Recombinant type IV collagen, preparation method and application thereof

Abstract

The present invention provides a recombinant type IV collagen protein and its preparation method and application, which relate to the field of biochemical engineering technology. The preparation method of recombinant type IV collagen protein comprises: obtaining codons through the amino acids of recombinant type IV collagen protein, and then obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid; transferring the plasmid into an expression host, screening positive transformants, and obtaining an expression strain; culturing the expression strain, expressing the target protein, and obtaining crude recombinant type IV collagen protein. High-purity recombinant type IV collagen protein is prepared by genetic engineering technology. Recombinant type IV collagen protein can significantly downregulate inflammatory factors and reduce inflammation; it can also reduce heat damage by upregulating heat shock proteins; after heat damage, recombinant type IV collagen protein can significantly downregulate metallomatrix proteases in heat-damaged cells, and can also significantly promote the expression of type I and type III collagen genes, improve skin elasticity and moisture content, and delay skin aging.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical engineering, and in particular to a recombinant type IV collagen and a preparation method and application thereof. Background Art

[0002] Collagen, a crucial structural protein, is widely present in animal tissues. Type IV collagen is a major component of the basement membrane and plays a crucial role in maintaining tissue structure and function. Traditionally, type IV collagen has been extracted primarily from animal tissue, which presents challenges such as limited sources, potential pathogens, and significant batch-to-batch variability. Summary of the Invention

[0003] The problem solved by the present invention is how to solve the problems of limited sources of extracted type IV collagen, possible pathogens and large differences between batches.

[0004] To solve the above problems, the present invention provides a recombinant type IV collagen and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a method for preparing recombinant type IV collagen, comprising the following steps:

[0006] S1: The codons are obtained by recombining the amino acid sequence of type IV collagen SEQ ID NO: 1, thereby obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid;

[0007] S2: The plasmid obtained in step S1 is transformed into an expression host, and positive transformants are screened to obtain an expression strain;

[0008] S3: Cultivate the expression strain obtained in step S2 to express the target protein to obtain crude recombinant type IV collagen.

[0009] Optionally, the method further comprises step S4: isolating and purifying the target protein obtained in step S3 to obtain recombinant type IV collagen.

[0010] Optionally, in step S4, the target protein obtained in step S3 is separated and purified by one or a combination of salting out, ultrafiltration, chromatography, isoelectric precipitation and membrane separation.

[0011] Optionally, in step S1, the amino acid sequence SEQ ID NO: 1 is optimized according to the codon preference of Escherichia coli and then amplified by PCR or whole gene synthesis to obtain the target gene.

[0012] Optionally, in step S1, the target gene is synthesized into the pKK223-3 expression vector to obtain the plasmid pKK223-3-IVNC1.

[0013] Optionally, in step S2, the expression host is a bacterial host Escherichia coli DE3 competent cell, Bacillus subtilis, Bacillus licheniformis or a eukaryotic host Pichia pastoris, Saccharomyces cerevisiae, animal cell, or plant cell.

[0014] Optionally, step S3 includes:

[0015] S31: inoculating the expression strain obtained in step S2 into a liquid culture medium supplemented with ampicillin antibiotic to obtain a seed solution;

[0016] S32: Take part of the seed liquid and inoculate it into liquid culture medium. When OD600 is 0.6-0.8, add inducer. After induction for 10-12 hours, collect the bacteria by centrifugation.

[0017] S33: The bacteria are placed in a buffer solution, the cells are disrupted, and the expressed target protein in the supernatant is collected by centrifugation to obtain crude recombinant type IV collagen.

[0018] Optionally, the inducing agent is isopropyl-β-D-thiogalactoside, lactose, galactose or methanol.

[0019] In a second aspect, the present invention provides a recombinant type IV collagen protein, which is prepared by the preparation method of recombinant type IV collagen protein as described in any one of the above items, wherein the amino acid sequence of the recombinant type IV collagen protein is

[0020] SVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAG

[0021] SCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRP

[0022] FISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGS

[0023] GQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFWLATIERSEMFKKPTPSTL KAGELRTHVSRCQVCMRRT (SEQ ID NO: 1).

[0024] In a third aspect, the present invention provides a use of the recombinant type IV collagen as described above in the fields of anti-aging, anti-inflammation and protection against thermal damage to the skin.

[0025] The beneficial effects of the recombinant type IV collagen, its preparation method, and its application are as follows: high-purity recombinant type IV collagen is produced through genetic engineering technology, is not restricted by its source, and is pathogen-free. The purity of the recombinant type IV collagen can reach over 80%. The genetic engineering technology produces stable results, with virtually no difference in the results of multiple preparations. The recombinant type IV collagen has excellent biocompatibility and biological activity, and can improve skin elasticity and moisture content, reduce wrinkles, and delay skin aging. In addition, recombinant type IV collagen can significantly downregulate inflammatory factors IL-1α, IL-6 and PGE2, reducing inflammation; it can also reduce thermal damage by upregulating heat shock proteins HSP32, HSP47 and HSP70; and after thermal damage, recombinant type IV collagen can significantly downregulate the metallomatrix proteinases (MMP1 and MMP3) of heat-damaged cells, while also significantly promoting the gene expression of type I collagen COL1A1 and type III collagen COL3A1. It can be seen that after thermal damage, recombinant type IV collagen can also inhibit collagen degradation and promote collagen secretion, improve skin elasticity and moisture content, reduce wrinkles, and delay skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flowchart of the method for preparing recombinant type IV collagen according to an embodiment of the present invention;

[0027] Figure 2 This is an SDS-PAGE electrophoresis diagram of the crude recombinant type IV collagen solution, the purified recombinant type IV collagen solution, and the marker in Example 1 of the present invention;

[0028] Figure 3 Schematic diagram showing Western Blot detection of heat shock protein expression in Hacat cells after intervention with full-length type IV collagen and recombinant type IV collagen at different concentrations according to an embodiment of the present invention;

[0029] Figure 4 This is a schematic diagram showing the up-regulation of heat shock protein HSP32 detected by Western Blot after intervention with full-length type IV collagen and recombinant type IV collagen at different concentrations according to an embodiment of the present invention;

[0030] Figure 5 This is a schematic diagram showing the up-regulation of heat shock protein HSP47 detected by Western Blot after intervention with full-length type IV collagen and recombinant type IV collagen at different concentrations according to an embodiment of the present invention;

[0031] Figure 6This is a schematic diagram showing the up-regulation of heat shock protein HSP70 detected by Western Blot after intervention with full-length type IV collagen and recombinant type IV collagen at different concentrations according to an embodiment of the present invention;

[0032] Figure 7 This is a schematic diagram of the down-regulation of the inflammatory factor IL-1α under the intervention of dexamethasone, full-length type IV collagen and recombinant type IV collagen at different concentrations detected by ELISA in an embodiment of the present invention;

[0033] Figure 8 This is a schematic diagram of the down-regulation of the inflammatory factor IL-6 detected by ELISA in an embodiment of the present invention under the intervention of dexamethasone, full-length type IV collagen and recombinant type IV collagen at different concentrations;

[0034] Figure 9 This is a schematic diagram of the down-regulation of the inflammatory factor PGE2 under the intervention of dexamethasone, full-length type IV collagen and recombinant type IV collagen at different concentrations detected by ELISA in an embodiment of the present invention;

[0035] Figure 10 This is a schematic diagram of the down-regulation of matrix metalloproteinase MMP1 after q-PCR detection of full-length type IV collagen and different concentrations of recombinant type IV collagen intervention in an embodiment of the present invention;

[0036] Figure 11 This is a schematic diagram of the down-regulation of matrix metalloproteinase MMP3 after q-PCR detection of full-length type IV collagen and different concentrations of recombinant type IV collagen intervention in an embodiment of the present invention;

[0037] Figure 12 This is a schematic diagram of the up-regulation of type I collagen gene expression after q-PCR detection of full-length type IV collagen and intervention with different concentrations of recombinant type IV collagen in an embodiment of the present invention;

[0038] Figure 13 This is a schematic diagram of the up-regulation of type III collagen gene expression after q-PCR detection of full-length type IV collagen and intervention with different concentrations of recombinant type IV collagen in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be construed as being limited to the embodiments described herein. Instead, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0040] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the present invention description are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0041] The term "including" and its variations used in this document are open inclusions, that is, "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "multiple" means two or more.

[0042] Traditional collagen production is primarily derived from animal tissue, which presents challenges such as limited sources, potential pathogens, and large batch-to-batch variability. Recombinant technology can overcome these shortcomings and produce high-purity, highly active collagen.

[0043] In today's society, people's pursuit of beauty is ever-increasing, and the beauty industry is booming. As an advanced cosmetic procedure, photoelectric cosmetic treatments have gained widespread popularity due to their remarkable results. However, while these procedures offer a beautiful transformation, they also raise a series of significant challenges. The use of technologies such as lasers and radiofrequency during photoelectric cosmetic procedures inevitably causes a degree of thermal damage to the skin. This thermal damage manifests as symptoms such as redness, pain, dryness, and scaling. In severe cases, it can lead to more complex conditions such as skin inflammation. This not only affects the long-term effectiveness of cosmetic treatments but can also cause physical and mental discomfort to patients, even disrupting their daily lives.

[0044] As the photoelectric beauty market continues to expand, the need to address the issue of thermal damage to the skin following photoelectric beauty treatments is becoming increasingly urgent. Consumers crave safe and effective methods to repair damaged skin, restore its health, and ensure long-term, stable results. Therefore, developing technologies or products that can specifically address this issue holds enormous market potential and social value.

[0045] In response to the problems existing in the above-mentioned related technologies, embodiments of the present invention provide a recombinant type IV collagen and a preparation method and application thereof.

[0046] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing recombinant type IV collagen, comprising the following steps:

[0047] S1: The codons are obtained by recombining the amino acid sequence of type IV collagen SEQ ID NO: 1, thereby obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid;

[0048] S2: The plasmid obtained in step S1 is transformed into an expression host, and positive transformants are screened to obtain an expression strain;

[0049] S3: Cultivate the expression strain obtained in step S2 to express the target protein to obtain crude recombinant type IV collagen.

[0050] In this example, high-purity recombinant type IV collagen was produced through genetic engineering techniques. This method is not restricted by its source and is pathogen-free. The purity of the recombinant type IV collagen can reach over 80%. The genetic engineering technique produces stable results, with virtually no discrepancies between multiple preparations. This recombinant type IV collagen exhibits excellent biocompatibility and biological activity, improving skin elasticity and moisture content, reducing wrinkles, and delaying skin aging. In addition, recombinant type IV collagen can significantly downregulate inflammatory factors IL-1α, IL-6 and PGE2, reducing inflammation; it can also reduce thermal damage by upregulating heat shock proteins HSP32, HSP47 and HSP70; and after thermal damage, recombinant type IV collagen can significantly downregulate the metallomatrix proteinases (MMP1 and MMP3) of heat-damaged cells, while also significantly promoting the gene expression of type I collagen COL1A1 and type III collagen COL3A1. It can be seen that after thermal damage, recombinant type IV collagen can also inhibit collagen degradation and promote collagen secretion, improve skin elasticity and moisture content, reduce wrinkles, and delay skin aging.

[0051] Specifically, after obtaining the codons of the amino acid sequence SEQ ID NO: 1, the target gene is obtained by PCR amplification or whole gene synthesis.

[0052] Optionally, the method further comprises step S4: isolating and purifying the target protein obtained in step S3 to obtain recombinant type IV collagen.

[0053] In this optional embodiment, the protein purity of the recombinant type IV collagen obtained after separation and purification can reach about 95%, which is high in purity.

[0054] Optionally, in step S4, the target protein obtained in step S3 is separated and purified by one or a combination of salting out, ultrafiltration, chromatography, isoelectric precipitation and membrane separation.

[0055] In this optional embodiment, the combination of chromatography and membrane separation is preferred, and the combination of ion exchange chromatography and membrane separation is more preferred, as the separation and purification effect is good.

[0056] Specifically, a combination of salting out, ion exchange chromatography and membrane separation is used to separate and purify the target protein, specifically including:

[0057] 1. Add NaCl solution to the target protein obtained in step S3, stir evenly and let it stand for more than 2 hours, collect the supernatant by centrifugation, and concentrate and desalt with an ultrafiltration membrane;

[0058] 2. Adjust the pH to 6-7 and elute with a cation exchange lipid;

[0059] 3. Concentrate and desalt again through an ultrafiltration membrane to obtain the purified recombinant type IV collagen.

[0060] Optionally, the ultrafiltration membrane uses a 3kd ultrafiltration membrane with a pore size of 3 kilodaltons. This pore size can filter out large molecular organic matter, microorganisms and suspended particles in the water.

[0061] Optionally, in step S1, the amino acid sequence SEQ ID NO: 1 is optimized according to the codon preference of Escherichia coli and then amplified by PCR or whole gene synthesis to obtain the target gene.

[0062] In this optional embodiment, protein expression can be increased after codon optimization.

[0063] Optionally, in step S1, the target gene is synthesized into the pKK223-3 expression vector to obtain the plasmid pKK223-3-IVNC1.

[0064] Specifically, the target gene is located in the NC1 region of the α1 chain of type IV collagen.

[0065] Optionally, in step S2, the expression host is a bacterial host Escherichia coli DE3 competent cell, Bacillus subtilis, Bacillus licheniformis or a eukaryotic host Pichia pastoris, Saccharomyces cerevisiae, animal cell, or plant cell.

[0066] Optionally, step S3 includes:

[0067] S31: inoculating the expression strain obtained in step S2 into a liquid culture medium supplemented with ampicillin antibiotic to obtain a seed solution;

[0068] S32: Take part of the seed liquid and inoculate it into liquid culture medium. When OD600 is 0.6-0.8, add inducer. After induction for 10-12 hours, collect the bacteria by centrifugation.

[0069] S33: The bacteria are placed in a buffer solution, the cells are disrupted, and the expressed target protein in the supernatant is collected by centrifugation to obtain crude recombinant type IV collagen.

[0070] Specifically, in step S32 , the seed liquid is inoculated into the liquid culture medium at an inoculum volume ratio of 1%, and cultured on a shaker at 37° C. and 220 rpm.

[0071] Optionally, the inducing agent is isopropyl-beta-D-thiogalactopyranoside (IPTG), lactose, galactose or methanol.

[0072] An embodiment of the present invention provides a recombinant type IV collagen protein, which is prepared by the preparation method of recombinant type IV collagen protein as described above. The amino acid sequence of the recombinant type IV collagen protein is SVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVS RCQVCMRRT (SEQ ID NO: 1).

[0073] In this embodiment, recombinant type IV collagen has good biocompatibility and biological activity, can improve skin elasticity and moisture content, reduce wrinkle formation, and delay skin aging. In addition, recombinant type IV collagen can significantly downregulate inflammatory factors IL-1α, IL-6, and PGE2, reducing inflammation; it can also reduce thermal damage by upregulating heat shock proteins HSP32, HSP47, and HSP70; and after thermal damage, recombinant type IV collagen can significantly downregulate metallomatrix proteinases (MMP1 and MMP3) in heat-damaged cells while significantly promoting type I collagen COL1A1 and type III collagen COL3A1. It can be seen that after thermal damage, recombinant type IV collagen can also inhibit collagen degradation and promote collagen secretion, improve skin elasticity and moisture content, reduce wrinkle formation, and delay skin aging.

[0074] An embodiment of the present invention provides an application of the recombinant type IV collagen as described above in the fields of anti-aging, anti-inflammation and protection against thermal damage to the skin.

[0075] The present invention is further described below with reference to specific embodiments.

[0076] Example 1, a method for preparing recombinant type IV collagen, specifically comprising the following steps:

[0077] 1. The amino acid sequence of recombinant type IV collagen SEQ ID NO: 1 was optimized according to the codon preference of Escherichia coli to obtain the target gene, which was synthesized into the pKK223-3 expression vector by whole gene synthesis to obtain the pKK223-3-IVNC1 plasmid.

[0078] 2. Transform pKK223-3-IVNC1 into DE3 competent cells by heat transformation, spread on resistance plates, screen positive transformants, and pick single colonies on the plates to obtain expression strains.

[0079] 3. Pick a single colony that has been successfully constructed by colony PCR verification, inoculate it into a test tube containing 3.0 ml of LB medium (with ampicillin antibiotics), and culture it at 37°C for 11 hours to obtain seed liquid;

[0080] The cultured seed liquid was inoculated into 100 ml of LB liquid medium at a 1% inoculum volume (i.e., 1 ml). The culture was shaken at 37°C and 220 rpm until OD600 = 0.6-0.8. Then, IPTG was added at a final concentration of 0.15 mM. The temperature was lowered to 28°C for induction. After induction for 11 hours, the cells were centrifuged at 9000 rpm for 10 minutes to collect the cells.

[0081] The bacteria were prepared into a 10% (g wet bacteria / mL PBS) suspension in PBS buffer at pH 6.0, homogenized under high pressure at 800 bar for 3 minutes to break the cells, and centrifuged at 9000 rpm for 10 minutes to collect the supernatant, which was the crude recombinant type IV collagen solution.

[0082] 4. Add NaCl to a final concentration of 60% to the crude recombinant type IV collagen supernatant collected by centrifugation, stir to dissolve, and then stand at room temperature for 2 hours. Centrifuge at 9000 rpm for 10 minutes, then collect the supernatant. Concentrate and desalt the collected supernatant through a 3KD ultrafiltration membrane;

[0083] The concentrated and desalted protein supernatant was adjusted to pH 6.5 with phosphoric acid, loaded onto a cation exchange resin, and eluted with 0.5 M NaCl to obtain the target protein;

[0084] The collected target protein solution is desalted by a 3KD ultrafiltration membrane to obtain the purified recombinant type IV collagen solution.

[0085] Effect embodiment

[0086] 1. The crude recombinant type IV collagen solution and the purified recombinant type IV collagen solution prepared in Example 1 were subjected to SDS-PAGE electrophoresis test. The test results are as follows: Figure 2 As shown in the figure, the target protein has a molecular weight of approximately 27 kDa. The first column on the left is the marker, the second column is the isolated and purified recombinant type IV collagen sample, and the third column is the crude recombinant type IV collagen solution. SDS-PAGE electrophoresis results show that the crude recombinant type IV collagen contains over 80% of the target protein, and the purity of the isolated and purified recombinant type IV collagen is approximately 95%.

[0087] 2. Test the effect of recombinant type IV collagen at different concentrations on heat shock proteins.

[0088] Test method: Hacat cells in good growth condition were trypsinized and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 2×10 5 Each well of a six-well plate was evenly seeded with 2 mL of cell suspension and incubated at 37°C in a 5% CO2 incubator. The experiment included a blank control group, a sample control group, and a sample control group. The cells were treated with 10% full-length type IV collagen and various concentrations of recombinant type IV collagen and incubated at 37°C in a 5% CO2 incubator.

[0089] After 48 hours, discard the culture medium and wash three times with PBS buffer. Add lysis buffer containing 1% PMSF to each well and incubate on ice for 30 minutes. Gently pipette to completely detach the cells and collect the samples. Centrifuge the samples at 12,000 rpm in a high-speed centrifuge at 4°C for 25 minutes, and collect the supernatant.

[0090] The total protein concentration of Hacat cells was determined using a BCA detection kit. The absorbance was detected at 562 nm and the protein concentration was calculated. The total protein concentration of each sample was adjusted to the same concentration using lysis buffer. The total protein concentration was mixed with 5× loading buffer at a ratio of 4:1 and boiled in 100°C boiling water for 8 minutes to denature the protein. After the sample returned to room temperature, it was placed in a -20°C refrigerator for use.

[0091] Thaw the sample to be tested in a 4°C refrigerator. Prepare the gel according to the preparation method of the separation gel and the stacking gel. After solidification, add the sample and perform electrophoresis at a voltage of 80V for 30 minutes. Then switch to a voltage of 120V and continue electrophoresis for 1.5 hours before stopping. Transfer the separated gel to a polyvinylidene fluoride (PVDF) membrane. After the transfer is completed, prepare 10% skim milk powder (dissolved in TBST) and block the PVDF membrane on a shaker at room temperature for 2 hours. Wash the blocked PVDF membrane with TBST 2-3 times, then place the PVDF membrane in the primary antibody prepared with skim milk powder and incubate it in a shaker at 4°C overnight. Wash the PVDF membrane 3-5 times with TBST, then place the membrane in the secondary antibody prepared with skim milk powder and incubate it on a shaker at room temperature for 2 hours. Then repeat the above membrane washing method, prepare the developer for development, and use ImageJ to analyze the grayscale value of the protein bands. The experimental results are shown in Table 1.

[0092] Table 1 Effects of full-length type IV collagen and recombinant type IV collagen at different concentrations on heat shock proteins

[0093]

[0094] * in the figures and tables indicates significant differences compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0095] Western Blot analysis of the effects of full-length type IV collagen and recombinant type IV collagen at different concentrations on heat shock proteins. Figure 3 As shown, the normal group was not affected by type IV collagen. The protein bands were analyzed by ImageJ software and the data were plotted using GraphPad. The effects on heat shock protein HSP32 are shown in Figure 4 As shown in (ordinate: relative protein expression), the effect on heat shock protein HSP47 is as follows Figure 5 As shown in (ordinate: relative protein expression), the effect on heat shock protein HSP70 is as follows Figure 6 As shown (ordinate: relative protein expression), data were analyzed using Excel, with STDEV and T-test calculations. Both full-length and recombinant type IV collagen upregulated HSP32, HSP47, and HSP70 to varying degrees. Recombinant type IV collagen upregulated heat shock proteins in a concentration-dependent manner, and at a similar concentration of 10%, recombinant type IV collagen was more effective than full-length type IV collagen in upregulating heat shock proteins.

[0096] 3. Test the inhibitory effect of recombinant type IV collagen at different concentrations on inflammatory factors.

[0097] Test method: Hacat cells in good growth condition were trypsinized and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 9×10 5 Each well of a 24-well plate was evenly inoculated with 1 mL of cell suspension and cultured in a 37°C, 5% CO2 incubator. The experiment set up blank, model, positive control, and sample groups. The model group was induced with 13 μg / mL of LPS, and the positive control group was treated with 100 μg / mL of dexamethasone. The sample group, based on the modeling, was treated with 10% full-length type IV collagen and different concentrations of recombinant type IV collagen, and the cells were incubated at 37°C, 5% CO2 in a 5% CO2 incubator.

[0098] Remove the cell culture plate from the incubator and gently aspirate the cell supernatant. To avoid aspirating cell debris, transfer the supernatant to a centrifuge tube and then centrifuge at 4°C, 1000 rpm for 20 minutes for subsequent ELISA testing. Select ELISA kits for IL-1α, IL-6, and PGE2. Follow the kit's operating procedures for sample addition, enzyme conjugate addition, incubation, washing, color development, and reaction termination. Draw a standard curve based on the concentration of the standard and the corresponding OD value. Calculate the concentration of inflammatory factors in the sample using the standard curve. The results are shown in Table 2.

[0099] Table 2 Concentrations of inflammatory factors under the intervention of dexamethasone, full-length type IV collagen and different concentrations of recombinant type IV collagen

[0100]

[0101]

[0102] Each group in the experiment had three parallel samples. The data were analyzed using EXCEL, and the data were plotted using GraphPad to show the decrease in IL-1α concentration relative to the model group. Figure 7 As shown, the GraphPad data of the IL-6 concentration reduction in the model group was plotted, as shown in Figure 8 As shown, the GraphPad data of the down-regulation of PGE2 concentration relative to the model group was drawn, as shown in Figure 9As shown in the figure, # indicates that the model group has a significant difference compared with the normal group, ###p<0.001; * indicates that there is a significant difference compared with the model group, *p<0.05, **p<0.01, ***p<0.001. The specific downregulation data are shown in Table 2. After Hacat cells were modeled with LPS, the inflammatory factors IL-1α, IL-6, and PGE2 in the model group were significantly upregulated compared with the normal group. It can be seen that different concentrations of recombinant type IV collagen can significantly downregulate these three inflammatory factors in a dose-dependent manner. The inhibitory effect of 10% recombinant type IV collagen is better than 10% full-length type IV collagen.

[0103] 4. Test the effects of recombinant type IV collagen at different concentrations on metallomatrix proteinases (MMP1 and MMP3) and type I and type III collagen in heat-damaged cells.

[0104] Test method: HSF cells in good growth condition were trypsinized and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 12×10 5 Cells were plated evenly with 2 mL of cell suspension per well of a 6-well plate and cultured in a 37°C, 5% CO2 incubator. The experiment included three groups: a blank group, a model group, and a sample group. The model group was induced at 47°C for 1 hour. The sample group, based on the modeling process, was treated with 10% full-length type IV collagen and different concentrations of recombinant type IV collagen, and incubated in a 37°C, 5% CO2 incubator.

[0105] Wash twice with PBS buffer, add 1 mL of Trizol on ice, lyse for 5-10 minutes, transfer to an enzyme-free EP tube, incubate at 25°C for 5-10 minutes, add 200 μL of chloroform, mix thoroughly, centrifuge at 12,000 g for 15 minutes at 4°C, collect 300-400 μL of the upper aqueous phase (do not aspirate the middle phase) and transfer to a new EP tube. Add 500 μL of isopropanol, shake well, centrifuge at 12,000 g for 10 minutes at 4°C, and pour the supernatant directly without aspirating. Prepare 75% ethanol in enzyme-free water, add 1 mL to the sample, mix thoroughly, centrifuge at 7,000 g for 5 minutes at 4°C, and discard the supernatant (air-dry). Dissolve the precipitate in enzyme-free water (add 50 μL if visible, 30 μL if invisible), synthesize cDNA, and measure RNA concentration using an ultramicrospectrophotometer. Samples are considered qualified if their concentration is greater than 20 ng and their A260 / A280 ratio is greater than 1.8. Fluorescence quantitative PCR was used to detect MMP1 (matrix metalloproteinase 1), MMP3 (matrix metalloproteinase 3), COL1A1 (type I collagen), and COL3A1 (type III collagen). The experimental results are shown in Table 3.

[0106] Table 3 Concentrations of MMP1, MMP3, type I collagen, and type III collagen under the intervention of full-length type IV collagen and recombinant type IV collagen at different concentrations

[0107]

[0108] Each group in the experiment had three parallel samples. The data were analyzed using EXCEL, and the data were plotted using GraphPad to show the MMP1 concentrations were down-regulated relative to the model group. Figure 10 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the model group down-regulating the MMP3 concentration is plotted, as shown in Figure 2 (ordinate: relative protein expression). Figure 11 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the up-regulated COL1A1 concentration in the model group was plotted, as shown in Figure 2 (ordinate: relative protein expression). Figure 12 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the up-regulated COL3A1 concentration in the model group was plotted, as shown in Figure 2 (ordinate: relative protein expression). Figure 13 As shown in the figure (ordinate: relative protein expression), the specific data are shown in Table 3. After HSF cells were thermally damaged, matrix metalloproteinases 1 and 3 were significantly upregulated, and type I and type III collagens were significantly downregulated. Compared with the model group, full-length type IV collagen significantly downregulated MMP1 and MMP3, and significantly promoted COL1A1 and COL3A1. Recombinant IV collagen significantly downregulated MMP1 and MMP3, and significantly promoted COL1A1 and COL3A1 in a concentration-dependent manner. 10% recombinant type IV collagen was superior to 10% full-length type IV collagen in inhibiting collagen degradation and promoting collagen secretion.

[0109] Therefore, recombinant type IV collagen can significantly downregulate the inflammatory factors IL-1α, IL-6 and PGE2, reduce inflammation, and has a concentration-dependent relationship; it can also reduce thermal damage by upregulating heat shock proteins HSP32, HSP47 and HSP70; and after thermal damage, recombinant type IV collagen can significantly downregulate the metallomatrix proteinases (MMP1 and MMP3) of heat-damaged cells, while also significantly promoting the gene expression of type I collagen COL1A1 and type III collagen COL3A1. It can be seen that after thermal damage, recombinant type IV collagen can also inhibit collagen degradation and promote collagen secretion, improve skin elasticity and moisture content, reduce wrinkles, and delay skin aging.

[0110] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A use of recombinant type IV collagen in the preparation of a drug for protecting against skin thermal damage, characterized in that: The amino acid sequence of the recombinant type IV collagen is SVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVS RCQVCMRRT (SEQ ID NO: 1).

2. Use of the recombinant type IV collagen according to claim 1 in preparing a drug for protecting against skin thermal damage, characterized in that: The recombinant type IV collagen is prepared by the following steps: S1: The codons are obtained by recombining the amino acid sequence of type IV collagen SEQ ID NO: 1, thereby obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid; S2: Transform the plasmid obtained in step S1 into an expression host, screen positive transformants, and obtain an expression strain; S3: culturing the expression strain obtained in step S2 to express the target protein to obtain the crude recombinant type IV collagen; comprising: S31: inoculating the expression strain obtained in step S2 into a liquid culture medium supplemented with ampicillin antibiotic to obtain a seed solution; S32: taking a portion of the seed solution, inoculating it into a liquid culture medium, culturing it to OD600 = 0.6-0.8, adding an inducer, inducing it for 10-12 hours, and collecting the bacteria by centrifugation; S33: placing the bacterial cells in a buffer, disrupting the cells, and collecting the expressed target protein in the supernatant by centrifugation to obtain the crude recombinant type IV collagen; S4: Separating and purifying the target protein obtained in step S3 by a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation to obtain the recombinant type IV collagen.

3. Use of the recombinant type IV collagen according to claim 2 in preparing a drug for protecting against skin thermal damage, characterized in that: In step S1, the amino acid sequence SEQ ID NO: 1 is optimized according to the codon preference of Escherichia coli and then amplified by PCR or whole gene synthesis to obtain the target gene.

4. Use of the recombinant type IV collagen according to claim 2 in preparing a drug for protecting against skin thermal damage, characterized in that: In the step S1, the target gene is synthesized into the pKK223-3 expression vector to obtain the plasmid pKK223-3-IVNC1.

5. Use of the recombinant type IV collagen according to claim 2 in preparing a drug for protecting against skin thermal damage, characterized in that: In step S2, the expression host is a bacterial host such as Escherichia coli DE3 competent cells, Bacillus subtilis, Bacillus licheniformis, or a eukaryotic host such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, or plant cells.

6. Use of the recombinant type IV collagen according to claim 2 in preparing a drug for protecting against skin thermal damage, characterized in that: The inducer is isopropyl-β-D-thiogalactoside, lactose, galactose or methanol.

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