Recombinant human VI-type collagen as well as coding gene and expression method thereof

Abstract

The invention belongs to the technical field of genetic engineering and recombinant collagen, and discloses recombinant human VI-type collagen as well as a coding gene and an expression method thereof, and the amino acid sequence of the recombinant human VI-type collagen is shown as SEQ ID NO.1. The recombinant human VI type collagen provided by the invention has good effects of promoting cell proliferation and adhesion, can protect cells and reduce oxidative stress injury, has a wide application prospect, and can be widely applied to the fields of medicines, medical instruments, biological materials, tissue engineering, cosmetics and the like.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and recombinant collagen technology, and specifically relates to a recombinant human type VI collagen, its coding gene, and an expression method thereof. Background Art

[0002] Type VI collagen is a component of the extracellular matrix of almost all connective tissues (including cartilage, bone, tendon, muscle, and cornea). In these tissues, it forms abundant and uniquely structured microfibrils and assembles in different ultrastructural forms. Type VI collagen has protective effects on a variety of cells: myofibroblasts, chondrocytes, neurons, fibroblasts, and cardiomyocytes. These protective effects include: anti-apoptosis and oxidative damage, regulation of autophagy, cell differentiation, and maintenance of stem cell characteristics. The absence of type VI collagen can trigger a series of pathological problems in muscles and other tissues, affecting key processes such as apoptosis and autophagy. As an adjustable binding platform, type VI collagen connects multiple collagens (I, II, IV, V, and XIV), various glycoproteins (fibronectin, fibulin-2, and matrilin), proteoglycans (decorin, perlecan, fibromodulin, laminin, biglycan, and aggrecan), hyaluronic acid, heparin, and other ECM factors and molecules. This extensive interaction network makes type VI collagen play an irreplaceable role in the organization and function of the extracellular matrix. The distribution and biological functions of type VI collagen in organisms are extensive, but the content is relatively low. Therefore, the development of recombinant human type VI collagen becomes necessary.

[0003] Currently, relevant patents have disclosed recombinant human type VI collagen and its preparation methods. For example, patent CN118206636 A discloses a type VI recombinant collagen, its preparation process, and uses. The obtained recombinant type VI collagen has certain biological activities and anti-aging effects, and has the best promotion effect on HSF (human skin fibroblasts) at a concentration of 0.5 mg / mL, with a cell absorbance of 120.63%. In addition, patent CN119409801A discloses a recombinant type VI collagen with antioxidant activity and its application. The obtained recombinant type VI collagen has a relatively obvious proliferation-promoting effect on HSF cells at a concentration of 5 mg / mL. The recombinant type VI collagen obtained in the above patents has certain biological activities at the said concentrations. However, to promote the further application of recombinant type VI collagen and meet the further needs of the market and scientific research, it is necessary to develop recombinant type VI collagen with higher performance.

[0004] Therefore, the present invention provides a method for efficiently obtaining type VI collagen, and the obtained recombinant human type VI collagen has good effects of promoting proliferation, promoting cell adhesion, and antioxidative stress, which will contribute to the research on the biological functions of type VI collagen and its application in the medical field. Summary of the Invention

[0005] The present invention analyzes the sequence of type VI collagen, selects the key sequence therein, optimizes the codons of the coding gene, synthesizes the coding gene, integrates it into a plasmid, and transfers it into a host bacterium. By screening, a strain expressing recombinant type VI collagen is provided, and through purification, purified type VI collagen is obtained. This protein has good cell activity, can protect cells from oxidative stress damage, and has the function of promoting the adhesion of human dermal papilla cells.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows: Technical Theme 1: The present invention provides a recombinant human type VI collagen, and its amino acid sequence is as shown in SEQ ID NO.1; SEQ ID NO.1: MHHHHHHGLDGEDGDKGLPGSSGEKGNPGRRGDKGPRGEKGERGDVGIRGDPGNPGQDSQERGPKGETGDLGPMGVPGRDGVPGGPGETGKNGGFGRRGPPGAKGNKGGPGQPGFEGEQGTRGAQGPAGPAGPPGLIGEQGISGPRGSGGAAGAPGERGRTGPLGRKGEPGEPGPKGGIGNRGPRGETGDDGRDGVGSEGRRGKKGERGFPGYPGPKGNPGEPGLNGTTGPKGIRGRRGNSGPPGIVGQKGDPGYPGPAGPKGNRGDSID.

[0007] Technical Theme 2: The present invention provides a nucleotide encoding the recombinant human type VI collagen described in Technical Theme 1, and the nucleotide sequence is as shown in SEQ ID NO.2; SEQ ID NO.2: CATATGCACCATCATCACCACCACGGGCTAGATGGAGAAGATGGCGACAAGGGCCTGCCGGGTTCTAGCGGTGAGAAGGGCAATCCGGGCCGTCGCGGTGACAAAGGGCCGCGTGGTGAGAAGGGCGAGCGCGGCGATGTTGGTATTCGCGGTGACCCGGGAAACCCGGGTCAAGATTCGCAAGAACGTGGCCCGAAGGGCGAGACTGGCGACCTCGGTCCTATGGGTGTCCCGGGCCGTGATGGCGTTCCGGGTGGTCCGGGTGAAACGGGTAAGAACGGTGGTTTTGGTCGCCGTGGCCCGCCGGGCGCTAAAGGCAACAAAGGTGGTCCGGGCCAACCGGGCTTCGAAGGTGAACAGGGCACCCGTGGTGCGCAGGGTCCGGCAGGCCCAGCGGGTCCACCGGGTCTGATTGGTGAGCAGGGTATCAGCGGTCCGCGTGGCTCCGGTGGCGCGGCTGGTGCCCCAGGCGAGCGCGGTCGCACCGGTCCACTGGGTCGTAAAGGTGAGCCGGGTGAACCGGGTCCCAAGGGGGGCATCGGTAATCGTGGTCCACGCGGCGAAACCGGCGACGATGGTAGAGACGGCGTGGGCAGCGAAGGTCGCCGTGGCAAGAAAGGTGAGCGTGGCTTCCCGGGCTATCCGGGTCCGAAAGGAAACCCGGGTGAGCCGGGTTTGAATGGCACCACCGGTCCGAAGGGCATCCGTGGCCGTAGAGGCAACAGCGGTCCGCCTGGCATCGTGGGTCAGAAAGGCGACCCGGGCTACCCGGGCCCGGCGGGTCCGAAAGGCAACCGTGGTGACTCCATTGATTAAGGTACC。

[0008] Technical Theme Three: The present invention provides an expression vector containing the nucleotide as described in Technical Theme Two.

[0009] Technical Theme Four: A host cell containing the nucleotide as described in Technical Theme Two or containing the expression vector as described in Technical Theme Three.

[0010] Technical Subject Five: A method for expressing recombinant human type VI collagen as described in Technical Subject One, which specifically includes the following steps: S1. Protein sequence design: Design an amino acid sequence based on human type VI collagen; S2. Gene design and synthesis: Reverse-design a coding nucleic acid sequence according to the amino acid sequence obtained in step S1, perform codon optimization to obtain a nucleotide sequence encoding recombinant human type VI collagen, and then perform gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type VI collagen fragment; S3. Construction of an expression vector: Connect the nucleic acid fragment obtained in S2 to a plasmid to obtain an expression vector; S4. Construction and screening of an expression strain: Transfer the expression vector obtained in step S3 into the competent host cell to obtain an expression strain; S5. Induced expression: Induce the expression of the expression strain obtained in step S4 and collect the bacterial solution; S6. Purification: Disrupt the bacteria in the bacterial solution collected in step S5, perform affinity chromatography, desalting, and lyophilization to obtain purified recombinant human type VI collagen.

[0011] Further, the plasmid described in S3 is pET30a(+).

[0012] Further, the nucleic acid fragment and the plasmid described in S3 are ligated through the multiple cloning sites NdeI and XhoI.

[0013] Further, the host cell is Escherichia coli BL21(DE3).

[0014] Further, the induced expression in S4 is carried out by adding an inducer IPTG to induce the strain to express.

[0015] Technical Subject Six: The present invention provides a use of the recombinant human type VI collagen as described in Technical Subject One in the preparation of pharmaceutical products, medical devices, biological materials, tissue engineering, or cosmetics.

[0016] The beneficial effects produced by adopting the above technical solutions are as follows: (1) The recombinant human type VI collagen provided by the present invention has been experimentally verified to have good cell migration-promoting and cell proliferation-promoting properties at the microgram level.

[0017] (2) The recombinant human type VI collagen provided by the present invention can promote the adhesion of dermal papilla cells and can produce significant effects at a concentration of 200 μg / mL.

[0018] (3) The recombinant human type VI collagen provided by the present invention has the ability to protect cells from oxidative stress damage.

[0019] (4) The recombinant human type VI collagen expression method provided by the present invention has a high yield, and the obtained recombinant human type VI collagen has a high purity, which is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the electrophoresis diagram of the purified sample of the recombinant human type VI collagen obtained by the present invention; Figure 2 It is the HPLC spectrum of the freeze-dried product of the recombinant human type VI collagen obtained by the present invention; Figure 3 It is the effect diagram of promoting cell migration of the recombinant human type VI collagen obtained by the present invention; Figure 4 It is the effect of promoting cell proliferation of the recombinant human type VI collagen obtained by the present invention; Figure 5 It is the effect diagram of promoting the adhesion of dermal papilla cells of the recombinant human type VI collagen obtained by the present invention; Figure 6 It is the antioxidant performance diagram of the recombinant human type VI collagen obtained by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0022] Example 1: Gene Design and Synthesis (1) Gene design: According to the sequence characteristics of human type VI collagen, the collagen sequence HC122 was designed, and its amino acid sequence is shown in SEQ ID NO.1. Its N-terminus starts with methionine to reduce the degradation rate of the protein; immediately followed by a His-Tag of 6 histidines for easy identification and purification. The amino acid sequence of the recombinant human type VI collagen fragment is shown below.

[0023] SEQ ID NO.1: MHHHHHHGLDGEDGDKGLPGSSGEKGNPGRRGDKGPRGEKGERGDVGIRGDPGNPGQDSQERGPKGETGDLGPMGVPGRDGVPGGPGETGKNGGFGRRGPPGAKGNKGGPGQPGFEGEQGTRGAQGPAGPAGPPGLIGEQGISGPRGSGGAAGAPGERGRTGPLGRKGEPGEPGPKGGIGNRGPRGETGDDGRDGVGSEGRRGKKGERGFPGYPGPKGNPGEPGLNGTTGPKGIRGRRGNSGPPGIVGQKGDPGYPGPAGPKGNRGDSID。

[0024] The coding nucleic acid sequence was reverse-designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host Escherichia coli. After the above optimization, the corresponding nucleic acid fragment encoding recombinant human type VI collagen was obtained, and Nde I and Kpn I restriction sites were added at the N-terminus and C-terminus, respectively. The nucleotide sequence corresponding to the above amino acid sequence is shown in SEQ ID NO.2.

[0025] SEQ ID NO.2: ATGGGGGTAAAAGGAGAAGCTGGTCTACCCGGAACCCCGGGTCCAACGGGTCCGGCAGGTCAGAAAGGCGAACCGGGTTCTGATGGTATCCCGGGCAGCGCAGGCGAAAAGGGCGAGCCGGGCCTCCCGGGACGCGGTTTCCCGGGTTTCCCGGGCGCGAAAGGCGACAAAGGCAGCAAGGGCGAGGTGGGCTTCGACGGTCGTAATGGTGAGAAGGGTGAAACCGGTGCGCCTGGTCTGAAAGGCGAGAATGGCTTGCCGGGCGAGAACGGCGCGCCGGGCCCGATGGGTCCGCGTGGTGCGCCAGGTGAGCGCGGTAGACCGGGCCTTCCGGGCGCTGCCGGCGCGCGTGGCGGGGCGGGCTCCCTGGGTGCCGGTGGCGCGTTTGGTGAAGCAGCGGGTGACCGTGGTCCGTACGGCACCGATATTGGTCCGGGCGGTGGTTATGGTGCCGCTGCTGAAGGTGGTATGTACGCGGGAAACGGCGGTTTGCTGGGTGCGGATTTTGCTGGCGACCTGGATTATAACGAACTGGCACATCATCACCACCACCATCACTAA。

[0026] (2) Gene synthesis: According to the above nucleic acid sequence, GenScript Biotech Corporation synthesized a nucleic acid fragment encoding a recombinant human type VI collagen fragment.

[0027] Example 2: Construction of expression vector pET30a(+)-HC122 The nucleic acid fragment obtained in Example 1 as shown in SEQ ID NO.2 was ligated to the pET30a(+) plasmid through the multiple cloning sites NdeI and KpnI to obtain the recombinant plasmid pET30a(+)-HC122.

[0028] Example 3: Construction of expression strain BL21(DE3) / pET30a(+)-HC122 The construction of the expression strain was carried out according to the method described in "Molecular Cloning: A Laboratory Manual (3rd Edition)" (written by J. Sambrook et al.). The specific steps are as follows: Pick a single colony of Escherichia coli BL21(DE3) into a test tube containing LB liquid medium. After inoculation, culture it overnight with shaking at 37°C; add 0.5 mL of the overnight LB culture to a 50-mL Erlenmeyer flask containing LB liquid medium, and culture it vigorously with shaking at 37°C for about 2 hours until the bacteria grow to the early logarithmic phase; transfer the bacteria to a 50-mL polypropylene tube pre-cooled with ice under sterile conditions, and place it on ice for 10 minutes; centrifuge at 4°C and 4000 rpm, pour out the supernatant, invert the tube to allow as much residual liquid to flow out as possible; add 6 mL of ice-precooled 0.1 mol / L CaCl2 to resuspend the pellet, and place it on ice for 30 minutes; centrifuge at 4°C and 3000 rpm, pour out the supernatant, invert the tube to allow as much residual liquid to flow out as possible; add 1.2 mL of ice-precooled 0.1 mol / L CaCl2 to resuspend the pellet (if preparing competent cells for storage at -70°C for later use, add 0.1 mol / L CaCl2 containing 20% glycerol to suspend the bacteria). After placing it at 4°C for 5 - 24 hours, pipette 200 μL of the competent cell suspension, add the recombinant plasmid pET30a(+)-HC122 prepared in Example 2 (volume < 10 μL, DNA < 50 ng), mix gently, and place it on ice for 30 minutes; heat shock at 42°C in a water bath for 90 seconds, and immediately cool it on ice; add 500 μL of liquid LB medium, mix well, and place it in a 37°C shaker and shake at low speed for 45 minutes to recover (or directly place it in a 37°C water bath for 1 hour after adding LB, and shake the tube in the middle to suspend the cells); pipette the transformed cells and spread them on a plate containing 50 μg / mL antibiotic (kanamycin), place it in an inverted position in a 37°C incubator for culture, and the grown colonies are the expression strain BL21(DE3) / pET30a(+)-HC122.

[0029] Example 4: Induced expression of the expression strain BL21(DE3) / pET30a(+)-HC122 Pick a single colony of the expression strain BL21(DE3) / pET30a(+)-HC122 prepared in Example 3 into an LB liquid medium containing 50 μg / mL Kan (kanamycin), culture it overnight at 37°C and 200 r / min to become an activated seed, and then inoculate it into a 5-L fermenter containing 3 L of complete medium at an inoculation amount of 3% for culture. Control the temperature at 37°C, dissolved oxygen at 30%, and pH at 7.0 during the fermentation process. When OD 600 reaches 60, add IPTG (isopropyl-β-D-thiogalactoside) with a final concentration of 0.5 mM for induced expression, and continue to culture for 12 hours, then centrifuge to collect the bacterial cells.

[0030] Example 5: Purification of recombinant human type VI collagen HC122 The specific purification steps of HC122 are as follows: 1. Bacterial lysis: The cells prepared in Example 4 were resuspended with an appropriate amount of cell-lysing solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH = 7.5). The ratio of cells to cell-lysing solution (m / v) was 1:5 - 1:20, and 1:10 was selected in this example. The cells were disrupted using a high-pressure homogenizer. The disrupted cell suspension was centrifuged at 12,000 g for 1 h, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane to obtain the supernatant solution.

[0031] 2. Affinity chromatography: (1) Column equilibration: The Ni Sepharose FF chromatography column was equilibrated with buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) until the baseline was stable.

[0032] (2) Sample loading: The supernatant solution obtained after filtration in step 1 was loaded onto the chromatography column, and the column retention time was controlled to be not less than 5 min. The flow-through was detected by electrophoresis.

[0033] (3) Column washing: The chromatography column was washed with buffer A until the baseline was stable, with a volume not less than 5 column volumes.

[0034] (4) Elution of target protein: Elution was performed with 80% Buffer A and 20% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole) to obtain the eluate. The eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human type VI collagen HC122.

[0035] 3. Desalting: The solution containing recombinant human type VI collagen HC122 obtained in step 2 was replaced with purified water using a 5 kDa ultrafiltration membrane device by equal-volume displacement to remove the salt in the solution. After filtration through a 0.22 μm filter membrane, it was freeze-dried to obtain the freeze-dried recombinant collagen product, with a yield of 8 g / L fermentation broth.

[0036] Test Example 1: SDS-PAGE protein electrophoresis detection Sample preparation: The cells prepared in Example 4 and the whole cells, supernatant, flow-through, and eluate in Example 5 were collected, and each was mixed evenly with loading buffer, heated in a boiling water bath for 10 min, and then cooled naturally for standby. A GenScript SurePAGE™ precast gel (4 - 12%) was selected for loading, and electrophoresis was performed at 140 V for 45 - 55 minutes until the bromophenol blue band ran to the bottom of the gel.

[0037] Coomassie Brilliant Blue R-250 Staining Using a Microwave Oven: (1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R250 at a final concentration of 0.1% (W / V) in a solution of 40% ethanol and 10% acetic acid. (2) Prepare the decolorizing solution: Dissolve ethanol at a final concentration of 10% (V / V) and acetic acid at 7.5% (V / V) together. (3) After electrophoresis, pry open the gel plate to remove the gel, and then place it in a staining container containing 100 mL of the staining solution. (4) Cover the container lid and place it in the microwave oven to heat at high power for 8 minutes. To avoid danger, note that the solution should not be allowed to boil. (5) Remove the staining container from the microwave oven and gently shake it on a decolorizing shaker at room temperature for 5 minutes. (6) Pour out the staining solution and carefully wash the gel with deionized water. (7) Pour out the deionized water and add 100 mL of the decolorizing solution. (8) Cover the lid and place it in the microwave oven to heat at high power for 8 minutes. (9) Pour out the decolorizing solution, add fresh decolorizing solution, and repeat step 8. (10) Remove it from the microwave oven and gently shake it on a decolorizing shaker at room temperature until the background is clear.

[0038] As Figure 1 shown, after one-step chromatography of recombinant type VI collagen, almost no bands of impurity proteins can be seen in electrophoresis, indicating that the recombinant human type VI collagen obtained in the present invention has a high purity.

[0039] Test Example 2: HPLC Detection Take 10 mg of the freeze-dried product of the recombinant collagen obtained in Example 5, dissolve and dilute it to 1 mg / mL with the mobile phase (0.1 M phosphate buffer PB + 0.2 M NaCl). Refer to the "Molecular Exclusion Chromatography" in the "Chromatography" section of the "Pharmacopoeia of the People's Republic of China" (2020 Edition) for determination. Detection conditions: Chromatographic column: TSKgel G3000SWxl, flow rate: 0.5 mL / min, column oven temperature: 25 °C, mobile phase: phosphate buffer solution, detector wavelength: 220 nm.

[0040] Preparation of the mobile phase (phosphate buffer solution PBS): 0.5 M NaH2PO4·2H2O solution: Weigh 78 g, add 800 mL of purified water, dissolve it with magnetic stirring, and make up the volume to 1000 mL with purified water; 0.5 M Na2HPO4·12H2O solution: Weigh 179.07 g, add 800 mL of purified water, dissolve it with magnetic stirring, and make up the volume to 1000 mL with purified water; Take 61 mL of the 0.5 M Na2HPO4·12H2O solution, 39 mL of the NaH2PO4·2H2O solution, and 5.84 g of NaCl, make up the volume to 500 mL with purified water, filter it through a 0.45 µm microporous membrane, and degas it by ultrasonic treatment to obtain a solution of (0.1 M PB + 0.2 M NaCl) with a pH of 6.85.

[0041] As Figure 2 shown, the purity of the sample was calculated by the area normalization method. It was calculated that the purity of the recombinant human type VI collagen obtained in the present invention was higher than 98%, reaching the purity ≥ 95% specified in "YY / T 1888-2023 Recombinant Humanized Collagen".

[0042] Effect Example 1: Recombinant human type VI collagen HC122 promotes cell migration test Use a marker pen to draw marks on the back of each well of a 6-well culture plate, dividing it into three equal parts horizontally and vertically. Seed about 10×10 5 L929 mouse fibroblasts in each well. When the cells reached 95% confluence, a cell scratch was formed with a 10 μL pipette tip. Rinse the cells 3 times with PBS to remove the scratched cells. Take a photo under the microscope with the intersection of the horizontal and vertical lines as the center, and record the scratch area at this time, denoted as the scratch area at 0 h.

[0043] Add 100 μL of medium to the blank control group, and add 100 μL of sample solution to the experimental group. The blank control group and the experimental group were continued to be cultured under the same conditions. After culturing for 24 h and 48 h, take photos under the microscope.

[0044] Among them, the sample solution of the experimental group was: the HC122 freeze-dried product obtained in Example 6 was dissolved with complete medium, filtered and sterilized with a 0.22 μm filter membrane, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL respectively.

[0045] As Figure 3 shown, the recombinant human type VI collagen HC122 obtained in the present invention has good cell migration-promoting performance at the microgram level.

[0046] Effect Example 2: Recombinant human type VI collagen HC122 promotes cell proliferation test After the HaCaT cells were digested with trypsin, they were seeded at 8×10 3 / well. After inoculating the cells in a 96-well culture plate, place it in an incubator at 37°C and 5% CO2 for 24 h. Discard the culture medium in the wells. Add 100 μL of medium to each well of the blank control group, and add 100 μL of sample solution to each well of the experimental group. Place the 96-well culture plate in an incubator at 37°C and 5% CO2 for 24 h. Add 10 μL of CCK8 reagent to each well and incubate in the incubator for 2 h. After incubation, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at a wavelength of 450 nm for each well. The absorbance value of the blank control group was set as 100%, and the ratio of the absorbance of each group to that of the blank control group × 100% was the cell viability of each group.

[0047] Among them, the sample solution of the experimental group was as follows: The freeze-dried product of HC122 obtained in Example 6 was dissolved in complete medium, filtered and sterilized with a 0.22 μm filter membrane, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL respectively.

[0048] As Figure 4 shown, the recombinant human type VI collagen HC122 obtained in the present invention has good cell proliferation effect at the microgram level. The absorbance of HaCaT cells at a concentration of 10 μg / mL HC122 is significantly different from that of the blank group.

[0049] Effect Example 3: Recombinant human type VI collagen HC122 promotes the adhesion test of dermal papilla cells Digest human dermal papilla cells HDPC and count them. Inoculate 1×10 5 cells per well in a 96-well plate, 100 μL per well. Aspirate the medium and wash once with PBS. The blank control group (BC) was added with 100 μL of basic medium, the experimental group was added with 150 μL of sample solution, and the positive control group was added with 100 μL of basic medium (the sample maximum enzyme activity control group, PC). Incubate for 24 h under the conditions of 5% CO2, 37 °C, and humidity greater than 90%.

[0050] One hour before detection, the positive control group was added with LDH release reagent, and the addition amount was 10% of the culture medium volume (10 μL / well). After adding the LDH release reagent, pipette and mix well, and continue to incubate for 1 h. Before detection, centrifuge the cell culture plate at 400 g for 5 min with a multi-well centrifuge. Take 120 μL of the supernatant into a new 96-well plate; add 60 μL of LDH detection solution to each well, mix well, incubate at room temperature in the dark for 30 min; measure the absorbance at 490 nm, and the ratio of the absorbance of each group to that of the blank control group is the LDH release ratio.

[0051] Among them, the sample solution of the experimental group was as follows: The freeze-dried product of HC122 obtained in Example 6 was dissolved in complete medium, filtered and sterilized with a 0.22 μm filter membrane, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL respectively. The LDH release agent and the LDH detection solution were from the LDH detection kit (Beyotime, C0016).

[0052] The results are as Figure 5 shown. Compared with the control group, the recombinant human type VI collagen HC122 obtained in the present invention can increase the LDH secretion level of human dermal papilla cells HDPC at a concentration of 100 μg / mL - 800 μg / mL, thereby improving the cell adhesion ability. HC122 has an obvious effect at a concentration of 200 μg / mL.

[0053] Effect Example 4: Antioxidant Property of Recombinant Type VI Collagen HC122 Adjust the density of human dermal papilla cells HDPC to 5×10 5 cells / mL. Use a multi-channel pipette to add 500 μL of human dermal papilla cell HDPC cell suspension to a 24-well tissue culture microtiter plate, with 3 replicate wells in each group. Incubate the cells for 24 h (5% CO2, 37 °C, >90% humidity) to form a semi-confluent monolayer.

[0054] Aspirate the culture medium, and add 500 μL of sample or complete culture medium to each well, and incubate for 24 h (5% CO2, 37 °C, >90% humidity).

[0055] Dilute DCFH-DA (2',7'-dichlorofluorescein diacetate) with serum-free medium at a ratio of 1:1000 to make the final concentration 10 μM. Aspirate the culture medium, and add an appropriate volume of the diluted DCFH-DA working solution to cover the cells. Incubate at 37 °C for 20 - 30 min. Wash the cells 1 - 2 times with serum-free medium to fully remove the DCFH-DA that has not entered the cells. Directly take pictures using fluorescence channel 1 (excitation wavelength 488 nm, emission wavelength 525 nm / FITC).

[0056] Among them, the sample of the blank control group is: complete culture medium; The samples of the experimental groups are: the lyophilized product of HC122 obtained in Example 6 is dissolved with complete culture medium, filtered and sterilized with a 0.22 μm filter membrane, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL respectively; DCFH-DA itself has no fluorescence, but can freely cross the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH (dichlorodihydrofluorescein), and DCFH cannot penetrate the cell membrane, so that the probe can be easily loaded into the cell. Reactive oxygen species in the cell can oxidize non-fluorescent DCFH to generate fluorescent DCF (2',7'-dichlorofluorescein). Detecting the fluorescence of DCF can know the level of reactive oxygen species in the cell.

[0057] The results are as Figure 6 shown. The fluorescence intensity of the experimental group added with recombinant human type VI collagen HC122 is significantly less than that of the negative control group without adding recombinant human type VI collagen HC122. Moreover, as the content of the added recombinant human type VI collagen increases, the fluorescence intensity gradually weakens, proving that the recombinant human type VI collagen obtained in the present invention has the ability of antioxidant stress.

[0058] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human type VI collagen, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

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

2.

3. An expression vector, characterized in that: Containing the nucleotide as claimed in claim 2.

4. A host cell, characterized in that Containing the nucleotide according to claim 2 or containing the expression vector according to claim 3.

5. A method for expressing recombinant human type VI collagen as claimed in claim 1, characterized in that: The specific steps include: S1. Protein sequence design: design the amino acid sequence based on human type VI collagen; S2. Gene design and synthesis: Reverse design of a coding nucleic acid sequence according to the amino acid sequence obtained in step S1, and codon optimization to obtain a nucleotide sequence encoding recombinant human type VI collagen, and then gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type VI collagen fragment; S3, construction of expression vector: connect the nucleic acid fragment obtained in S2 to the plasmid to obtain an expression vector; S4, construction and screening of expression strains: the expression vector obtained in step S3 is transferred into a competent host cell to obtain an expression strain; S5, inducing expression: inducing expression of the expression strain obtained in step S4, and collecting bacterial liquid; S6. Purification: The bacterial liquid collected in step S5 is subjected to bacterial cell disruption, affinity chromatography, desalting and freeze-drying to obtain recombinant human type VI collagen.

6. The expression method according to claim 5, characterized in that: The plasmid S3 is pET30a(+).

7. The expression method according to claim 5, characterized in that: The nucleic acid fragment and plasmid S3 are connected via the multiple cloning sites NdeI and XhoI.

8. The expression method according to claim 5, characterized in that: The host cell is Escherichia coli BL21 (DE3).

9. The expression method according to claim 5, characterized in that: The induced expression described in S5 is induced by adding the inducer IPTG to induce the strain to express.

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