Recombinant IV type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant IV-type collagen as well as a preparation method and application thereof, and relates to the technical field of biochemical engineering. The preparation method of the recombinant IV-type collagen comprises the following steps: obtaining a codon from amino acid of the recombinant IV-type collagen, further obtaining a target gene, and synthesizing the target gene onto an expression vector to obtain a plasmid; transferring the plasmid into an expression host, and screening a positive transformant to obtain an expression strain; and culturing the expression strain, and expressing the target protein to obtain the crude recombinant IV-type collagen. The high-purity recombinant IV-type collagen is prepared through a genetic engineering technology, and the recombinant IV-type collagen can significantly down-regulate inflammatory factors and reduce inflammation; heat injury can be reduced by up-regulating heat shock protein; after heat injury, the recombinant IV-type collagen can obviously lower metal matrix protease of heat injury cells, and also can obviously promote gene expression of type I collagen and type III collagen, 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] As an important structural protein, collagen is widely present in animal tissues. Type IV collagen is one of the main components of the basement membrane and plays an important role in maintaining the structure and function of tissues. Traditional type IV collagen is mainly extracted from animal tissues, which has problems such as limited sources, possible pathogens and large differences between batches. 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] In order 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: obtaining codons by recombining the amino acid sequence of type IV collagen SEQ ID NO: 1, and then obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid;

[0007] S2: The plasmid obtained in step S1 is transferred into an expression host, positive transformants are screened, and expression strains are obtained;

[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, inoculate it into the liquid culture medium, and culture it until OD600 = 0.6-0.8, then add the inducer, and after induction for 10-12 hours, collect the bacteria by centrifugation;

[0017] S33: placing the bacterial cells in a buffer, breaking the cells, and collecting the expressed target protein in the supernatant 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, which is prepared by the method for preparing recombinant type IV collagen as described in any one of the above items, wherein the amino acid sequence of the recombinant type IV collagen 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 protein and its preparation method and application of the present invention are: high-purity recombinant type IV collagen protein is prepared by genetic engineering technology, which is not limited by the source and does not carry pathogens. The purity of the recombinant type IV collagen protein can reach more than 80%, and the preparation results of the genetic engineering technology are stable, and the results of multiple preparations can be almost indistinguishable. The recombinant type IV collagen protein has good biocompatibility and biological activity, can improve skin elasticity and moisture content, reduce wrinkle generation, 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 metal matrix proteinases (MMP1 and MMP3) in heat-damaged cells, while 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 This is a flow chart of a method for preparing recombinant type IV collagen according to an embodiment of the present invention;

[0027] Figure 2 The 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 It is a schematic diagram showing the expression of heat shock proteins in Hacat cells after Western Blot detection of full-length type IV collagen and intervention of recombinant type IV collagen at different concentrations according to an embodiment of the present invention;

[0029] Figure 4 It 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 It 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 6It 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 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] Fig. 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] Fig.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] Fig.11 This is a schematic diagram of the down-regulation of matrix metalloproteinase MMP3 detected by q-PCR in an embodiment of the present invention after intervention with full-length type IV collagen and recombinant type IV collagen at different concentrations;

[0037] Fig.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 of recombinant type IV collagen at different concentrations in an embodiment of the present invention;

[0038] Fig.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 of recombinant type IV collagen at different concentrations in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with 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 interpreted as being limited to the embodiments described herein. On the contrary, 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 only for exemplary purposes 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 specification of the present invention 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 article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part 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 only for descriptive purposes 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, the meaning of "multiple" is two or more.

[0042] In related technologies, traditional collagen is mainly extracted from animal tissues, which has problems such as limited sources, possible pathogens, and large differences between batches. Recombinant technology can overcome these shortcomings and produce high-purity and high-activity collagen.

[0043] In today's society, people's pursuit of beauty is growing, and the beauty industry is booming. As an advanced beauty method, photoelectric beauty projects have been widely welcomed for their remarkable effects. However, while these projects bring beautiful transformation to people, they also cause a series of problems that cannot be ignored. In the process of photoelectric beauty, the application of technologies such as laser and radio frequency will inevitably cause a certain degree of thermal damage to the skin. This thermal damage manifests as symptoms such as redness, pain, dryness, and desquamation of the skin. In severe cases, it may also lead to more complicated conditions such as skin inflammation. This not only affects the durability of the beauty effect, but may also cause physical and mental discomfort to the patient, and even cause trouble to his daily life.

[0044] As the photoelectric beauty market continues to expand, the need to solve the problem of skin thermal damage after photoelectric beauty projects has become more urgent. Consumers are eager to have a safe and effective way to repair damaged skin, restore the skin's health, and ensure the long-term stability of the beauty effect. Therefore, the development of a technology or product that can specifically solve the problem of skin thermal damage after photoelectric beauty has huge market potential and social value.

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

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

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

[0048] S2: The plasmid obtained in step S1 is transferred into an expression host, positive transformants are screened, and expression strains are obtained;

[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 embodiment, high-purity recombinant type IV collagen is prepared by genetic engineering technology, which is not limited by the source and does not carry pathogens. The purity of recombinant type IV collagen can reach more than 80%, and the preparation results of genetic engineering technology are stable, and the results of multiple preparations can be almost indistinguishable. The recombinant type IV collagen has good biocompatibility and biological activity, 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 metal matrix proteinases (MMP1 and MMP3) in heat-damaged cells, while 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 the codons of the amino acid sequence SEQ ID NO: 1 are obtained, 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, which has a good separation and purification effect.

[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 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 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 will 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, inoculate it into the liquid culture medium, and culture it until OD600 = 0.6-0.8, then add the inducer, and after induction for 10-12 hours, collect the bacteria by centrifugation;

[0069] S33: placing the bacterial cells in a buffer, breaking the cells, and collecting the expressed target protein in the supernatant by centrifugation to obtain crude recombinant type IV collagen.

[0070] Specifically, in step S32, the seed solution is inoculated into the liquid culture medium at an inoculation rate of 1% by volume, 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] A recombinant type IV collagen provided in an embodiment of the present invention is prepared by the preparation method of the recombinant type IV collagen as described above, and the amino acid sequence of the recombinant type IV collagen 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 wrinkles, and delay skin aging. In addition, recombinant type IV collagen can significantly downregulate inflammatory factors IL-1α, IL-6 and PGE2, reduce inflammation; 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 metal matrix proteinases (MMP1 and MMP3) of heat-damaged cells, while also 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 wrinkles, 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 in conjunction with specific embodiments.

[0076] Embodiment 1, the preparation method of recombinant type IV collagen comprises 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. Transfer 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 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 LB liquid medium at a 1% (i.e. 1 ml) inoculation volume, and cultured at 37°C, 220 rpm in a shaking incubator until OD600 = 0.6-0.8, then the inducer IPTG was added at a final concentration of 0.15 mM, and the temperature was lowered to 28°C for induction. After induction for 11 hours, the bacteria were collected by centrifugation at 9000 rpm for 10 minutes;

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

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

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

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

[0085] Effect Example

[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. The molecular weight of the target protein is about 27kDa. The first column on the left is the marker, the second column is the recombinant type IV collagen sample after separation and purification, and the third column is the crude recombinant type IV collagen solution. The results of SDS-PAGE electrophoresis showed that the crude recombinant type IV collagen target protein accounted for more than 80%, and the purity of the recombinant type IV collagen target protein after separation and purification was about 95%.

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

[0088] Test method: Hacat cells with good growth status were digested with trypsin 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 inoculated with 2 mL of cell suspension and cultured in a 37°C, 5% CO2 incubator. The experiment set up a blank group and a sample group. The sample group was treated with 10% full-length type IV collagen and different concentrations of recombinant type IV collagen, and the cells were incubated in a 37°C, 5% CO2 incubator.

[0089] After 48 hours, discard the culture medium, wash with PBS buffer solution three times, add lysis solution containing 1% PMSF to each well, place on ice for 30 minutes, blow gently to completely remove the cells, and collect the samples. Centrifuge the samples at 4℃ high-speed centrifuge at 12000rpm for 25 minutes, and collect the supernatant.

[0090] The total protein concentration of Hacat cells was determined by 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, and then the total protein concentration was mixed with 5× loading buffer at a ratio of 4:1. The samples were boiled in 100°C boiling water for a total of 8 minutes to denature the protein. After the samples returned to room temperature, they were placed in a -20°C refrigerator for use.

[0091] The samples to be tested were placed in a 4°C refrigerator to thaw. Prepare the gel according to the preparation method of the separation gel and the concentrated gel. After solidification, add the sample, electrophoresis at a voltage of 80V for 30min, and then switch to a voltage of 120V to continue electrophoresis for 1.5h and stop. Transfer the separated gel to a polyvinylidene fluoride (PVDF) membrane. After the transfer is completed, 10% skim milk powder (TBST dissolves) is prepared, and the PVDF membrane is blocked on a shaker at room temperature for 2h. The blocked PVDF membrane is washed 2-3 times with TBST, and then the PVDF membrane is placed in the primary antibody prepared with skim milk powder and incubated in a 4°C refrigerator on a shaker overnight. Wash the PVDF membrane 3-5 times with TBST, and after washing, place the membrane in the secondary antibody prepared with skim milk powder and incubate it on a shaker at room temperature for 2h. Then repeat the above membrane washing method, prepare the developer for development, and use ImageJ to analyze the gray value of the protein band. 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] The * in the figures and tables indicates significant difference compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0095] Western Blot detection 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), EXCEL was used to analyze the data, and STDEV and T test were calculated. Full-length type IV collagen and recombinant type IV collagen upregulated HSP32, HSP47, and HSP70 to varying degrees. Recombinant type IV collagen upregulated heat shock protein in a concentration-dependent manner, and at the same concentration of 10%, recombinant type IV collagen had a better effect on heat shock protein upregulation than full-length type IV collagen.

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

[0097] Test method: Hacat cells with good growth status were digested with trypsin and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 9×10 5 / mL, 1mL of cell suspension was evenly inoculated in each well of the 24-well plate and placed in a 37°C, 5% CO2 incubator for culture. The experiment set up a blank group, a model group, a positive control group, and a sample group. The model group was induced by 13μg / mL of LPS, and the positive control group was intervened by 100μg / mL of dexamethasone. On the basis of modeling, the sample group 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 incubator.

[0098] Take the cell culture plate out of the incubator and gently aspirate the cell supernatant. To avoid aspirating cell debris, the supernatant can be transferred to a centrifuge tube first, and then centrifuged at 4°C, 1000rpm for 20 minutes for subsequent ELISA testing. Select ELISA kits for IL-1α, IL-6, and PGE2. According to the operating steps of the kit, add samples, add enzyme conjugates, incubate, wash, color, terminate the reaction readings, and draw a standard curve based on the concentration of the standard and the corresponding OD value. The concentration of inflammatory factors in the sample is calculated by 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 GraphPad was used to draw a data graph of the IL-1α concentration that was downregulated relative to the model group. Figure 7 As shown, the GraphPad data of the down-regulation of IL-6 concentration relative to the model group is drawn, as shown Figure 8 As shown, the GraphPad data of the down-regulation of PGE2 concentration relative to the model group is drawn, as shown in Fig. 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 down-regulation 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, and there is a dose-dependent relationship. The effect of 10% recombinant type IV collagen in inhibiting inflammation is better than 10% full-length type IV collagen.

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

[0104] Test method: HSF cells with good growth status were digested with trypsin and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 12×10 5 / mL, 2mL of cell suspension was evenly inoculated in each well of a 6-well plate and cultured in a 37°C, 5% CO2 incubator. The experiment set up a blank group, a model group, and a sample group. The model group was induced at 47°C for 1h. On the basis of modeling, the sample group 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 1mL Trizol on ice, lyse for 5-10min, aspirate into enzyme-free EP tube, bathe at 25℃ for 5-10min, add 200μL chloroform, mix, centrifuge at 4℃, 12000g for 15min, collect 300-400μL of upper aqueous phase (do not aspirate the middle layer) into a new EP tube. Add 500μL isopropanol, shake evenly, centrifuge at 4℃, 12000g for 10min, pour the supernatant directly without aspirating. Prepare 75% ethanol with enzyme-free water, add 1mL to the sample, mix, centrifuge at 4℃, 7000g for 5min, and discard the supernatant (air dry). Dissolve the precipitate with enzyme-free water (add 50μL if visible to the naked eye, add 30μL if invisible), synthesize cDNA, and measure the RNA concentration with an ultra-micro spectrophotometer. The concentration is greater than 20ng and the A260 / A280 value is greater than 1.8 for qualified samples. 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 different concentrations of recombinant type IV collagen

[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 reduction in MMP1 concentration relative to the model group. Fig.10 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the down-regulated MMP3 concentration in the relative model group is plotted, as shown in Figure 2 Fig.11 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the up-regulated COL1A1 concentration in the relative model group is plotted, as shown in Figure 2 Fig.12 As shown in Figure 2 (ordinate: relative protein expression), the GraphPad data of the up-regulated COL3A1 concentration in the relative model group is plotted, as shown in Figure 2 Fig.13 As shown in the figure (ordinate: relative protein expression), the specific data are shown in Table 3. After the HSF cells were modeled by thermal injury, 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, and there was a concentration-dependent relationship. 10% recombinant type IV collagen inhibited collagen degradation and promoted collagen secretion better than 10% full-length type IV collagen.

[0109] Therefore, recombinant type IV collagen can significantly downregulate 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 metal matrix proteinases (MMP1 and MMP3) of heat-damaged cells, and can also significantly promote 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 method for preparing recombinant type IV collagen, characterized in that: The following steps are involved: S1: obtaining codons by recombining the amino acid sequence of type IV collagen SEQ ID NO: 1, and then obtaining the target gene, synthesizing it into an expression vector, and obtaining a plasmid; S2: transferring the plasmid obtained in step S1 into an expression host, screening positive transformants, and obtaining an expression strain; S3: Cultivate the expression strain obtained in step S2 to express the target protein to obtain the crude recombinant type IV collagen.

2. The method for preparing recombinant type IV collagen according to claim 1, characterized in that: The method also includes step S4: isolating and purifying the target protein obtained in step S3 to obtain the recombinant type IV collagen.

3. The method for preparing recombinant type IV collagen according to claim 2, characterized in that: In the 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.

4. The method for preparing recombinant type IV collagen according to claim 1, characterized in that: In the 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.

5. The method for preparing recombinant type IV collagen according to claim 1, 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.

6. The method for preparing recombinant type IV collagen according to claim 1, 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.

7. The method for preparing recombinant type IV collagen according to claim 1, characterized in that: The step S3 comprises: 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 until 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.

8. The method for preparing recombinant type IV collagen according to claim 7, characterized in that: The inducing agent is isopropyl-β-D-thiogalactoside, lactose, galactose or methanol.

9. A recombinant type IV collagen, characterized in that: The recombinant type IV collagen is prepared by the preparation method of any one of claims 1 to 8, wherein the amino acid sequence of the recombinant type IV collagen is SVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVS RCQVCMRRT (SEQ ID NO: 1).

10. Use of the recombinant type IV collagen as claimed in claim 9 in the fields of anti-aging, anti-inflammation and protection against thermal damage to the skin.

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