Recombinant VI type collagen with antioxidant activity and application thereof

Abstract

The invention discloses a recombinant VI-type collagen with antioxidant activity and application thereof, the recombinant VI-type collagen is a truncated protein of human VI-type collagen, and the amino acid sequence of the recombinant VI-type collagen is as shown in SEQ ID NO: 1. The recombinant type VI collagen can be expressed in an escherichia coli expression system, the expression yield is high, the tested biological activity (antioxidant activity, cell proliferation promoting activity and cell adhesion promoting activity) is obviously superior to that of natural type VI collagen, and the recombinant type VI collagen has excellent antioxidant activity, is an effective free radical scavenger, and can be used for preparing a biological preparation. The polypeptide can remove free radicals in the body, provides a molecular basis for some important physiological and pathological processes related to oxidation resistance, can be widely applied to the fields of food, health food, biological medicine and the like, and has good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant type VI collagen with antioxidant activity and application thereof. Background Art

[0002] Antioxidant is an important topic that has attracted much attention in the health field today. As people's health awareness increases, more and more studies are focusing on the effects of antioxidant components in food, medicine and other natural substances on human health. Antioxidants can neutralize free radicals and reduce the damage caused by oxidative stress, thereby delaying cell aging, preventing diseases and maintaining health. Among them, natural antioxidants are safer and more effective than industrially synthesized additives used in food, medicine, cosmetics and feed on the market, making them a hot research topic internationally. Screening natural resources with strong antioxidant activity has become a new trend in food science, biology, medicine and other research.

[0003] Collagen is the most abundant protein in mammalian tissues, accounting for 30% of the total protein content of the body. It is also an important component of the extracellular matrix, maintaining the structure and function of tissues and organs such as skin, cartilage, tendons, ligaments and internal organs. It has the characteristics of biodegradability, low immunogenicity and promoting cell proliferation and differentiation, and is an ideal biomedical and beauty skin care material.

[0004] Type VI collagen is a beaded, filamentous collagen that is usually associated with the basement membrane. It has been observed to form a lattice-like structure that functions to connect cells to other basement membrane components and organize the three-dimensional tissue structure of skeletal muscle, tendons, bones, and cartilage. By binding to type IV collagen and other collagens in the basal layer, type VI collagen can establish a tight connection between muscle cells and the ECM. As the main component of the extracellular matrix, type VI collagen can promote the adhesion of fibroblasts in wound repair and enhance the regeneration of chondrocytes, playing an important role in wound healing. However, there is little research on the antioxidant properties of type VI collagen. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant type VI collagen with antioxidant activity and its application in view of the above-mentioned deficiencies in the prior art. The recombinant collagen can remove free radicals in the body, provide a molecular basis for certain important physiological and pathological processes related to antioxidant, and can be widely used in the fields of food, health food, biomedicine, etc.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a recombinant type VI collagen with antioxidant activity, characterized in that the recombinant type VI collagen is a truncated protein of human type VI collagen, and its amino acid sequence is shown in SEQ ID NO: 1;

[0007] SEQ ID NO:1

[0008] GPPGSEGARGAPGPAGPPGDPGLMGERGEDGPAGNGTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDDNNDIAPRGVKGAKGYRGPEGPQGPPGHQGPPGPDECEILDIIMKMCSCCECKCGPIDLLFVLDSSSESIGLQNFEIAKDFVVK.

[0009] Furthermore, the present invention provides a nucleic acid encoding the above-mentioned recombinant type VI collagen.

[0010] Furthermore, the present invention provides an expression vector comprising the above-mentioned nucleic acid, characterized in that the expression vector is pET28a.

[0011] 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, Pichia pastoris or Bacillus subtilis, preferably Escherichia coli.

[0012] Furthermore, the present invention provides a method for producing the above-mentioned recombinant type VI collagen, characterized in that it includes culturing host cells and collecting cell culture.

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

[0014] Furthermore, the present invention provides a product, characterized in that it contains the above-mentioned recombinant type VI collagen, and the product is a cosmetic, a health food or a medical device.

[0015] Furthermore, the present invention provides a use of the above-mentioned recombinant type VI collagen in the preparation of cosmetics, health foods or medical devices.

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

[0017] 1. The recombinant type VI collagen of the present invention can be expressed in an E. coli expression system with a high expression yield, and the biological activities (antioxidant activity, cell proliferation promoting activity and cell adhesion promoting activity) that have been tested are significantly better than those of natural type VI collagen.

[0018] 2. The recombinant type VI collagen of the present invention has excellent antioxidant activity and is an effective free radical scavenger that can remove free radicals in the body and provide a molecular basis for certain important physiological and pathological processes related to antioxidant activity. It can be widely used in the fields of food, health food, biomedicine, etc. and has good development and application prospects.

[0019] 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

[0020] Figure 1 This is an SDS-PAGE analysis diagram of the recombinant type VI collagen after expression and purification of the present invention. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1: Construction and expression of recombinant type VI collagen

[0023] (1) Construction of recombinant type VI collagen bacteria

[0024] 1) Construction of recombinant plasmid

[0025] According to the amino acid sequence shown in SEQ ID NO: 1, the codon optimization of the Escherichia coli expression system was carried out to obtain the target gene sequence. The obtained target gene sequence was entrusted to BGI for gene synthesis. The synthesized gene was ligated into the pET28a plasmid to obtain the pET28a-VI plasmid.

[0026] 2) Construction of E. coli expression strain

[0027] pET28a-VI was introduced into BL21 (DE3) competent cells purchased from Qingke Biotechnology. The operation process was referred to the instructions attached to the competent cells. Kanamycin resistance was used as a screening marker to screen transformants to obtain Escherichia coli expression strains.

[0028] (2) Induced expression of target protein

[0029] 1) Pick a single colony of the constructed E. coli expression strain and inoculate it into 20 mL LB liquid culture medium, and culture it at 37°C, 200 rpm for 18 h for activation;

[0030] 2) Transfer the inoculum to a 1000 mL conical flask (containing 300 mL of LB culture medium) at 1% of the inoculum volume and culture at 37°C, 200 rpm for 24 hours as the seed for the upper tank;

[0031] 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 37° C., adjust the pH to 6.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0032] 4) When the OD600 reaches 30, 50% glycerol is added. When the OD600 reaches about 40, the addition is stopped. When the dissolved oxygen rebounds to 100%, IPTG is added for induction.

[0033] 5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 6.0. The induction was carried out for 24 hours, and then the fermentation was terminated. The culture solution was centrifuged at 12000 rpm for 20 minutes, and the precipitate was taken and crushed with an ultrasonic crusher, and then the protein yield was detected by SDS-PAGE method.

[0034] The measurement results are shown in Table 1 below.

[0035] Table 1 Test results of recombinant type VI collagen after fermentation

[0036]

[0037] The results in Table 1 show that the yield of the recombinant type VI collagen of the present invention reaches 11.0 g / L, the recombinant expression amount is high, and can meet the requirements of fermentation production.

[0038] Example 2: Purification of recombinant type VI collagen

[0039] (1) Collect the bacterial sludge of the recombinant type VI collagen from the Escherichia coli after fermentation. Release the target protein from the microbial cells by ultrasonic or homogenization. After the disruption, remove the cell fragments and unbroken cells by centrifugation, filtration, etc.

[0040] (2) The fermentation broth from which the debris has been removed is first subjected to a crude purification by salting out, and then is refined using ion exchange technology. The buffer is configured according to the characteristics of the protein: 20mM potassium phosphate buffer (liquid A, pH 6.0), and 20mM potassium phosphate buffer and 1mol / L NaCl (liquid B, pH6.0) are used as eluents. The collected supernatant is adjusted to pH, filtered, and then loaded onto a hydrophobic cation exchange chromatography column. Before loading, the column is balanced with liquid A, and after loading, impurities are washed with 30% liquid B, and finally eluted with liquid B. The eluted protein is the target protein. The SDS-PAGE electrophoresis of the purified recombinant type VI collagen of SEQ ID NO: 1 is shown in Figure 2. Figure 1 shown.

[0041] (3) 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.

[0042] (4) 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. The protein was then transferred to a vacuum freeze dryer for freeze-drying. After 48 h, the freeze-dried protein was collected and stored in a -80°C refrigerator for later use.

[0043] Example 3: Determination of the antioxidant activity of recombinant type VI collagen

[0044] 1. DPPH free radical scavenging activity assay

[0045] Prepare 2 × 10 -4 mol / L DPPH solution, stored at 4°C in the dark. 2 mL of the antioxidant recombinant type VI collagen, GSH, natural full-length human type VI collagen (commercially available, obtained by extraction method) and DPPH solution prepared in Example 2 were added to the stoppered test tube, mixed and placed in a dark place for reaction for 30 minutes, and the absorbance at 517 nm was measured and recorded as A; the sample solution was replaced with anhydrous ethanol in the control tube, and other conditions were the same. After mixing, the reaction was carried out in the dark for 30 minutes, and the absorbance at 517 nm was measured as Aj; 2 mL of the antioxidant recombinant type VI collagen solution and 2 mL of anhydrous ethanol were added to the stoppered test tube, mixed and reacted for 30 minutes, and the absorbance at 517 nm was measured as A0.

[0046] The scavenging rate of recombinant type VI collagen on DPPH free radicals was calculated according to the following formula:

[0047]

[0048] 2. ABTS free radical scavenging activity assay

[0049] Prepare a mixed solution of ABTS (7mmol / L) and potassium persulfate (2.45mmol / L) with ultrapure water and place it at room temperature in the dark for 12 to 16h to prepare ABTS free radical cation base liquid. Dilute the ABTS free radical cation base liquid with ultrapure water so that its absorbance value measured at 734nm is 0.700±0.050 to obtain ABTS free radical cation working liquid. Prepare the antioxidant recombinant type IV collagen, GSH and natural full-length human type VI collagen (commercially available, obtained by extraction method) prepared in Example 2 with PBS solution to 1mg / mL, take 0.1mL and 3.9mL ABTS free radical cation working liquid and mix them evenly, place them at room temperature in the dark for 6min, and measure their absorbance at 734nm, which is recorded as Ai. The absorbance value measured at 734nm after 0.1mL ultrapure water and 3.9mL ABTS free radical cation working liquid are used as a control, which is recorded as Ac. 0.1 mL of ultrapure water and 3.9 mL of antioxidant recombinant type VI collagen (1 mg / mL) were mixed evenly and the absorbance value measured at 734 nm was recorded as Aj.

[0050] The scavenging rate of ABTS free radicals by antioxidant recombinant type VI collagen was calculated according to the following formula:

[0051]

[0052] 3. Superoxide Anion Scavenging Activity Assay

[0053] The antioxidant recombinant type VI collagen, GSH and natural full-length human type VI collagen (commercially available, obtained by extraction method) prepared in Example 2 were prepared into 1 mg / mL PBS solution, 0.2 mL was taken respectively, and then 4.5 mL of pH 8.2, 50 mmol / L Tris-HCl buffer and 4 mL of deionized water were added, mixed, and placed at 27°C for 10 min as reagent I; 0.3 mL of pyrogallol (3 mmol / L) was taken and kept at 25°C for 10 min as reagent II; reagent I and 0.3 mL of 10 mmol / L HCl solution were used as blank control group for zero adjustment, and then reagent I and reagent II were quickly mixed and measured with ultraviolet spectrophotometer at 325 nm absorbance A0, and recorded every 30 seconds. Repeat the above operation with deionized water instead of sample to measure its absorbance Aj.

[0054] The scavenging rate of superoxide anions by antioxidant recombinant type VI collagen was calculated according to the following formula:

[0055]

[0056] The results are shown in Table 2 below.

[0057] Table 2 Antioxidant activity of recombinant type VI collagen

[0058]

[0059] The results in Table 2 show that the recombinant type VI collagen of the present invention has good antioxidant activity, which is significantly better than natural human type VI collagen and other truncated proteins, and even significantly better than the recognized strong antioxidant GSH.

[0060] Example 4: Determination of the proliferation effect of recombinant type VI collagen

[0061] To evaluate the cell activity induced by recombinant collagen, HSF (human dermal fibroblasts) in logarithmic growth phase were seeded in 96-well tissue culture plates and cultured at 37°C under 5% CO 2 Incubate for 1 day in a biochemical incubator. Prepare standard extracts of 0.5 mg / L, 1 mg / L, 3 mg / L and 5 mg / L with PBS solution, filter and sterilize. After incubation for 24 hours, discard the old culture medium, add 100 μL of fresh complete culture medium to the control group, and 100 μL of different protein solutions of equal volume to the experimental group.

[0062] Subsequently, cell proliferation was verified using the MTT assay and the absorbance was measured at 490 nm.

[0063]

[0064] Compared with the control group, the cell survival rate of HSF in the experimental group was higher than that in the control group, and there was no significant cytotoxicity, indicating that the recombinant type VI collagen at a concentration of 0.5 to 5 mg / mL had no inhibitory effect on the growth of human skin fibroblasts. This property proves the safety of using the recombinant type VI collagen of the present invention as a biomaterial in various applications, and its proliferation-promoting effect is significantly better than that of natural human type VI collagen.

[0065] Example 5: Determination of the cell adhesion promoting effect of recombinant type VI collagen

[0066] Normally culture HSF (human skin fibroblast) cells, take the purified lyophilized recombinant type VI collagen and the control natural full-length human type VI collagen (commercially available, obtained by extraction method), prepare 0.25mg / L, 0.5mg / L, 1mg / L with PBS solution, and act on HSF (skin fibroblast) cells to promote cell adhesion experiment. The specific steps are as follows: human HSF cells are inoculated in a sterile 96-well plate, 200μL culture medium per well, and only PBS is added to the blank control group. After being placed at 37℃ for 2h, each group is washed 3 times with PBS to wash away the non-adherent cells, 100μL culture medium and 50μL MTT solution are added to each well, shaken, and incubated in an incubator for 4h, the mixed solution is aspirated, 150μL DMSO is added to dissolve the purple crystal formazan, shake for 10min, put into the microplate reader, and the detection wavelength is 570nm to measure the absorbance value of each well.

[0067] The data of recombinant type VI collagen and natural full-length human type VI collagen (commercially available) promoting HSF (human skin fibroblast) cell adhesion are shown in Table 3, where:

[0068]

[0069] Table 3 Effects of different concentrations of recombinant collagen on the adhesion of skin fibroblasts

[0070]

[0071] The results in Table 3 show that the recombinant type VI collagen of the present invention can significantly promote HSF cell adhesion, and its adhesion promoting effect is significantly better than that of natural human type VI collagen.

[0072] 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 VI collagen with antioxidant activity, characterized in that: The recombinant type VI collagen is a truncated protein of human type VI collagen, and its amino acid sequence is shown in SEQ ID NO: 1; SEQ ID NO:1 GPPGSEGARGAPGPAGPPGDPGLMGERGEDGPAGNGTEGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDPGDDNNDIAPRGVKGAKGYRGPEGPQGPPGHQGPPGPDECEILDIIMKMCSCCECKCGPIDLLFVLDSSSESIGLQNFEIAKDFVVK.

2. A nucleic acid encoding the recombinant type VI collagen as claimed in claim 1.

3. An expression vector comprising the nucleic acid according to claim 2, characterized in that The expression vector is pET28a.

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

5. A method for producing the recombinant type VI collagen as claimed in claim 1, characterized in that: This involves culturing the host cells and harvesting the cell culture.

6. The method according to claim 5, characterized in that The method further comprises separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation and membrane separation, preferably a combination of salting out and ion exchange chromatography.

7. A product, characterized in that The product comprises the recombinant type VI collagen as claimed in claim 1, wherein the product is a cosmetic, a health food or a medical device.

8. Use of the recombinant type VI collagen as claimed in claim 1 in the preparation of cosmetics, health foods or medical devices.

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