Recombinant IV type collagen with anti-inflammatory activity as well as preparation method and application thereof

Abstract

The invention discloses a recombinant IV-type collagen with anti-inflammatory activity and a preparation method and application thereof, the recombinant IV-type collagen is a truncated protein of natural human IV-type collagen, and the amino acid sequence of the recombinant IV-type collagen is shown as SEQ ID NO: 1. The recombinant IV-type collagen has a remarkable anti-inflammatory effect, can regulate inflammatory factors, relieve and improve inflammatory symptoms, has anti-inflammatory and soothing effects, and can be used as a biomedical material to be applied to preparation of skin care products, medicines or medical instruments with anti-inflammatory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant type IV collagen with anti-inflammatory activity and a preparation method and application thereof. Background Art

[0002] Inflammation is a protective mechanism that aims to control harmful factors and remove damaged tissues, which helps the body recover. However, inflammatory reactions are often accompanied by an increase in reactive oxygen and proinflammatory cytokines. If they accumulate excessively in tissues, they may cause persistent inflammation and hinder tissue regeneration. Although inflammation is an early immune response to trauma, excessive reactions can lead to a variety of diseases. Typical inflammatory reactions of the skin are manifested as redness, swelling, heat, and pain: redness and heat are caused by capillary dilation, while swelling and pain are caused by congestion and nerve compression caused by inflammatory substances. Inflammation weakens the skin barrier, making it more susceptible to external stimuli, forming a vicious cycle, and aggravating sensitivity and inflammation. Therefore, controlling the activity of inflammatory factors is crucial to maintaining skin health. Moreover, traditional anti-inflammatory products mostly add hormones to inhibit inflammation, and long-term use can also damage the skin barrier and damage the skin.

[0003] Collagen is one of the most abundant proteins in the human body and the main component of the extracellular matrix (ECM). It plays a vital role in maintaining the normal physiological functions of cells, tissues and organs and repairing damage. In recent years, collagen has become a safe and widely used biomedical material in the fields of medicine, health products and cosmetics due to its good biocompatibility and low antigenicity. So far, there are as many as 29 known types of collagen, which are distributed in different tissues and organs and have their own unique biological functions.

[0004] Among them, type IV collagen is mainly distributed in the basement membrane. It is a collagen with a non-fibrous reticular triple helix structure, which can play a key role in the support and stability of the basement membrane structure. The basement membrane is one of the core components of the extracellular matrix and plays an important regulatory role in cell growth, differentiation, migration and other processes. Type IV collagen is the main component of the basement membrane to maintain its structural and functional integrity. Studies have shown that the lack or abnormality of type IV collagen can lead to the disorder of the basement membrane structure, thereby affecting the normal physiological function of cells and may even cause a series of diseases. Therefore, type IV collagen has extremely high application value in maintaining the stability of the basement membrane structure, tissue repair and biocompatibility. However, despite the outstanding performance of type IV collagen in supporting tissue structure and maintaining the stability of the basement membrane, its anti-inflammatory activity has not been reported and widely used.

[0005] Traditional collagen is mainly extracted from animal connective tissue by acid and alkali hydrolysis. It is inevitable that some biological activity will be lost during the extraction. If it is applied to the human body, it will produce a xenobiotic rejection reaction and there is a great risk of viruses. In addition, due to the extremely low content of type IV collagen in animals, traditional extraction methods are difficult to meet the needs of large-scale production. Therefore, recombinant expression of type IV collagen through genetic engineering technology has become a better choice. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant type IV collagen with anti-inflammatory activity and its preparation method and application in view of the deficiencies of the above-mentioned prior art. The recombinant type IV collagen of the present invention has a significant anti-inflammatory effect, can regulate inflammatory factors, alleviate and improve inflammatory symptoms, and play an anti-inflammatory and soothing role.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is: a recombinant type IV collagen with anti-inflammatory activity, characterized in that the recombinant type IV collagen is a truncated protein of natural human type IV collagen, and its amino acid sequence is shown in SEQ ID NO:1.

[0008] Furthermore, the present invention provides a nucleic acid encoding the above-mentioned recombinant type IV collagen, characterized in that the nucleotide sequence of the nucleic acid is shown in SEQ ID NO:2.

[0009] Furthermore, the present invention provides an expression vector comprising the above-mentioned nucleic acid, characterized in that the expression vector is pPICZαA.

[0010] Furthermore, the present invention provides a host cell into which the above-mentioned expression vector is introduced, characterized in that the host cell is Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Pichia pastoris, Saccharomyces cerevisiae, animal cells or plant cells.

[0011] The above-mentioned host cell is characterized in that the host cell is Escherichia coli or Pichia pastoris, preferably Pichia pastoris.

[0012] Furthermore, the present invention provides a method for producing the above-mentioned recombinant type IV collagen, characterized in that it includes culturing host cells to secrete the recombinant type IV collagen into a culture medium, and separating and purifying the protein in the culture medium to obtain the recombinant type IV collagen.

[0013] The above method is characterized in that the separation and purification adopts one or a combination of salting out, acid-base precipitation, chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography and membrane separation.

[0014] Furthermore, the present invention provides a use of the above-mentioned recombinant type IV collagen in the preparation of skin care products, health foods, tissue engineering materials or medical devices.

[0015] Furthermore, the present invention provides a use of the recombinant type IV collagen as described above as a biomaterial in the preparation of skin care products, medicines or medical device products with anti-inflammatory activity.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] 1. The recombinant type IV collagen constructed by the present invention is a truncated protein of natural human type IV collagen. The amino acid sequence of the obtained recombinant type IV collagen has a typical GXY triplet structure, which is 100% consistent with the sequence of human type IV collagen. It will not produce an immune response when applied to the human body and has good safety. In addition, the present invention avoids the cost and risk of exogenous protein residue caused by the use of protease cleavage in vitro, and can also shorten the time and cost of the subsequent purification process.

[0018] 2. The host cell of the present invention is preferably Pichia pastoris, which can secrete proteins for extracellular expression, and can effectively avoid problems such as impurities caused by lysing bacteria when extracting products. As a eukaryotic organism, it can perform post-translational modifications on the secreted recombinant proteins, such as glycosylation and phosphorylation, and the produced recombinant proteins do not contain pathogens, viral inclusion bodies or heat sources, have high safety, and low fermentation costs.

[0019] 3. Experimental verification shows that the recombinant type IV collagen of the present invention has a significant anti-inflammatory effect, can regulate inflammatory factors, reduce and improve inflammatory symptoms, and play an anti-inflammatory and soothing role. It can be used as a biomedical material to prepare skin care products, medicines or medical device products with anti-inflammatory activity.

[0020] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis diagram of the rhColIV-P protein expression in shake flask fermentation in Example 1 of the present invention.

[0022] Figure 2 This is the electrophoresis diagram of protein expression of rhColIV-P protein.

[0023] Figure 3 This is the protein electrophoresis diagram of the purified rhColIV-P protein.

[0024] Figure 4 These are the results of the rhColIV-P protein inhibition test on cell proliferation.

[0025] Figure 5 To detect the TNF-α content in the supernatant of LPS-induced macrophage model cells.

[0026] Figure 6 To detect the IL-6 content in the supernatant of LPS-induced macrophage model cells.

[0027] Figure 7 To detect the TGF-β1 content in the supernatant of LPS-induced macrophage model cells.

[0028] Figure 8 To detect the IL-10 content in the supernatant of LPS-induced macrophage model cells. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] Example 1: Construction and expression of recombinant type IV collagen (rhColIV-P) engineering strain 1. Construction of recombinant plasmid

[0031] According to the amino acid sequence shown in SEQ ID NO: 1, the codon optimization of the yeast expression system was performed to obtain the target gene sequence (SEQ ID NO: 2). The obtained target gene sequence was commissioned to BGI for gene synthesis, and the synthesized gene was ligated into the pPICZαA plasmid to obtain the pPICZαA-rhColIV plasmid.

[0032] SEQ ID NO:1

[0033] GYQGPDGPRGPKGEAGDPGPPGLPAYSPHPSLAKGARGDPGFPGAQGEPGSQGEPGDPGLPGPPGLSIGDGDQRRGLPGEMGPKGFIGDPGIPALYGGPPGPDGKRGPPGPPGLPGPPGPDGFLFGLKGAKGRAGFPGLP GSPGARGPKGWKGDAGECRCTEGDEAIKGLPGLPPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKGVPGNIGAPGPKGAKGDSRTITTKGERGQPGVPGVPGMKGDDGSPGRDGLDGFPGLPGPPGDGIKGPPGDPGYP

[0034] SEQ ID NO:2

[0035] TCATCCATGCCGCAACTTTCCAAACATCCCCTGCCTGCCTACCTGCCACCTCCGAGAAAAGGGACAGGACCACATACTGGGGCCTCAGTTGGAGCTGGGAGCTCTTTTTTAACACTGGTCTCCTAAACCCTGCCCTTCCAATTCTTTCCACCCCTCTGAGAGCAAAGTTCAGCCACCAAGATTGTTAATCACAGAAACAATGACATTTTGGTATCTCAAAGGAGCAAACTTGAAGGTCATCTTTCAGTGCAAAGACCCTTAGACTTGAGAGCAGACAGGCAGAATGACCATGTCTTCTGTGTATAGTACACCATCACAGTGCACATGGAAATGACATGCTGGTGTATAGTTCCTATTTCTCCCTCATATTTCAGAGGCGGAAGCCCCTCCCAGAAGTTTCCTCTGAGTTCCCCAAAGCTGGATTCAGTGTTCCTATAGCCCTTCGATTCCCCTGATTGTGACATTTACCAGCCTGACTTGGATTCGTCTGTTTCTTTTCCAATATTTCTCACCTGACCTTCCTGAGGACAGAGAGTGGGGGTGAGTCCCTGCCGCATGGCCAGCACCTGGCAAGGTCATGGCAGGAAGGAGGTGTTCCCGATGTGTGTGTGGAATGAAGGAAGGCACAAACGGGTCCGTGTGAGGAAGGGACAGACCTGCCAGGGAAACTGAGGTATAGAGTTTGATGAGTACCCCCAAATCATCTGCTTAAAAGCAGGTATAAAATGTAAGGTCCTCTGGTGCCTAAAGCACACTCATCCTGTGTACACGTGACTATTAATAATATATAGCATTGATAATTCTGTGAATCTAGTCAGAGTACATGAACATTGTGTCTTCTGG

[0036] 2. Construction of engineering strains

[0037] pPICZαA-rhColIV was linearized with Sac I and then transferred into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a screening marker to screen transformants. The expression of positive strains was verified by shake flask verification (10 mL culture system). A strain with high yield and stable expression was further screened from several strains, and a genetically engineered strain was successfully constructed.

[0038] 3. Shake flask fermentation of recombinant type IV collagen

[0039] For the genetically engineered bacteria of recombinant type IV humanized collagen obtained by screening, BMGY was used as the growth medium (30 mL) and cultured on a shaker at 30°C and 220 rpm until OD600 was 6-10. The bacteria were collected by centrifugation at room temperature and resuspended in 30 mL BMMY medium to make OD600 about 1.0. The resulting bacterial suspension was then placed in a 250 mL Erlenmeyer flask and continued to be cultured on a shaker at 28-30°C and 200-220 rpm. Anhydrous methanol was added to the culture solution every 24 hours to a final concentration of 1% to induce the expression of recombinant type IV collagen. The culture was stopped after 72 hours of induction, and the shake flask fermentation was completed. The electrophoresis diagram of protein expression in shake flask fermentation is shown below. Figure 1 shown.

[0040] 4. Fermentation of recombinant type IV collagen

[0041] (1) Pick a single colony of the constructed yeast expression strain and add it to 10 mL of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 220 rpm for 48 h for activation;

[0042] (2) Transfer the inoculum to a 500-ml Erlenmeyer flask (containing 200 mL of YPD culture medium) at 1% of the inoculum and culture at 30°C and 200 rpm for 24 h to serve as the seed for the tank.

[0043] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0044] (4) When the OD600 reaches 70, 50% glycerol is added. When the OD600 reaches about 120, the addition is stopped. When the dissolved oxygen rebounds to 100%, methanol is added for induction at a temperature of 27°C.

[0045] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 hours. The fermentation was terminated and the culture medium was centrifuged at 12000 rpm for 3 minutes to collect the supernatant. Figure 2shown.

[0046] Example 2: Purification of recombinant type IV collagen (rhColIV-P)

[0047] (1) The supernatant collected by centrifugation is filtered through a hollow fiber filter to remove impurities, and then ultrafiltered to 10% of the initial volume.

[0048] (2) Prepare the equilibrium buffer according to the isoelectric point of the protein: 20mmol / L PBS buffer (liquid A, pH7.0), and use 20mmol / L PBS buffer + 1.0mol / LNaCl (liquid B, pH6.0) as the eluent. Dilute the protein concentrate from the previous step with liquid A to prepare the loading solution, which is then filtered and loaded onto a 30mL Q Seplife XL anion exchange chromatography column. Equilibrate the column with the equilibrium buffer before loading. After loading, rinse 3 column volumes with liquid A, then use liquid B for gradient elution at a flow rate of 8mL / min. Collect the eluted fractions and detect them using SDS-PAGE.

[0049] (3) According to the distribution range of protein molecular weight obtained after ion exchange chromatography, Q Sephadex FF was selected to further purify the target protein. AKTA operation process: first rinse with equilibrium buffer (0.01mol / L PBS, 0.05mol / L NaCl) until the baseline is stable, and then the recombinant type IV collagen protein eluted from the ion exchange column obtained in the previous step is loaded onto a gel filtration chromatography column filled with QSephadex FF, eluted with eluent, the flow rate is set to 10mL / min, and the UV detection wavelength is 215nm. Finally, the target protein components that flow out are collected after SDS-PAGE electrophoresis detection. The electrophoresis diagram of the purified protein is shown in the figure. Figure 3 shown.

[0050] (4) Desalting: Desalting is done by using a G25 desalting column, i.e., using 25 mL of G25 filler. The operation process is similar to the gel filtration chromatography step. 6.5 mL of sample is loaded each time, and about 8 mL is collected. Desalting is completed 10 minutes after loading.

[0051] (5) Ultrafiltration: The protein was concentrated to 20-30% of the initial volume by ultrafiltration, and then placed in a -20°C refrigerator for pre-freezing for 4 h. It was then transferred to a vacuum freezer and dried for freezing. After 72 h, the frozen protein was collected and stored in a -80°C refrigerator for later use.

[0052] Example 3: In vitro cell proliferation assay

[0053] Used to evaluate the cell activity induced by recombinant collagen. HACAT cells in the logarithmic growth phase were inoculated on a 96-well culture plate and cultured for 1 day in a biochemical incubator at 37°C and 5% CO2. Recombinant type IV collagen was dissolved in HACAT complete medium and prepared into working solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2.5 mg / mL and 5 mg / mL, filtered and sterilized. After incubation for 24 hours, the old culture medium was discarded, and 100 μL of fresh complete culture medium was added to the control group, while the experimental group was 100 μL of equal volumes of protein solutions of different concentrations. Subsequently, cell proliferation was verified using the CCK8 method, and the absorbance was measured at 450 nm.

[0054] The relative cell viability was calculated as follows: relative cell viability (%) = (OD to be tested - OD blank) / (OD control - OD blank) X 100. The values ​​are mean ± SD (n = 3), compared with the control group * p<0.05, compared with the control group ** p<0.01, compared with the control group *** p<0.001.

[0055] Table 1 Cell viability

[0056]

[0057] Compared with the control group, the cell survival rate of the experimental group was higher than that of the control group, and there was no significant cytotoxicity, indicating that the recombinant type IV collagen at a concentration of 0.05-5 mg / mL had no inhibitory effect on the growth of human immortalized keratinocytes. This property proves the safety of using the recombinant type IV collagen as a biomaterial in various applications.

[0058] Example 4: Anti-inflammatory activity of recombinant type IV collagen

[0059] The present invention uses the RAW 264.7 cell anti-inflammatory model to evaluate the anti-inflammatory activity of recombinant type IV collagen. RAW264.7 cells are a mouse-derived macrophage cell line that is widely used in in vitro inflammatory response studies. This model can effectively evaluate the regulatory effect of recombinant type IV collagen on inflammatory response.

[0060] When the skin is stimulated by external stimuli, the body's defense mechanism begins to accelerate and secrete a large number of inflammatory factors, which manifest as redness, swelling, heat, and pain; TNF-α and IL-6 work synergistically in the inflammatory response. TNF-α mainly acts as an initiator of inflammation, triggering a series of inflammatory cascade reactions, while IL-6 plays an important role in maintaining inflammation and tissue damage. Overexpression of both can lead to aggravated inflammatory responses, causing cell damage and tissue dysfunction. TGF-β1 and IL-10 are both important anti-inflammatory cytokines that play a synergistic role in immunoregulation and inflammation inhibition, helping to maintain the balance of the immune system and prevent excessive inflammation and autoimmune reactions. Therefore, regulating the levels of TNF-α, IL-6, TGF-β1, and IL-10 is an important strategy for treating inflammatory diseases.

[0061] (1) Take RAW 264.7 cells in the logarithmic growth phase and calculate 1x10 5 The cells were seeded at a density of 1000 / well in a 24-well plate and incubated overnight in an incubator (37°C, 5% CO2).

[0062] (2) Liquid preparation: Prepare the working solution according to the experimental design table 2

[0063] Table 2 Experimental design

[0064]

[0065] (3) LPS stimulation: According to the experimental design, 100 μL of 10x LPS working solution was added to the drug-treated wells, and the wells were shaken left and right to mix the drugs in the wells. The final concentration of LPS was 1 μg / mL, and the wells were cultured in an incubator (37°C, 5% CO2) for 24 h.

[0066] (4) 24 hours after LPS stimulation, the mice were divided into groups and given medication.

[0067] (5) Sample collection: After the incubation is completed, the cell culture supernatant is collected in an EP tube (Note: the amount of sample collected is determined based on the test indicators). After collection, the sample is placed in a -80°C refrigerator for storage.

[0068] (6) Detection of TNF-α content: Detection was performed according to the operating instructions of the Mouse TNF-α ELISA kit.

[0069] (7) Detection of IL-6 content: Detection was performed according to the operating instructions of the Mouse IL-6 ELISA kit.

[0070] (8) TGF-β1 content detection: The detection was performed according to the operating instructions of the Mouse TGF-β1 ELISA kit.

[0071] (9) Detection of IL-10 content: Detection was performed according to the operating instructions of the Mouse IL-10 ELISA kit.

[0072] The experimental results showed that LPS stimulated giant cell RAW264.7, and the secretion of TNF-α and IL-6 in the model group was significantly increased compared with the control group; the secretion of TGF-β1 and IL-10 was significantly decreased, and there was a statistical difference compared with the model group, and the model was successfully established. Based on the LPS-stimulated giant cell RAW264.7 model, when the concentration of the sample recombinant type IV collagen (rhColIV-P) was 1 mg / mL, the secretion of inflammatory factors TNF-α and IL-6 by giant cells RAW264.7 was significantly reduced, and there was a statistical difference compared with the model group, and the inhibitory effect on inflammatory factors TNF-α and IL-6 was obvious. On the contrary, when the concentration of the sample recombinant type IV collagen (rhColIV-P) was 1 mg / mL, the secretion of inflammatory factors TGF-β1 and IL-10 by giant cells RAW264.7 was significantly increased, and there was a statistical difference compared with the model group, and the promoting effect on inflammatory factors TGF-β1 and IL-10 was obvious. The above experimental results show that recombinant type IV collagen has a significant anti-inflammatory effect, can regulate inflammatory factors, reduce and improve inflammatory symptoms, and play an anti-inflammatory and soothing role. (Values ​​are mean ± SD (n = 3), compared with the control group # p<0.05, compared with the control group ## p<0.01, compared with the control group ### p<0.001; compared with the model group * p<0.05, compared with the model group * p<0.01, compared with the model group *** p<0.001).

[0073] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant type IV collagen with anti-inflammatory activity, characterized in that: The recombinant type IV collagen is a truncated protein of natural human type IV collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the recombinant type IV collagen according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid is shown in SEQ ID NO:

2.

3. An expression vector comprising the nucleic acid according to claim 2, characterized in that The expression vector is pPICZαA.

4. A host cell introduced with the expression vector according to claim 3, characterized in that: The host cell is Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Pichia pastoris, Saccharomyces cerevisiae, animal cell or plant cell.

5. The host cell according to claim 4, characterized in that The host cell is Escherichia coli or Pichia pastoris, preferably Pichia pastoris.

6. A method for producing the recombinant type IV collagen as claimed in claim 1, characterized in that: The method comprises culturing host cells to secrete the recombinant type IV collagen into a culture medium, and separating and purifying the protein in the culture medium to obtain the recombinant type IV collagen.

7. The method according to claim 6, characterized in that The separation and purification adopts one or a combination of salting out, acid-base precipitation, chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography and membrane separation.

8. Use of the recombinant type IV collagen as claimed in claim 1 in the preparation of skin care products, health foods, tissue engineering materials or medical devices.

9. Use of the recombinant type IV collagen as claimed in claim 1 as a biomaterial in the preparation of skin care products, medicines or medical devices with anti-inflammatory activity.

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