A recombinant collagen xvii and expression method and application thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a recombinant collagen XVII with oxidation and anti-aging, and an expression method and application thereof, wherein the amino acid sequence of the collagen XVII comprises n amino acid residue repeating units, the sequence of the repeating units is shown as SEQ ID NO. 1, and 1 < n < 60. The recombinant collagen XVII provided by the present application has anti-oxidation and anti-aging activities, a large molecular weight, and is easy to prepare.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a recombinant collagen XVII with antioxidant and anti-aging effects, and an expression method and application thereof. BACKGROUND

[0002] Collagen is the most abundant functional protein in mammals, accounting for 25-30% of the total protein, and even more than 80% in some organisms. It is widely distributed in tissues such as skin, bone, tendon, blood vessels, etc., and has the functions of supporting, connecting and repairing tissues. Due to its unique triple helix structure, collagen has excellent biocompatibility, low immunogenicity and degradability, and has wide application prospects in the fields of medical treatment, beauty, food, etc.

[0003] Skin, as the largest organ of the human body, its aging process is the result of the combined action of endogenous factors (such as genetics, telomere loss) and exogenous factors (such as ultraviolet light, oxidative stress). With age, the skin shows characteristics such as thinning of the epidermis, loss of collagen in the dermis, and breaking of elastic fibers, leading to deepening of wrinkles, relaxation, and impaired barrier function. Among them, the excessive accumulation of reactive oxygen species (ROS) is a key factor in initiating skin oxidative stress, which accelerates collagen degradation by destroying collagen structure and activating matrix metalloproteinases (MMPs), and induces the release of inflammatory factors (such as IL-6, TNF-α), forming a vicious cycle of “oxidative damage-inflammation-collagen loss”.

[0004] Collagen XVII (COL17 / BP180 / BPAG2) is an important component of the basement membrane zone and is a heterotrimer composed of three 180kDa α1 (XVII) chains (COL17A1). It is mainly distributed in the basement membrane zone (BMZ) at the junction of the epidermis and the dermis, and plays a key role in maintaining the structural stability and functional integrity of the skin. COL17A1 contains 15 collagen domains, which are located in the extracellular domain (AA 489-1497). This extracellular collagen domain is likely to be the structural part that interacts with extracellular matrix components to stabilize the connection between the epidermis and the dermis.

[0005] Collagen XVII binds to cell surface receptors such as integrins through its specific functional domains (such as KGD site), mediates cell adhesion to extracellular matrix (ECM), and thus supports the anchoring, proliferation and differentiation of epidermal stem cells. These functions make Collagen XVII play an important role in skin regeneration, repair and anti-aging. With age, the expression of COL17A1 decreases significantly, leading to decreased function of epidermal stem cells, loose basement membrane structure, and weakened skin barrier function, and thus causing skin relaxation, increased wrinkles, decreased repair capacity and other signs of aging. The Lys-Gly-Asp (KGD) motif is a new integrin binding site in Collagen XVII that is independent of the classic collagen receptor, and its function is similar to that of Arg-Gly-Asp (RGD), but its structure is unique. The RGD-dependent integrin pathway has been proven to be related to tissue regeneration and aging regulation. Studies have found that after the denaturation of Collagen XVII, part of the KGD domain activates integrins (such as α5β1 and αVβ1) through the KGD site, and promotes the migration efficiency of HaCaT cells. This mechanism may accelerate skin damage repair and delay skin aging. However, the expression amount of natural Collagen XVII is low and difficult to extract, which limits its application. SUMMARY

[0006] To solve the above technical problems, the present application provides a recombinant Collagen XVII with antioxidant and anti-aging activity, as well as its expression method and application.

[0007] A recombinant Collagen XVII with antioxidant and anti-aging activity, which is composed of n times of repeating units of the amino acid sequence from the extracellular C1 region of Col17A1 gene, wherein the amino acid sequence of the repeating unit is shown as SEQ ID NO. 1, and 1 < n ≤ 60.

[0008] The present application obtains a recombinant Collagen XVII by designing and splicing the sequence of human Collagen XVII, which has antioxidant and anti-aging activity, and has a larger molecular weight and is easy to prepare.

[0009] A nucleic acid molecule encoding the recombinant Collagen XVII.

[0010] An expression vector comprising the nucleic acid molecule.

[0011] Preferably, the nucleic acid molecule is inserted into the pPIC9K vector EcoR I and Not I site.

[0012] A genetically engineered bacterium comprising the expression vector.

[0013] Preferably, the genetically engineered bacteria are Pichia pastoris, Escherichia coli, yeast or Bacillus subtilis.

[0014] Preferably, the expression vector is transformed into Pichia pastoris.

[0015] The method for expressing the type XVII collagen protein by using the genetically engineered bacteria comprises the following steps:

[0016] The genetically engineered bacteria are inoculated into a liquid culture medium for culture to obtain a fermentation seed liquid; the fermentation seed liquid is inoculated into a fermentation tank for culture, and the dissolved oxygen is controlled at 30% to 40%, pH is controlled at 4.0 to 5.0, and when glycerol is consumed, glycerol feeding culture is started, and induction culture is started at the same time.

[0017] The recombinant type XVII collagen protein is used for preparing skin repair, anti-aging cosmetics or medicines.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application designs and splices the sequence of human type XVII collagen to obtain a recombinant type XVII collagen protein, which has antioxidant and anti-aging activities, and has a large molecular weight and is easy to prepare.

[0020] The recombinant type XVII collagen protein of the present application is preferably prepared by using Pichia pastoris as a host cell, and the protein is secreted and expressed outside the cell, which can effectively avoid the problems of impurities and the like caused by lysing the bacterial cells during extraction of the product. As a eukaryote, the recombinant protein secreted and expressed by the present application can be post-translationally modified, such as glycosylation and phosphorylation, and the produced recombinant protein does not contain pathogens, viral inclusion bodies or heat sources, has high safety, and has low fermentation cost.

[0021] The sequence of the recombinant type XVII collagen protein with antioxidant and anti-aging activities is obtained by ultrafiltration, immobilized metal ion affinity chromatography separation of the recombinant protein with antioxidant and anti-aging activities, and mass spectrometry sequencing.

[0022] Experimental verification shows that the recombinant type XVII collagen protein of the present application has significant antioxidant and anti-aging effects, can remove senescent cells and regulate important factors in oxidative stress, has a soothing effect, and can be used as a biological medical material to prepare skin care products, medicines or medical device products with antioxidant and anti-aging activities. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a protein expression electrophoresis graph of shake flask fermentation of Col17-1810 in Example 1 of the present application.

[0024] Figure 2 Figure 6 is a protein expression electrophoretogram of Col17-1810 protein.

[0025] Figure 3 Figure 9 is a protein electrophoretogram of Col17-1810 protein after purification.

[0026] Figure 4 Figure 10 is the result of cell proliferation inhibition test of Col17-1810 protein.

[0027] Figure 5 Figure 11 is the effect of Col17-1810 protein on the removal of β-galactosidase produced by D-gal induced HaCat cells.

[0028] Figure 6 Figure 12 is the migration effect of Col17-1810 protein on cell scratch test.

[0029] Figure 7 Figure 13 is a statistical chart of the result of cell scratch test of Col17-1810 protein. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified.

[0031] A recombinant collagen type XVII protein with antioxidant and anti-aging activity and an expression strain thereof, wherein the sequence of the recombinant collagen type XVII protein is obtained by splicing and repeating 10 times the functional domain of human collagen type XVII protein that binds to cell integrin as a repeating unit, the selected repeating unit sequence is located in the first collagen domain of human collagen type XVII protein, and the high-copy Pichia pastoris genetic engineering bacteria obtained by transforming the codon-optimized sequence into Pichia pastoris and screening by G418 resistance gradient can stably express the recombinant collagen type XVII protein. The expression recombinant collagen type XVII protein of the present application has the effects of promoting HaCat cell proliferation, migration, removing senescent cells, and antioxidant, and can be used in the fields of skin repair and anti-aging cosmetics and medicine.

[0032] Example 1

[0033] Preparation of Col17-1810 protein

[0034] The present application is based on the extracellular C1 region of Col17A1, designs a spliced amino acid sequence (amino acid sequence as shown in SEQ ID NO. 1), and obtains a recombinant protein after repeating. In a specific embodiment, the amino acid sequence is repeated 10 times and named Col17-1810, the amino acid sequence thereof is shown as SEQ ID NO. 2, the nucleotide sequence thereof is shown as SEQ ID NO. 3, and the sequence information is as follows:

[0035] Taking the amino acids at positions 1450-1455 and 1471-1482 of Col17A1 gene as a basic unit, the amino acid sequence of the basic unit is: GPKGDRGHKGEKGDKGDQ, denoted as SEQ ID NO. 1;

[0036] The amino acid sequence of Col17-1810 is: GPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQ, denoted as SEQ ID NO. 2;

[0037] Col17-1810 nucleotide sequence: , denoted as SEQ ID NO.3;

[0038] The sequence shown in SEQ ID NO.3 was cloned into the Pichia pastoris expression vector pPIC9K to construct the recombinant expression vector pPIC9K-COL17-1810. The pPIC9K vector contains the following key elements:

[0039] AOX1 promoter: for efficient methanol-induced expression.

[0040] α-factor signal peptide: used to guide the secretion of recombinant proteins into the culture medium.

[0041] G418 resistance gene: used for screening high-copy transformants.

[0042] Use S al The constructed recombinant expression vector pPIC9K-COL17-1810 was linearized using an I restriction enzyme. This step was performed according to the NEB instructions, utilizing S... alI Endonuclease cut 5 μg of plasmid in 100 μL system. After linearization, 4 μL of DNA was taken for gel electrophoresis, and then 4 μL of sterile water was added to complete the system, 2.5 times the volume of anhydrous ethanol and 5% x 3.5 times the volume of 5 mol / L Nacl were added to the -80°C refrigerator alcohol recovery for 20 min. After alcohol precipitation, the plasmid was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the residual ethanol was blown dry in the fume hood to complete the plasmid recovery.

[0043] Pichia pastoris GS115 single colony was picked from the non-resistant YPD-GS115 plate and inoculated into a 10 mL YPD (peptone 2%, yeast extract 1%, glucose 1%) shake flask, 30°C, 220r shaking overnight, OD 600 about 10. The cell concentration was observed, and the cells with OD 600 10 were inoculated into 100 mL YPD medium to make their OD 600 =0.5, 30°C, 220r culture to OD 600 1.2-1.5. The cell solution with OD 600 1.2-1.5 was centrifuged in a 50 mL centrifuge tube at 8000r for 5 min, and the cells were collected. Each tube of centrifuge tube was added with 40 mL of pre-configured LDST solution to resuspend the cells and incubated in a 30°C incubator for 30 min. After collecting the cells, 1 mL of pre-cooled 1M sorbitol solution was added to resuspend the cells and wash the residual LDST. Then the cells were collected by centrifugation at 12000r for 5 min, and the above resuspension and cell collection steps were repeated 4 times. The cells were resuspended with 400 μL of pre-cooled 1M sorbitol solution, aliquoted into cryotubes, 80 μL per tube, to obtain GS115 competence, which was stored in a -80°C refrigerator.

[0044] The linearized plasmid was mixed with the GS115 competence and transferred into the pre-iced electric transformation cup, and placed on ice for 5 min. The parameters were voltage 1.5Kv, capacitance 25 μF, resistance 200Ω, and pulse time 5msec. After electric shock, 1 mL of ice-precooled 1M sorbitol solution was immediately added, and the cells were incubated at 30°C, 220rpm for 1h to obtain the culture solution. 100 μL and 200 μL of the culture solution were taken and plated on MD plates, and incubated in a 30°C incubator until single colonies grew. All single colonies were picked and inoculated on 2, 4, and 6 mg / ml resistance plates for high copy colony selection under pressure.

[0045] The G418 plates were observed, and the slightly better growing transformants were selected and inoculated into 50 ml of BMGY medium (250 ml culture bottle) at 30°C, 220r shaking to OD 600The bacterial cells were collected by centrifugation at 6000 rpm for 10 min and transferred to 100 mL BMMY medium (500 mL culture flask) until the OD reached 1.0. Samples were taken every 24 h, and 1% methanol was added. SDS-PAGE was performed on the samples to identify the expression, yielding Pichia pastoris genetically engineered strains. Pichia pastoris with good expression performance will be used for large-scale culture to prepare recombinant type XVII collagen. The shake-flask results are as follows: Figure 1 As shown.

[0046] After obtaining the Pichia pastoris genetically engineered strain, the specific culture conditions in the 5L fermenter are as follows: The Pichia pastoris genetically engineered strain was inoculated into BMGY medium and cultured at 30℃ and 220 rpm until OD. 600 =8-11, used as the seed culture for the upper tank, after expanding the seed culture, is inoculated into an inorganic salt culture medium fermenter with an initial volume of 5L at a 10% inoculation rate. The culture temperature is 30℃, pH = 4.0-5.0, and dissolved oxygen is controlled at 30%-40%. After the glycerol is depleted, glycerol feeding culture begins. The feeding stop time can be referenced from OD. 600 The concentration was 170-190 g / L. After stopping feeding and starving for 1 hour, methanol was added to induce the target protein. The results in the upper tank are as follows. Figure 2 As shown.

[0047] After loading the culture medium into the container, centrifuge at 10000 rpm for 10 min and collect the supernatant; filter through a 0.22 μm membrane. Equilibrate the Ni-NTA column with 5 column volumes of equilibration buffer (Tris 20 mM, NaCl 250 mM, completely dissolved, pH adjusted to 7.5 with hydrochloric acid, and finally brought to 1 L, stored at room temperature) at a flow rate of 10 ml / min. Load the culture supernatant onto the column and collect the flow-through. Wash the column with elution buffer (Tris 20 mM, NaCl 250 mM, imidazole 300 mM, completely dissolved, pH adjusted to 7.5 with hydrochloric acid, and finally brought to 1 L, stored at room temperature) to remove contaminating proteins, collect the eluent, and analyze the purified target protein solution using SDS-PAGE. After determining the conditions, perform multiple purification cycles to collect the target protein solution. The purification results are as follows. Figure 3 As shown.

[0048] The purified protein was dialyzed to reduce its conductivity to below 300 μs / cm, and then lyophilized using a low-temperature vacuum drying method to obtain recombinant protein lyophilized powder, which was then stored.

[0049] Example 2

[0050] Cell experiments

[0051] 1. HaCat cell culture

[0052] HaCat cells were cultured in DMEM (5.5 mM glucose) containing 10% fetal bovine serum (FBS), 100 IU / mL penicillin and 100 μg / mL streptomycin at 37 °C and 5% CO2 in an incubator. The cells were subcultured every 2 days and grew well with high survival rate, high activity and stability.

[0053] 2. HaCat cell proliferation experiment

[0054] The effect of Col17-1810 on HaCat cell activity was detected by CCK-8 method.

[0055] Logarithmic growth phase HaCat cells were washed with PBS, digested with trypsin, collected, centrifuged, resuspended with an appropriate amount of culture solution, blown evenly, counted with a cell counting plate, and the cell suspension concentration was adjusted to about 1 × 10 5 6 / mL, then inoculated into a 96-well plate, 100 μL of cell suspension per well. The 96-well plate was cultured in a 37 °C and 5% CO2 incubator. When the cells grew to about 70%, different concentrations of recombinant collagen (0, 0.125, 0.25, 0.5, 1, 2.5, 5 and 10 mg) were added. The recombinant collagen was dissolved in 10% FBS and serum-free medium for cell culture, respectively. After 48 hours of action of recombinant collagen and D-galactose, 10 μL of CCK-8 was added to each well, and incubated in a 37 °C incubator for 0.5-4 hours. The OD value at 450 nm of the 96-well plate was detected by a microplate reader, and the cell survival rate was calculated. The results are shown in Figure 4 .

[0056] The results show that in the presence of 10% FBS medium, even at a low concentration of 0.125 mg / mL, Col17-1810 promotes cell proliferation. Compared with the relevant blank control group, HaCaT cells exposed to 0.125 mg / mL Col17-1810 increased the number of cells by 1.39 times after 48 hours (P<0.001), and at 1 mg / mL, the promotion of proliferation was the best, reaching 1.72 times (P<0.001). The proliferation potential of HaCaT cells cultured with Col17-1810 in serum-free medium. The results show that 1.0 mg / mL of Col17-1810 significantly enhances HaCaT cell proliferation, increasing by 1.5 times in 48 hours (P<0.001). In summary, these findings indicate that rhCOL17 can positively regulate cell proliferation in HaCaT cells, especially in the presence of serum. In the absence of serum, the promoting effect of rhCOL17 will be weakened. The optimal Col17-1810 concentration is 1.0 mg / mL.

[0057] 3. β-galactosidase staining

[0058] Establishing a senescent cell model using D-galactose.

[0059] HaCat cells in logarithmic growth phase were taken, and a prepared D-galactose solution (20 g / L) was added to the culture medium, thereby obtaining a senescent cell model, wherein the D-galactose solution was prepared by weighing D-galactose solids into a certain amount of prepared DMEM to prepare a D-galactose solution with a concentration of 20 g / L, and then filtering with a 0.22 μm filter membrane.

[0060] The HaCat senescent cell model was inoculated into a 6-well plate at a density of 5 x 10 5 cells / well, 2 mL of DMEM medium was added to each well, and after the cells adhered, the medium was replaced with DMEM medium containing 20 g / L D-galactose and different concentrations of Col17-1810 (0.5 mg / mL, 1 mg / mL), and a blank control group was set up. When the cells in the 6-well plate reached about 60%, the medium and cells were washed twice with PBS, each time for 3 minutes. During this process, 1 mL of β-galactosidase staining fixative was added, and the cells were fixed at room temperature for 15 min. After the PBS was aspirated, 1 mL of staining working solution was added to each well, and incubated overnight at 37°C. To prevent evaporation, the 6-well plate was covered with plastic wrap. Then, observation was performed under a conventional optical microscope. The results are shown in Figure 5 .

[0061] The results showed that the number of senescent cells in the D-galactose group was significantly increased compared with the control group, and after 48 h of Col17-1810 intervention, the number of senescent cells in the low-dose group (0.5 mg / mL) and the high-dose group (1 mg / mL) was significantly reduced.

[0062] 4. Cell scratch experiment

[0063] 5 x 10 5 HaCaT cells were added to each well of a 6-well plate, and when a layer of cells was observed to have grown on the plate bottom under a microscope, a sterilized 10 μL gun tip was used to lightly scratch the cells, and a steel ruler was used to ensure that the scratches were straight and evenly spaced, which facilitated subsequent observation. Then, the cell fragments were washed away with PBS, and serum-free medium containing 0.125 mg / mL and 1 mg / mL of Col17-1810 protein was added to the experimental groups, and serum-free medium alone was used as the control group. During the culture process, the changes in the scratches were observed under a microscope, and photographs were taken to record the cell state at 0, 12, and 24 h. The scratch area was quantitatively analyzed by ImageJ software to evaluate the effect of Col17-1810 protein on HaCaT cell migration. The results are shown in Figure 6 andFigure 7 As shown in the figure.

[0064] The results show that after 24 hours of intervention by Col17-1810, the low-dose group (0.5 mg / mL) and the high-dose group (1 mg / mL) can significantly promote the proliferation of HaCat cells.

[0065] 5. Antioxidant activity determination of Col17-1810

[0066] The present application determines the ability of Col17-1810 protein to scavenge DPPH, ABTS, superoxide anion, and hydroxyl radicals to evaluate its own antioxidant activity. In the experiment, the Col17-1810 protein is first dissolved with the extraction solution in the corresponding detection kit, and samples with a low concentration of 0.5 mg / mL and a high concentration of 1 mg / mL are prepared, and then the determination is carried out according to the instructions. The results are shown in Table 1.

[0067] Table 1: Scavenging rate of different concentrations of Col17-1810 protein on different free radicals

[0068]

[0069] In order to study the effect of Col17-1810 on the oxidative damage of HaCat cells, the effects of Col17-1810 on the oxidative stress related indicators T-AOC, SOD, MDA, and GSH-PX of HaCat cells were detected according to the instructions of the related kit, and a blank control group and a D-gal model group were set. The results are shown in Table 2.

[0070] Table 2: Effect of different concentrations of Col17-1810 protein on oxidative stress factors in D-gal induced HaCat cells

[0071]

[0072] It should be noted that when the present application claims involve numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0073] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0074] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A recombinant collagen type XVII having antioxidant and anti-aging activity, characterized in that, The amino acid sequence of the collagen is shown as SEQ ID NO.

2.

2. A nucleic acid molecule encoding the recombinant collagen type XVII according to claim 1.

3. An expression vector, characterized by, comprising the nucleic acid molecule according to claim 2.

4. The expression vector of claim 3, wherein, is inserting the nucleic acid molecule into a pPIC9K vector EcoR I and Not I site.

5. A genetically engineered bacterium, characterized by, The genetically engineered bacteria comprise the expression vector according to any one of claims 3 to 4. 6.The genetically engineered bacterium of claim 5, characterized in that, is obtained by transforming the expression vector into Pichia pastoris.

7. A method for expressing collagen type XVII using the genetically engineered bacteria according to claim 6, characterized by, comprising the following steps: The genetically engineered bacteria are inoculated into a liquid medium for culture to obtain a fermentation seed liquid; The fermentation seed liquid is inoculated into a fermentation tank for culture, the dissolved oxygen is controlled at 30% to 40%, pH = 4.0 to 5.0, and when the glycerol is consumed, the glycerol feeding culture is started, and the induction culture is started at the same time.

8. The recombinant collagen type XVII according to claim 1 for use in the preparation of a skin repair medicament.

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