Recombinant XVII type collagen as well as expression method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to recombinant XVII type collagen with oxidation and aging resistance and an expression method and application thereof, the amino acid sequence of the collagen comprises n amino acid residue repetitive units, the sequence of the repetitive units is shown as SEQ ID NO.1, and n is larger than 1 and smaller than or equal to 60. The recombinant XVII type collagen provided by the invention has antioxidant and anti-aging activity, and is large in molecule quantity and 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: 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.

[0016] The recombinant type XVII collagen protein is used for preparing skin repair, anti-aging cosmetics or medicines.

[0017] Compared with the prior art, the present application has the following beneficial effects: 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, a large molecular weight and is easy to prepare.

[0018] The recombinant type XVII collagen protein of the present application is preferably prepared by using Pichia pastoris as a host cell, can be secreted and expressed extracellularly, can effectively avoid impurities and other problems caused by lysing bacteria when extracting products, can be post-translationally modified, such as glycosylation and phosphorylation, as a eukaryote, and the produced recombinant protein does not contain pathogens, virus inclusion bodies or heat sources, has high safety and low fermentation cost.

[0019] 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.

[0020] 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 instrument products with antioxidant and anti-aging activities. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a protein expression electrophoresis graph of shake flask fermentation of Col17-1810 protein in the embodiment 1 of the present application.

[0022] Figure 2Electrophoresis diagram of Col17-1810 protein expression on a plate.

[0023] Figure 3 This is an electrophoresis image of the purified Col17-1810 protein.

[0024] Figure 4 The results are from the assay of cell proliferation inhibition by Col17-1810 protein.

[0025] Figure 5 The effect of Col17-1810 protein on the clearance of β-galactosidase produced by D-gal-induced HaCat cells.

[0026] Figure 6 The migration effect of Col17-1810 protein on cell scratch assay.

[0027] Figure 7 This is a statistical graph showing the results of the cell scratch assay using Col17-1810 protein. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0029] A recombinant type XVII collagen with antioxidant and anti-aging activities and its expression strain are disclosed. The recombinant type XVII collagen sequence is obtained by splicing and repeating the cytoin-binding functional domain of human type XVII collagen as a repeating unit 10 times. The selected repeating unit sequence is located in the first collagen domain of human type XVII collagen. After codon optimization, it was transformed into Pichia pastoris, and the high-copy Pichia pastoris genetically engineered strain obtained through G418 resistance gradient selection can stably express the recombinant type XVII collagen. The recombinant type XVII collagen expressed by this invention has the effects of promoting HaCat cell proliferation and migration, clearing senescent cells, and anti-oxidation, and can be used in skin repair and anti-aging cosmetics and pharmaceuticals.

[0030] Example 1 Preparation of Col17-1810 protein This invention designs a spliced ​​amino acid sequence (amino acid sequence as shown in SEQ ID NO. 1) based on the extracellular C1 region of Col17A1, and obtains a recombinant protein by repeating it. In one specific embodiment, this amino acid sequence is repeated 10 times and named Col17-1810, the amino acid sequence of which is shown in SEQ ID NO. 2, and the nucleotide sequence of which is shown in SEQ ID NO. 3. The sequence information is as follows: The basic unit is amino acids 1450-1455 and 1471-1482 of the Col17A1 gene. The amino acid sequence of the basic unit is: GPKGDRGHKGEKGDKGDQ, denoted as SEQ ID NO.1. Col17-1810 amino acid sequence: GPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKG DKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQGPKGDRGHKGEKGDKGDQ, denoted as SEQ ID NO.2; Col17-1810 nucleotide sequence: , denoted as SEQ ID NO.3; 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:

[0031] AOX1 promoter: for efficient methanol-induced expression.

[0032] α-factor signal peptide: used to guide the secretion of recombinant proteins into the culture medium.

[0033] G418 resistance gene: used for screening high-copy transformants.

[0034] 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... alThe plasmid was digested with an enzyme I at a volume of 100 μL. After linearization, 4 μL of the plasmid was subjected to DNA gel electrophoresis. Then, 4 μL of sterile, enzyme-free water was added to complete the reaction. 2.5 volumes of anhydrous ethanol and 3.5 volumes of 5 mol / L NaCl were added, and the mixture was precipitated at -80°C for 20 min. After precipitation, the plasmid was centrifuged at 12,000 rpm for 10 min, the supernatant was discarded, and residual ethanol was dried in a fume hood to complete plasmid recovery.

[0035] Pick a single colony of Pichia pastoris GS115 from a non-resistant YPD-GS115 plate and inoculate it into a shake flask containing 10 mL of YPD (2% peptone, 1% yeast extract, and 1% glucose). Incubate overnight at 30°C and 220 rpm until OD reaches 100%. 600 Approximately 10. Observe the bacterial cell concentration and take the OD value. 600 10 cells were inoculated into 100 mL of YPD medium to allow OD to be adjusted. 600 =0.5, 30℃, 220r incubation until OD 600 The value is 1.2-1.5. OD 600 The bacterial culture with a concentration of 1.2-1.5 was centrifuged in 50 mL centrifuge tubes at 8000 rpm for 5 min to collect the bacterial cells. For each centrifuge tube, 40 mL of pre-prepared LDST solution was added to resuspend the bacterial cells, and the tubes were incubated at 30 °C for 30 min. After collecting the bacterial cells, 1 mL of pre-chilled 1M sorbitol solution was added to resuspend the bacterial cells and wash away any residual LDST. The cells were then collected by centrifugation at 12000 rpm for 5 min. This resuspension and centrifugation process was repeated four times. The bacterial cells were resuspended in 400 μL of pre-chilled 1M sorbitol solution and aliquoted into 80 μL cryovials to obtain GS115 competent cells, which were stored at -80 °C.

[0036] The linearized plasmid was mixed with GS115 competent cells and transferred into an electroporation vessel pre-chilled on ice, where it was placed for 5 min on ice. Electroporation was performed at parameters of 1.5 kV, 25 μF, 200 Ω, and 5 msec. Immediately after electroporation, 1 mL of 1 M sorbitol solution pre-chilled on ice was added, and the mixture was incubated at 30 °C and 220 rpm for 1 h to obtain the culture medium. 100 μL and 200 μL of the culture medium were plated onto MD plates and incubated at 30 °C until single colonies appeared. All single colonies were picked and transferred to 2, 4, and 6 mg / mL antibiotic plates for pressure screening to identify high-copy colonies.

[0037] Observe the G418 plates, select the better-growing transformants and inoculate them into 50ml of BMGY medium (250ml culture flasks), incubate at 30℃ and shake at 220r until 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 collected samples to identify 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Example 2 Cell experiments 1. HaCat cell culture HaCat cells were cultured in DMEM (5.5 mM glucose) medium containing 10% fetal bovine serum (FBS), 100 IU / mL penicillin, and 100 μg / mL streptomycin at 37°C in an incubator with 5% CO2. The cells were passaged every 2 days and showed good growth, high survival rate, and high activity and stability.

[0042] 2. HaCat cell proliferation experiment The effect of Col17-1810 on HaCat cell viability was detected using the CCK-8 assay.

[0043] HaCat cells in the logarithmic growth phase were washed with PBS, digested with trypsin, collected, centrifuged, resuspended in an appropriate amount of culture medium, and counted using a cell counting chamber. The cell suspension concentration was adjusted to 1×10⁻⁶. 5 After reaching a cell density of approximately [number] cells / mL, 100 μL of cell suspension was seeded into each well of a 96-well plate. The 96-well plates were incubated at 37°C with 5% CO2. When the cells reached approximately 70% confluence, 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. After 48 hours of treatment with recombinant collagen and D-galactose, 10 μL of CCK-8 was added to each well, and the plates were incubated at 37°C for 0.5–4 hours. The OD value at 450 nm of the 96-well plates was measured using a microplate reader, and cell viability was calculated. The results are shown in [see attached table]. Figure 4 .

[0044] The results showed that even at a low concentration of 0.125 mg / mL, Col17-1810 promoted cell proliferation in medium containing 10% FBS. Compared with the relevant blank control group, HaCaT cells exposed to 0.125 mg / mL Col17-1810 showed a 1.39-fold increase in cell number after 48 hours (P < 0.001), with the best proliferation-promoting effect at 1 mg / mL, reaching 1.72-fold (P < 0.001). The proliferative potential of HaCaT cells cultured with Col17-1810 in serum-free medium was also examined. The results showed that 1.0 mg / mL Col17-1810 significantly enhanced HaCaT cell proliferation, increasing it to 1.5-fold after 48 hours (P < 0.001). In conclusion, these findings indicate that rhCOL17 can positively regulate cell proliferation in HaCaT cells, especially in the presence of serum. The promoting effect of rhCOL17 is weakened in the absence of serum. The optimal concentration of Col17-1810 was determined to be 1.0 mg / mL.

[0045] 3. β-galactosidase staining A model of senescent cells was established using D-galactose.

[0046] HaCat cells in the logarithmic growth phase were taken, and a prepared D-galactose solution (20 g / L) was added to their culture medium to obtain a senescent cell model. The D-galactose solution was prepared by weighing D-galactose solid and adding it to a certain amount of prepared DMEM to prepare a D-galactose solution with a concentration of 20 g / L. The solution was then filtered through a 0.22 μm filter membrane and prepared fresh each time it was used.

[0047] The HaCat senescent cell model was used at a density of 5×10⁻⁶. 5 Cells were seeded per well into 6-well plates, with 2 mL of DMEM medium added to each well. After cell attachment, the medium was replaced with DMEM containing 20 g / L D-galactose and different concentrations of Col17-1810 (0.5 mg / mL, 1 mg / mL). A blank control group was also included. When the cells reached approximately 60% confluence, both the medium and cells were washed twice with PBS for 3 minutes each time. During this process, 1 mL of β-galactosidase staining fixative was added, and the cells were fixed at room temperature for 15 minutes. After removing the PBS, 1 mL of staining working solution was added to each well, and the plates were incubated overnight at 37°C. To prevent evaporation, the 6-well plates were sealed with plastic wrap. The results were then observed under a standard optical microscope. Figure 5 As shown.

[0048] The results showed that, compared with the control group, the number of senescent cells in the D-galactose group was significantly increased. After 48 hours of intervention with Col17-1810, the number of senescent cells in both the low-dose group (0.5 mg / mL) and the high-dose group (1 mg / mL) was significantly reduced.

[0049] 4. Cell scratch test Add 5×10 to each well of the 6-well plate 5 HaCaT cells were cultured under a microscope until they formed a confluent layer at the bottom of the plate. Then, sterile 10 μL pipette tips were used to gently scratch the cells, ensuring straight and evenly spaced scratches for easy observation. Cell debris was then washed away with PBS. The experimental groups were cultured in serum-free medium containing 0.125 mg / mL and 1 mg / mL Col17-1810 protein, respectively, while the control group received only serum-free medium. During culture, scratch changes were observed under a microscope, and cell status was recorded at 0, 12, and 24 hours. The scratch area was quantitatively analyzed using ImageJ software to assess the effect of Col17-1810 protein on HaCaT cell migration. Results are as follows: Figure 6 and Figure 7 As shown.

[0050] The results showed that after 24 hours of intervention with Col17-1810, both the low-dose group (0.5 mg / mL) and the high-dose group (1 mg / mL) significantly promoted the proliferation of HaCat cells.

[0051] 5. Determination of antioxidant activity of Col17-1810 This invention evaluates the antioxidant activity of the Col17-1810 protein by measuring its ability to scavenge DPPH, ABTS, superoxide anions, and hydroxyl radicals. In the experiment, the Col17-1810 protein was first dissolved in the extraction solution of the corresponding detection kit to prepare samples at low concentrations of 0.5 mg / mL and high concentrations of 1 mg / mL, which were then measured according to the instructions. The results are shown in Table 1.

[0052] Table 1: Scavenging rates of different free radicals by different concentrations of Col17-1810 protein To investigate the effects of Col17-1810 on oxidative damage in HaCat cells, the effects of Col17-1810 on oxidative stress-related indicators T-AOC, SOD, MDA, and GSH-PX in HaCat cells were measured according to the instructions of the relevant kit. A blank control group and a D-gal model group were also included. The results are shown in Table 2.

[0053] Table 2: Effects of different concentrations of Col17-1810 protein on oxidative stress factors induced by D-gal in HaCat cells. It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A recombinant collagen type XVII having antioxidant and anti-aging activity, characterized in that, The amino acid sequence of the collagen comprises n amino acid residue repeating units, the sequence of the repeating units is shown as SEQ ID NO. 1, 1 < n ≤ 60.

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. Use of the recombinant collagen type XVII according to any one of claims 1 to 2 in the preparation of skin repair, anti-aging cosmetics or medicines.

Images