g for 5 min, and discard the supernatant; add 150 μl of melanin extraction solution to each tube, and place in a 90℃ water bath for 1 h to completely dissolve the melanin.

[0189] e. Pipette 100 μl of solution from each EP tube into a 96-well plate, using melanin extract as a zero control. Measure the absorbance at 405 nm using a microplate reader, and record the OD value. 405 The size of the value indicates the amount of melanin. The melanin synthesis inhibition rate is calculated using the following formula:

[0190]

[0191] In the formula: T is the absorbance of the model group, positive control group or experimental group; C is the absorbance of the blank control group; C0 is the absorbance of the melanin extract.

[0192] Table 7. Sampling Information for Well Plates

[0193]

[0194] Note: Three parallel samples are set up for each group.

[0195] The results are as follows Figure 8 As shown, Figure 8The figure shows the effect of recombinant humanized type III collagen and β-arbutin on melanin content in B16-F10 cells. Arbutin is β-arbutin, and TD-hrHC is recombinant humanized type III collagen. The bars from left to right are the positive control group, the experimental group with 250 μg / ml TD-hrHC, the experimental group with 125 μg / ml TD-hrHC, and the experimental group with 63 μg / ml TD-hrHC.

[0196] Depend on Figure 8 It can be seen that recombinant humanized type III collagen can reduce melanin synthesis in B16-F10 cells, and the inhibitory effect increases with increasing concentration. Even at a concentration as low as 63 μg / ml (i.e., a content of 0.0063%), the inhibitory effect of recombinant humanized type III collagen on melanin is still superior to that of the positive control group of 0.01 wt% β-arbutin. This indicates that the recombinant humanized type III collagen described in this invention has a good whitening effect.

[0197] b. Quantitative detection method for tyrosinase activity inhibition

[0198] Experimental group: B16F10 cells were seeded into 96-well plates and cultured for 16 h. Then, they were cultured in a medium containing 0.0063% and 0.0125% recombinant humanized type III collagen for 48 h. Cells were then lysed using Triton X-100, and 10 μL of substrate solution (2 mL DOPA solution) was thoroughly mixed with 90 μL of lysate supernatant and incubated at 37°C for 1 h. The absorbance of the reaction solution at 450 nm was then measured using a microplate reader, and the tyrosinase activity in the cells was calculated.

[0199] Positive control group: 1 mg / mL α-arbutin was added to the culture medium, but no collagen sample was added. The remaining steps were the same as those for the experimental group.

[0200] Blank control group: The culture medium did not contain collagen samples, and the remaining steps were the same as the experimental group.

[0201] The results are shown in Table 8.

[0202] Table 8 Results of Tyrosinase Activity Test

[0203]

[0204] As shown in Table 8, the tyrosinase activity in B16F10 melanoma cells of mice in both the positive control group and the experimental group was lower than that in the blank control group; the tyrosinase activity in the experimental group was lower than that in the positive control group; indicating that the recombinant humanized type III collagen of the present invention has a good whitening effect.

[0205] Example 9

[0206] Recombinant humanized type III collagen hyaluronidase inhibition assay

[0207] Hyaluronidase is involved in type I hypersensitivity reactions. Hyaluronidase is strongly correlated with inflammation and allergies. Studies have reported that various drugs that release histamine from mast cells can regulate hyaluronidase activity. Some anti-allergy drugs have strong inhibitory effects on hyaluronidase activity. Therefore, inhibiting hyaluronidase activity is used as an indicator for studying soothing and repair effects.

[0208] Hyaluronidase hydrolyzes hyaluronic acid to produce β-N-acetylglucosamine. Under alkaline conditions, β-N-acetylglucosamine condenses with acetylacetone to form a 2-methyl-3-diacetylpyrrole derivative. This 2-methyl-3-diacetylpyrrole derivative reacts with p-dimethylaminobenzaldehyde in concentrated hydrochloric acid and ethanol, exhibiting characteristic absorption at a specific wavelength. Furthermore, the change in absorbance is linearly correlated with the degree of hyaluronidase inhibition. By measuring the change in absorbance, the hyaluronidase inhibition rate is calculated, thereby assessing whether recombinant humanized type III collagen has a soothing and repairing effect.

[0209] Take 100 μl each of recombinant humanized type III collagen and positive control vitamin C, add 100 μl of calcium chloride solution (12.5 mmol / L) and 50 μl of 5000 U / ml hyaluronidase respectively, and incubate at 37℃ for 40 min. Then add 200 μl of sodium hyaluronate solution (2 mg / ml) and incubate at 37℃ for 30 min. Then add 100 μl of sodium hydroxide solution (0.4 mol / L) and 100 μl of acetylacetone solution (1 mol / L), respectively, boil in a water bath for 15 min, cool immediately, add 500 μl of p-dimethylaminobenzaldehyde, mix thoroughly, incubate at 37℃ for 10 min, and measure the absorbance at 540 nm. Calculate the inhibitory effect of the samples on hyaluronidase, and plot the inhibition rate curve. The results are as follows: Figure 9 As shown.

[0210] The results are as follows Figure 9 As shown, Figure 9 The curve shows the hyaluronidase inhibition rate of recombinant humanized type III collagen, where TD-HrHC is recombinant humanized type III collagen and VC is the positive control vitamin C.

[0211] Depend on Figure 9 It can be seen that recombinant humanized type III collagen has a good inhibitory effect on hyaluronidase, and this effect is concentration-dependent, indicating potential soothing and repairing effects.

[0212] Example 10

[0213] Recombinant humanized type III collagen promotes HACAT cell migration assay

[0214] (1) Seed cells in 6-well culture dishes (3 ml DMEM + 10% FBS per dish), 6 × 10⁶ cells / well. 4 Cells / well;

[0215] (2) Culture the cells until a monolayer of cells is formed. Use a 200 μl pipette tip to gently draw a straight line from the middle to form a scratch. At the same time, change the culture medium to serum-free DMEM medium and add different concentrations (5, 10, 20 μg / ml) of recombinant humanized type III collagen to the experimental group. The negative control group is added with an equal amount of PBS.

[0216] (3) The culture dish was placed in a constant temperature incubator at 37℃ and 5% CO2. The migration of cells was observed at time points of 6h, 12h and 24h, and the observation and photographs were recorded by optical microscope.

[0217] (4) Based on the image records at different time points, measure the cell migration speed and migration distance, and statistically analyze the experimental results.

[0218] The formula for calculating cell migration rate is as follows:

[0219]

[0220] Test results as follows Figure 10 As shown, Figure 10 The results are for cell migration rate detection. In the figure, a is an optical microscope image, b is the calculated cell migration rate, PBS is the negative control group, NC is the negative control group, and 5, 10, and 20 μg / ml are the experimental groups with different concentrations of recombinant humanized type III collagen.

[0221] Depend on Figure 10 It can be seen that recombinant humanized type III collagen has been shown to have a strong ability to promote wound repair. The test results showed that in serum-free DMEM medium containing 20 μg / ml recombinant humanized type III collagen, the cell migration rate reached 59.2±0.9% after 24 h of culture, which was much higher than the 32.3±0.8% of the negative control group.

[0222] Example 11

[0223] Transdermal performance testing of recombinant humanized type III collagen

[0224] To assess the transdermal absorption capacity of recombinant humanized type III collagen, we used the static diffusion cell method to determine its transdermal absorption capacity and compared it with commercially available collagen peptides. The specific steps were as follows:

[0225] Healthy adult mice were euthanized by cervical dislocation; the fur on the back of the mice was shaved clean with a razor, taking care not to damage the skin; the skin on the back was cut off, and the inner layer of fat tissue was scraped clean with the back of a scalpel in physiological saline; the skin surface moisture was dried with absorbent paper and placed in clean physiological saline for later use. Skin samples were prepared just before use.

[0226] Prepare mouse skin samples, then clamp the samples with the stratum corneum facing upwards and the dermis downwards. Install the diffusion cells (see diagram of diffusion cells). Figure 11 As shown in the figure, mouse skin samples were kept in contact with the liquid surface of the receiving chamber; 6.5 ml of PBS was added to the receiving chamber, and 1 ml of each of the solutions shown in Table 9 was added to the supply chamber; the diffusion instrument was started, and the duration was set to 24 h and 48 h for two groups, with three parallel samples in each group; the collagen content in the receiving chamber at each time point was detected by BCA method.

[0227] Table 9 Solution information for each group

[0228] Grouping Adding samples negative control group PBS Control group 1 200 μg / ml fish collagen peptides (molecular weight ≤ 3000 Da) Control group 2 200 μg / ml porcine skin collagen peptides (molecular weight ≤750 Da) experimental group 200 μg / ml recombinant humanized type III collagen

[0229] Test results are as follows Figure 12 As shown, Figure 12 The results show the transdermal performance of recombinant humanized type III collagen. In this data, B represents the transdermal performance of recombinant humanized type III collagen at different time points, C represents the transdermal performance of each collagen at the same time point, and rHC represents recombinant humanized type III collagen.

[0230] Depend on Figure 12 It can be seen that: no protein was detected in the negative control group PBS; in the experimental group, the content of recombinant humanized type III collagen in the receiving pool was 122.1±62.8 ng / ml after 24 h, and 241.1±55.5 ng / ml after 48 h; in controls 1-2, the content of fish-derived collagen peptides in the receiving pool remained at a low level of 12.7±1.9 ng / ml after 48 h, and the content of only porcine skin-derived collagen peptides in the receiving pool remained at a low level of 12.5±0.7 ng / ml after 48 h, which is about 5.3% and 5.2% of that of recombinant humanized type III collagen, respectively. The results suggest that the transdermal permeability of hydrolyzed collagen peptides from animal sources is very weak, while the transdermal permeability of recombinant humanized type III collagen is significantly increased compared to them.

[0231] In summary, this invention screens suitable functional fragments of human type III collagen α1 chain and uses 4-proline hydroxylase to achieve the formation of a triple helix structure of the human type III collagen α1 chain functional fragments in vitro, obtaining collagen with a similar degree of stability to naturally occurring human type III collagen. Its structure is more similar to human type III collagen, and it has better bioactivity and good stability. Its bioactivity and stability are superior to collagen peptides, which facilitates the development and preservation of cosmetic, medical device and other products. Furthermore, it can achieve the self-assembly of collagen fibers.

[0232] This invention links the amino acid sequence of transdermal peptide TD-1 to the N-terminus of the amino acid sequence of the functional fragment of human type III collagen α1 chain, thereby giving the prepared recombinant humanized type III collagen good transdermal ability. Its transdermal performance is significantly better than that of animal-derived collagen, solving the problem of difficult transdermal absorption of large molecular weight recombinant collagen. In addition, the recombinant humanized type III collagen of this invention has a small molecular weight, which can further promote transdermal absorption.

[0233] The recombinant humanized type III collagen obtained in this invention also has good antioxidant capacity, anti-carbonylation capacity, anti-skin aging capacity, ability to inhibit tyrosinase activity and inhibit melanin production, can reduce melanin synthesis in B16-F10 cells, and also has the activity of promoting keratinocyte migration, and has the potential to promote wound repair.

[0234] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0235] sequence list

[0236] Applicants: Hefei Kadir Biotechnology Co., Ltd., and the Advanced Technology Research Institute of the University of Science and Technology of China

[0237] SEQ ID NO.1

[0238] GGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPG

[0239] SEQ ID NO.2

[0240] ACSSPSSKHCG

[0241] SEQ ID NO.3

[0242] GGAGGCCCTGGAGGTCCTGGCCCGCAGGGTCCCCCAGGAAAGAATGGTGAAACCGGACCTCAAGGTCCCCCAGGGCCAACTGGGCCGGGTGGAGACAAAGGAGACAGGACCCCCTGGTCCACCAAGGATTACAA GGCTTGCCAGGTACGGGTGGACCTCCAGGAGAAAACGGAAAACCTGGGGAGCCAGGTCCAAAGGGTGATGCCGGTGCACCAGGAGCTCCAGGCAAGGGTGATGCTGGTGCCCCTGGTGAACGTGGACCACCGG GATTGGCAGGGGCCCCAGGACTTAGAGGTGGAGCTGGTCCCCCTGGTCCCGAGGGAGGTAAGGGTGCTGCAGGTCCACCCGGGCCACCTGGTGCTGCGGGTACTCCTGGTCTGCAAGGAATGCCTGGAGAAAGAGGG AGGTCTAGGAAGTCCTGGTCCAAAGGGTGACAAGGGTGAGCCAGGCGGTCCAGGTGCTGGTGTTCCAGGGAAAGATGGCCCAAGGGGTCCTACTGGTCCTATTGGTCCTCCGGGCCCAGCTGGCCAGCCTGGA

[0243] SEQ ID NO.4

[0244] GCTTGTTCTTCCTCACCTTCTAAACATTGCGGT

[0245] SEQ ID NO.5

[0246] HHHHHH

[0247] SEQ ID NO.6

[0248] CATCACCATCACCATCAC

[0249] SEQ ID NO.7

[0250] GCTTGTTCTTCCTCACCTTCTAAACATTGCGGTGGAGGCCCTGGAGGTCCTGGCCCGCAGGGTCCCCCAGGAAAGAATGGTGAAACCGGACCTCAAGGTCCCCCAGGGCCAACTGGGCCGGGTGGAGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGATTACAAGGCTTGCCAGGTACGGGTGGACCTCCAGGAGAAAACGGAAAACCTGGGGAGCCAGGTCCAAAGGGTGATGCCGGTGCACCAGGAGCTCCAGGAGGCAAGGGTGATGCTGGTGCCCCTGGTGAACGTGGACCACCGGGATTGGCAGGGGCCCCAGGACTTAGAGGTGGAGCTGGTCCCCCTGGTCCCGAGGGAGGTAAGGGTGCTGCAGGTCCACCCGGGCCACCTGGTGCTGCGGGTACTCCTGGTCTGCAAGGAATGCCTGGAGAAAGAGGAGGTCTAGGAAGTCCTGGTCCAAAGGGTGACAAGGGTGAGCCAGGCGGTCCAGGTGCTGATGGTGTTCCAGGGAAAGATGGCCCAAGGGGTCCTACTGGTCCTATTGGTCCTCCGGGCCCAGCTGGCCAGCCTGGA

[0251] SEQ ID NO.8

[0252] GCTTGTTCTTCCTCACCTTCTAAACATTGCGGTGGAGGCCCTGGAGGTCCTGGCCCGCAGGGTCCCCCAGGAAAGAATGGTGAAACCGGACCTCAAGGTCCCCCAGGGCCAACTGGGCCGGGTGGAGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGATTACAAGGCTTGCCAGGTACGGGTGGACCTCCAGGAGAAAACGGAAAACCTGGGGAGCCAGGTCCAAAGGGTGATGCCGGTGCACCAGGAGCTCCAGGAGGCAAGGGTGATGCTGGTGCCCCTGGTGAACGTGGACCACCGGGATTGGCAGGGGCCCCAGGACTTAGAGGTGGAGCTGGTCCCCCTGGTCCCGAGGGAGGTAAGGGTGCTGCAGGTCCACCCGGGCCACCTGGTGCTGCGGGTACTCCTGGTCTGCAAGGAATGCCTGGAGAAAGAGGAGGTCTAGGAAGTCCTGGTCCAAAGGGTGACAAGGGTGAGCCAGGCGGTCCAGGTGCTGATGGTGTTCCAGGGAAAGATGGCCCAAGGGGTCCTACTGGTCCTATTGGTCCTCCGGGCCCAGCTGGCCAGCCTGGACATCACCATCACCATCAC

[0253] SEQ ID NO.9

[0254]

[0255] SEQ ID NO.10

[0256] ACSSSPSKHCGGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGHHHHHH 。

Claims

1. A gene encoding a gene, characterized in that, The coding gene can encode a functional fragment of human type III collagen α1 chain and transdermal peptide TD-1, wherein the coding gene for the functional fragment of human type III collagen α1 chain and the coding gene for transdermal peptide TD-1 are expressed in tandem. The amino acid sequence of the transdermal peptide TD-1 is located at the N-terminus of the amino acid sequence of the functional segment of the human type III collagen α1 chain; The amino acid sequence of the transdermal peptide TD-1 is directly linked to the amino acid sequence of the functional fragment of the human type III collagen α1 chain; The amino acid sequence of the functional fragment of the human type III collagen α1 chain is shown in SEQ ID NO.1; The amino acid sequence of the transdermal peptide TD-1 is shown in SEQ ID NO.

2.

2. The encoding gene according to claim 1, characterized in that, The coding gene can also encode a His tag, wherein the coding gene for the His tag, the coding gene for the functional fragment of human type III collagen α1 chain, and the coding gene for transdermal peptide TD-1 are expressed in tandem.

3. The encoding gene according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the functional fragment of human type III collagen α1 chain is shown in SEQ ID NO.

3.

4. The encoding gene according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the transdermal peptide TD-1 is shown in SEQ ID NO.

4.

5. The encoding gene according to claim 2, characterized in that, The amino acid sequence of the His tag is shown in SEQ ID NO.

5.

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

6.

7. The encoding gene according to claim 2, characterized in that, The His tag's amino acid sequence is located at the C-terminus of the functional segment of the human type III collagen α1 chain, and the His tag's amino acid sequence is directly linked to the functional segment of the human type III collagen α1 chain.

8. A recombinant vector, characterized in that, The recombinant vector is inserted with the coding gene as described in any one of claims 1-7.

9. The recombinant vector according to claim 8, characterized in that, The recombinant vector is a recombinant expression vector.

10. A recombinant bacterium, characterized in that, The recombinant bacteria contain the encoding gene of any one of claims 1-7 and the encoding gene of human 4-proline hydroxylase.

11. The recombinant bacteria according to claim 10, characterized in that, The recombinant strain was Pichia pastoris.

12. The recombinant bacteria according to claim 10, characterized in that, The recombinant strain is Pichia pastoris (Pichia pastoris). Pichia pastoris PX21 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231948.

13. A method for constructing a recombinant bacterium as described in any one of claims 10-12, characterized in that, The process includes the following steps: co-expressing the encoding gene of any one of claims 1-7 and the encoding gene of human 4-proline hydroxylase in the starting strain to obtain a recombinant strain that efficiently expresses recombinant humanized type III collagen with high transdermal performance; Alternatively, the recombinant vector described in claim 8 or 9 and a recombinant vector containing the encoding gene for human 4-proline hydroxylase can be co-expressed in the starting strain to obtain a recombinant strain capable of efficiently expressing recombinant humanized type III collagen with high transdermal performance.

14. The use of the encoding gene as described in any one of claims 1-7, the recombinant vector as described in claim 8 or 9, and the recombinant bacteria as described in any one of claims 10-12 in the production of recombinant humanized type III collagen with high transdermal permeability.

15. A recombinant humanized type III collagen, characterized in that, The gene encoding the recombinant humanized type III collagen is the gene encoding as described in any one of claims 1-7.

16. A method for preparing recombinant humanized type III collagen as described in claim 15, characterized in that, The method includes the following steps: performing protein separation and purification on the fermentation broth of the recombinant bacteria according to any one of claims 10-12 to obtain recombinant humanized type III collagen.

17. The application of the recombinant humanized type III collagen as described in claim 15 in the preparation of cosmetics.

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