Recombinant type IV collagen with antioxidant activity and application thereof

Abstract

The present application discloses a recombinant human type IV collagen with antioxidant activity and its application. The recombinant human type IV collagen with antioxidant activity is a truncated protein of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO: 1. The recombinant type IV collagen with antioxidant activity can be expressed in a Pichia pastoris expression system with high expression yield, and its biological activity is significantly better than that of natural type IV collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a recombinant type IV collagen with antioxidant activity, a preparation method and application thereof. Background Art

[0002] Natural antioxidants are a class of biologically active proteins that have the functions of scavenging free radicals, inhibiting lipid peroxidation, and chelating metal ions. They have antioxidant and anti-aging effects and can be used to prevent and treat diseases, delay aging, and extend the shelf life of food. Natural antioxidants have broad application prospects in medicine, cosmetics, health products, and food and feed additives. Compared with industrially synthesized additives used in food, medicine, cosmetics, and feed on the market, they are safer and more effective, 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] Type IV collagen is an important member of the collagen family, widely present in tissue matrix, and plays an important role in the formation of extracellular matrix and the regulation of cell function. Type IV collagen (IV-C) is the main collagen found in the extracellular basement membrane separating various epithelial cells and endothelial cells. It is the main component of the dermal-epidermal junction and is mainly present in the dense layer.

[0004] Traditionally, type IV collagen is isolated from human placenta and purified using a multi-step process. This collagen extraction method has certain limitations, such as low extraction efficiency and low purity. On the other hand, those skilled in the art have tried many methods to recombinantly express type IV collagen in prokaryotic or eukaryotic expression systems, but unfortunately encountered the following problems: (1) Recombinant expression of full-length type IV collagen has not been successful; (2) Although some truncated proteins of type IV collagen can be recombinantly expressed, the yield is low and / or the biological activity is much worse than that of full-length type IV collagen, and therefore they are not suitable for industrial-scale production. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a recombinant type IV collagen with high recombinant expression yield in Pichia pastoris and significantly better biological activity than natural type IV collagen, and a preparation method and application thereof.

[0006] That is, the present invention includes:

[0007] 1. A recombinant human type IV collagen with antioxidant activity, which is a truncated protein of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO: 1.

[0008] 2. A nucleic acid encoding the recombinant human type I collagen described in item 1.

[0009] 3. An expression vector comprising the nucleic acid described in item 2.

[0010] 4. A host cell introduced with the expression vector according to item 3. The host cell may be Escherichia coli, Pichia pastoris, or Bacillus subtilis, preferably Pichia pastoris.

[0011] 5. A method for producing the recombinant human type I collagen described in item 1, comprising the steps of culturing the host cell described in item 4 and collecting the cell culture.

[0012] 6. The method according to item 5 further comprises the step of 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 ion exchange chromatography and hydrophobic chromatography.

[0013] 7. A product comprising the recombinant human type IV collagen described in item 1, wherein the product is a cosmetic, a health product or a medical device.

[0014] 8. Use of the recombinant human type IV collagen described in item 1 in the preparation of cosmetics, health products or medical devices.

[0015] The recombinant type IV collagen of the present invention can be expressed in a Pichia pastoris expression system with high expression yield, and its tested biological activities (antioxidant activity, cell adhesion promoting activity) are significantly better than those of natural type IV collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an SDS-PAGE analysis chart of the recombinant type IV collagen prepared in the example after expression and purification in X-33. DETAILED DESCRIPTION

[0017] Example 1: Construction and expression of recombinant human type IV collagen

[0018] (1) Construction of recombinant human type IV collagen

[0019] 1) Construction of recombinant plasmid

[0020] According to the amino acid sequences shown in SEQ ID No: 1, 2, 3, the codon optimization of the yeast expression system was performed to obtain the target gene sequence. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-IV plasmid.

[0021] 2) Construction of yeast expression strain

[0022] pPicZαA-IV was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0023] (2) Induced expression of target protein

[0024] 1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C, 200 rpm for 48 h for activation;

[0025] 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, 200 rpm for 24 hours as the seed for the upper tank;

[0026] 3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, cool to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0027] 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.

[0028] 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, and the fermentation was terminated. The culture solution was centrifuged at 12000 rpm for 2 minutes, and the supernatant was taken for detection of protein yield by SDS-PAGE.

[0029] The measurement results are shown in Table 1 below.

[0030] Table 1 Test results of each recombinant type IV collagen after fermentation

[0031]

[0032] The results in Table 1 show that the collagen production of IV-1 reached 15 g / L, its recombinant expression level was high and could meet the requirements of fermentation production.

[0033] Example 2: Purification of recombinant human type IV collagen

[0034] Collect the obtained engineered bacteria fermentation liquid, use a Thermo Fisher desktop centrifuge to separate the bacterial liquid and the bacterial body, and collect the supernatant. Prepare the buffer according to the characteristics of the protein: 20mM / L potassium phosphate buffer (liquid A, pH 6.0), and use 20mM / L potassium phosphate buffer + 1mol / L Nacl (liquid B, pH 6.0) as the eluent. The collected supernatant is adjusted to pH, filtered, and then loaded onto a hydrophobic cation exchange chromatography column. Before loading, balance the column with liquid A, wash with 20% liquid B after loading, and finally elute with liquid B. The eluted protein is the target protein. The SDS-PAGE electrophoresis of the purified recombinant human type IV collagen of SEQ ID No: 1, 2, and 3 is shown in the figure. Figure 1 shown.

[0035] Example 3: Determination of the antioxidant activity of recombinant type IV collagen

[0036] 1. DPPH free radical scavenging activity assay

[0037] Take DPPH and dissolve it in 95% ethanol to prepare a DPPH solution with a final concentration of 0.1mmo / L. Prepare the antioxidant recombinant type IV collagen, GSH and natural full-length human type IV collagen (commercially available, obtained by extraction method) prepared in Example 2 with PBS solution to 1mg / mL, take 2mL and mix them with 2mL DPPH solution, place them at room temperature away from light for 30min, and detect the absorbance at 517nm, which is recorded as Ai. The absorbance measured at 517nm after mixing 2mL 95% ethanol + 2mL DPPH solution is used as a control, which is recorded as Ac. The absorbance measured at 517nm after mixing 2mL 95% ethanol + 2mL antioxidant recombinant type IV collagen (1mg / mL) is recorded as Aj.

[0038] The scavenging rate of recombinant type IV collagen on DPPH free radicals was calculated according to the following formula:

[0039] DPPH free radical scavenging rate (%) = [1-(Ai-Aj) / Ac]X100%.

[0040] 2. ABTS free radical scavenging activity assay

[0041] Prepare a mixed solution of ABTS (7mmol / L) and potassium persulfate (2.45mmol / L) with ultrapure water and place it in room temperature and dark for incubation for 12-16h to prepare ABTS free radical cation base liquid. Dilute the ABTS free radical cation base liquid with ultrapure water so that its absorbance measured at 734nm is 0.700±0.050, and obtain ABTS free radical cation working liquid. The antioxidant recombinant type IV collagen, GSH and natural full-length human type IV collagen (commercially available, obtained by extraction method) prepared in Example 2 are prepared into 1mg / mL with PBS solution, and 0.1mL and 3.9mL ABTS free radical cation working liquid are taken respectively and mixed evenly, and placed at room temperature and dark for 6min, and its absorbance is measured at 734nm, which is recorded as Ai. The absorbance measured at 734nm after mixing 0.1mL ultrapure water + 3.9mL ABTS free radical cation working liquid is used as a control, which is recorded as Ac. 0.1 mL ultrapure water + 3.9 mL antioxidant recombinant type IV collagen (1 mg / mL) were mixed evenly and the absorbance value measured at 734 nm was recorded as Aj.

[0042] The scavenging rate of ABTS free radicals by antioxidant recombinant type IV collagen was calculated according to the following formula:

[0043] ABTS free radical scavenging rate (%) = [1-(Ai-Aj) / Ac] X 100%.

[0044] The results are shown in Table 2 below.

[0045] Table 2. Antioxidant activity of recombinant type IV collagen

[0046]

[0047] The results in Table 1 show that recombinant human collagen IV-1 has good antioxidant activity, which is significantly better than natural human type IV collagen and other truncated proteins, and even significantly better than the recognized strong antioxidant substance GSH.

[0048] Example 4: Determination of the cell adhesion promoting effect of recombinant type IV collagen

[0049] Normally culture HSF (human skin fibroblast) cells, take the purified lyophilized product of recombinant human type IV collagen and the control natural full-length human type IV collagen (commercially available, obtained by extraction method), prepare 0.25mg / L, 0.5mg / L, 1mg / L with PBS solution, and act on HSF (human 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°C 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 an ELISA reader, and the detection wavelength is 570nm to measure the absorbance value of each well.

[0050] The data of HSF (human skin fibroblast) cell adhesion promoted by recombinant human type IV collagen and natural full-length human type IV collagen (commercially available) are shown in Table 3, where:

[0051] Cell adhesion promotion rate = (OD of experimental group - OD of blank group) × 100% / blank group.

[0052] Table 3 Cell adhesion promoting effect of different concentrations of recombinant collagen on skin fibroblasts

[0053]

[0054] The results in Table 3 show that recombinant human collagen IV-2 can significantly promote HSF cell adhesion, and its adhesion-promoting effect is significantly better than other recombinant type IV collagens, and even significantly better than natural human type IV collagen.

[0055] Sequence information

[0056] SEQ ID NO1:

[0057] GPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGPKGFPGLPGQPGPPGLPVPGQAGAPG FPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPGPPGQP GYTNGIVECQPGPPGDQGPP GIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGR DGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLKGDKGDPGFPGQPGMPGRAGSPG RDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDTGPPGPP

[0058] SEQIDN0.2:

[0059] GLPGLPGQQGAPGIPGFPGSKGEMGVMGTPGQPGSPGPVGAPGLPGEKGDHGFPGSSGPR GDPGLKGDKGDVGLPGKPGSMDKVDMGSMKGQKGDQGEKGQIGPIGEKGSRGDPGTPG VPGKDGQAGQPG QPGPKGDPGISGTPGAPGLPGPKGSVGGMG LPGTPGEKGVPGIPGPQ GSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSP GLKGSPGSVGY PGSPG

[0060] SEQIDN0.3:

[0061] GIPGSKGEQG FMGPPGPQGQ PGLPGSPGHATEGPKGDRGP QGQPGLPGLP GPMGPPGLPGIDGVKGDKGN PGWPGAPGVP GPKGDPGFQG MPGIGGSPGI TGSKGDMGPP GVPGFQGPKG LPGLQGIKGDQGDHGVPGAK GLPGPPGPPG PYDIIKGEPG LPGPEGPPGL KGLQGLPGPK GQQGVTGLVG IPGPPGIPGFDGAPGQKGEM GPAGPTGPRG FPGPPGPDGL PGSMGPPGTP SVDHGFLVTR HSQ

Claims

1. A recombinant human type IV collagen with antioxidant activity, which is a truncated protein of human type IV collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid encoding the recombinant human type IV collagen according to claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced.

5. A method for producing the recombinant human type IV collagen according to claim 1, comprising: The step of culturing the host cell according to claim 4 and collecting the cell culture.

6. The method according to claim 5, further comprising the step of 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.

7. A product comprising the recombinant human type IV collagen according to claim 1, wherein the product is a cosmetic, a health product or a medical device.

8. Use of the recombinant human type IV collagen according to claim 1 in the preparation of cosmetics, health products or medical devices.

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