A recombinant human collagen type VI, its encoding gene and expression method

Abstract

The application belongs to the technical field of genetic engineering and recombinant collagen, and discloses a recombinant human VI type collagen, a coding gene and an expression method thereof, and an 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 application has good effects of promoting cell proliferation and adhesion, and can protect cells and reduce oxidative stress damage, has a wide application prospect, and can be widely applied to the fields of medicine, medical devices, biological materials, tissue engineering, cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and recombinant collagen, and particularly relates to a recombinant human collagen type VI, a coding gene and an expression method thereof. BACKGROUND

[0002] Collagen type VI is a component of the extracellular matrix of almost all connective tissues, including cartilage, bone, tendon, muscle and cornea, where it forms abundant and structurally unique microfibrils and assembles in different ultrastructural forms. Collagen type VI has protective effects on a variety of cells: myofibroblasts, chondrocytes, neurons, fibroblasts and cardiomyocytes. These protective effects include: anti-apoptotic and oxidative damage, regulation of autophagy and maintenance of cell differentiation and stem cell properties, etc. The absence of collagen type VI triggers a series of pathological problems in muscle and other tissues, affecting key processes such as apoptosis and autophagy. Collagen type VI serves as a tunable binding platform that connects a variety of collagens (I, II, IV, V and XIV), various glycoproteins (fibronectin, perlecan-2 and matrilin), proteoglycans (core proteoglycan, basement membrane proteoglycan, fibromodulin, basement membrane proteoglycan, biglycan and aggrecan), hyaluronic acid and heparin, as well as other ECM factors and molecules. This extensive network of interactions makes collagen type VI irreplaceable in the organization and function of the extracellular matrix. Collagen type VI is widely distributed and has a wide range of biological functions in the body, but its content is relatively low. Therefore, the development of recombinant human collagen type VI is necessary.

[0003] At present, there are related patents that disclose recombinant human collagen type VI and its preparation method. For example, patent CN118206636 A discloses a collagen type VI recombinant protein, its preparation process and use, and the obtained recombinant collagen type VI has certain biological activity and anti-aging effect, and the promotion effect on HSF (human skin fibroblasts) is best at a concentration of 0.5 mg / mL, and the cell absorbance is 120.63%. In addition, patent CN119409801 A discloses a recombinant collagen type VI with antioxidant activity and its application, and the obtained recombinant collagen type VI has a relatively obvious proliferation effect on HSF cells at a concentration of 5 mg / mL. The recombinant collagen type VI obtained in the above patents has certain biological activity at the concentration, however, in order to promote the further application of recombinant collagen type VI and cater to the further needs of market and scientific research, it is necessary to develop recombinant collagen type VI with higher performance.

[0004] Therefore, the application provides a method for efficiently obtaining collagen VI, and the obtained recombinant human collagen VI has good effects of promoting proliferation, promoting cell adhesion and resisting oxidative stress, and will help the research on the biological function of collagen VI and the application of collagen VI in the medical field. SUMMARY

[0005] The application selects a key sequence in collagen VI by analyzing the sequence of collagen VI, optimizes the coding gene by codon, synthesizes the coding gene, integrates the coding gene into a plasmid, and transfers the plasmid into a host bacterium, thereby providing a strain for expressing recombinant collagen VI protein, and obtaining purified collagen VI protein through purification. The protein has good cell activity, can protect cells from oxidative stress damage, and has the function of promoting human dermal hair papilla cell adhesion.

[0006] To achieve the above object, the technical scheme adopted by the application is as follows:

[0007] Technical subject one:

[0008] The application provides a recombinant human collagen VI, and the amino acid sequence of the recombinant human collagen VI is shown as SEQ ID NO. 1.

[0009] SEQ ID NO. 1:

[0010] MHHHHHHGLDGEDGDKGLPGSSGEKGNPGRRGDKGPRGEKGERGDVGIRGDPGNPGQDSQERGPKGETGDLGPMGVPGRDGVPGGPGETGKNGGFGRRGPPGAKGNKGGPGQPGFEGEQGTRGAQGPAGPAGPPGLIGEQGISGPRGSGGAAGAPGERGRTGPLGRKGEPGEPGPKGGIGNRGPRGETGDDGRDGVGSEGRRGKKGERGFPGYPGPKGNPGEPGLNGTTGPKGIRGRRGNSGPPGIVGQKGDPGYPGPAGPKGNRGDSID.

[0011] Technical subject two:

[0012] The application provides a nucleic acid for coding the recombinant human collagen VI in technical subject one, and the nucleic acid sequence is shown as SEQ ID NO. 2.

[0013] SEQ ID NO. 2:

[0014] CAT ATG CAC CAT CAT CAC CAC CAC GGG CTA GAT GGA G AAG ATG GCG ACA AGG GCCTGCCGGGTTCTAGCGGTGAGAAGGGCAATCCGGGCCGTCGCGGTGACAAAGGGCCGCGTGGTGAGAAGGGCGAGCGCGGCGATGTTGGTATTCGCGGTGACCCGGGAAACCCGGGTCAAGATTCGCAAGAACGTGGCCCGAAGGGCGAGACTGGCGACCTCGGTCCTATGGGTGTCCCGGGCCGTGATGGCGTTCCGGGTGGTCCGGGTGAAACGGGTAAGAACGGTGGTTTTGGTCGCCGTGGCCCGCCGGGCGCTAAAGGCAACAAAGGTGGTCCGGGCCAACCGGGCTTCGAAGGTGAACAGGGCACCCGTGGTGCGCAGGGTCCGGCAGGCCCAGCGGGTCCACCGGGTCTGATTGGTGAGCAGGGTATCAGCGGTCCGCGTGGCTCCGGTGGCGCGGCTGGTGCCCCAGGCGAGCGCGGTCGCACCGGTCCACTGGGTCGTAAAGGTGAGCCGGGTGAACCGGGTCCCAAGGGGGGCATCGGTAATCGTGGTCCACGCGGCGAAACCGGCGACGATGGTAGAGACGGCGTGGGCAGCGAAGGTCGCCGTGGCAAGAAAGGTGAGCGTGGCTTCCCGGGCTATCCGGGTCCGAAAGGAAACCCGGGTGAGCCGGGTTTGAATGGCACCACCGGTCCGAAGGGCATCCGTGGCCGTAGAGGCAACAGCGGTCCGCCTGGCATCGTGGGTCAGAAAGGCGACCCGGGCTACCCGGGCCCGGCGGGTCCGAAAGGCAACCGTGGTGACTCCATTGATTAAGGTACC.

[0015] Technical subject three:

[0016] The present invention provides an expression vector comprising the nucleotide as described in technical subject two.

[0017] Technical subject four:

[0018] A host cell comprising the nucleotide as described in technical subject two or comprising the expression vector as described in technical subject three.

[0019] Technical subject five:

[0020] A technical subject one expression method of the recombinant human collagen type VI, which specifically comprises the following steps:

[0021] S1, protein sequence design: according to human collagen type VI, design amino acid sequence;

[0022] S2, gene design and synthesis: according to the amino acid sequence obtained in step S1, the reverse design coding nucleic acid sequence is carried out, and the codon optimization is carried out, the nucleotide sequence coding the recombinant human collagen type VI is obtained, then the gene synthesis is carried out, the nucleic acid fragment coding the recombinant human collagen type VI fragment is obtained;

[0023] S3, construction of expression vector: the nucleic acid fragment obtained in S2 is connected to the plasmid to obtain the expression vector;

[0024] S4, construction and screening of expression strain: the expression vector obtained in step S3 is transformed into host cell competent cell to obtain expression strain;

[0025] S5, induction expression: the expression strain obtained in step S4 is induced to express, and the bacterial liquid is collected;

[0026] S6, purification: the bacterial liquid collected in step S5 is broken, affinity chromatography, desalting and freeze-drying, and the purified recombinant human collagen type VI is obtained.

[0027] Further, the plasmid in S3 is pET30a (+).

[0028] Further, the nucleic acid fragment and the plasmid in S3 are connected through the multiple cloning sites NdeI and Kpn I.

[0029] Further, the host cell is E. coli BL21 (DE3).

[0030] Further, the induction expression in S4 is induced by adding inducer IPTG to induce the strain to express.

[0031] Technical subject six:

[0032] The present application provides a kind of technical subject one described recombinant human collagen type VI in the use of preparation medicine product, medical device, biological material, tissue engineering or cosmetics.

[0033] The beneficial effects produced by the above technical scheme are as follows:

[0034] (1) the recombinant human collagen type VI provided by the present application has good cell migration and cell proliferation performance in microgram level verified by experiment.

[0035] (2) The recombinant human collagen VI provided by the application can promote dermal hair papilla cell adhesion, and can produce a significant effect at a concentration of 200 μg / mL.

[0036] (3) The recombinant human collagen VI provided by the application has the ability to protect cells from oxidative stress damage.

[0037] (4) The recombinant human collagen VI provided by the application has a high yield, and the obtained recombinant human collagen VI has high purity, and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Electrophoretogram of a purified sample of the recombinant human collagen VI obtained by the application;

[0039] Figure 2 HPLC chromatogram of a lyophilized product of the recombinant human collagen VI obtained by the application;

[0040] Figure 3 Diagram of the cell migration promoting effect of the recombinant human collagen VI obtained by the application;

[0041] Figure 4 Diagram of the cell proliferation promoting effect of the recombinant human collagen VI obtained by the application;

[0042] Figure 5 Diagram of the dermal hair papilla cell adhesion promoting effect of the recombinant human collagen VI obtained by the application;

[0043] Figure 6 Diagram of the antioxidant performance of the recombinant human collagen VI obtained by the application. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the application more clear, the application will be described clearly and completely below in combination with specific examples.

[0045] Example 1: Gene design and synthesis

[0046] (1) Gene design:

[0047] According to the sequence characteristics of human collagen VI, a collagen protein sequence HC122 is designed, and the amino acid sequence is shown as SEQ ID NO. 1. The N-terminus starts with methionine to reduce the degradation rate of the protein; followed by a His-Tag of 6 histidines to facilitate identification and purification. The amino acid sequence of the recombinant human collagen VI fragment is as follows.

[0048] SEQ ID NO. 1:

[0049] MHHHHHHGLDGEDGDKGLPGSSGEKGNPGRRGDKGPRGEKGERGDVGIRGDPGNPGQDSQERGPKGETGDLGPMGVPGRDGVPGGPGETGKNGGFGRRGPPGAKGNKGGPGQPGFEGEQGTRGAQGPAGPAGPPGLIGEQGISGPRGSGGAAGAPGERGRTGPLGRKGEPGEPGPKGGIGNRGPRGETGDDGRDGVGSEGRRGKKGERGFPGYPGPKGNPGEPGLNGTTGPKGIRGRRGNSGPPGIVGQKGDPGYPGPAGPKGNRGDSID.

[0050] The nucleic acid sequence encoding the amino acid sequence of the recombinant human collagen type VI was designed by 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 nucleic acid fragment encoding the recombinant human collagen type VI was obtained, and Nde I and Kpn I enzyme cutting 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.

[0051] SEQ ID NO. 2:

[0052] CAT ATG CAC CAT CAT CAC CAC CAC GGG CTA GAT GGA G AAG ATG GCG ACA AGG GCCTGCCGGGTTCTAGCGGTGAGAAGGGCAATCCGGGCCGTCGCGGTGACAAAGGGCCGCGTGGTGAGAAGGGCGAGCGCGGCGATGTTGGTATTCGCGGTGACCCGGGAAACCCGGGTCAAGATTCGCAAGAACGTGGCCCGAAGGGCGAGACTGGCGACCTCGGTCCTATGGGTGTCCCGGGCCGTGATGGCGTTCCGGGTGGTCCGGGTGAAACGGGTAAGAACGGTGGTTTTGGTCGCCGTGGCCCGCCGGGCGCTAAAGGCAACAAAGGTGGTCCGGGCCAACCGGGCTTCGAAGGTGAACAGGGCACCCGTGGTGCGCAGGGTCCGGCAGGCCCAGCGGGTCCACCGGGTCTGATTGGTGAGCAGGGTATCAGCGGTCCGCGTGGCTCCGGTGGCGCGGCTGGTGCCCCAGGCGAGCGCGGTCGCACCGGTCCACTGGGTCGTAAAGGTGAGCCGGGTGAACCGGGTCCCAAGGGGGGCATCGGTAATCGTGGTCCACGCGGCGAAACCGGCGACGATGGTAGAGACGGCGTGGGCAGCGAAGGTCGCCGTGGCAAGAAAGGTGAGCGTGGCTTCCCGGGCTATCCGGGTCCGAAAGGAAACCCGGGTGAGCCGGGTTTGAATGGCACCACCGGTCCGAAGGGCATCCGTGGCCGTAGAGGCAACAGCGGTCCGCCTGGCATCGTGGGTCAGAAAGGCGACCCGGGCTACCCGGGCCCGGCGGGTCCGAAAGGCAACCGTGGTGACTCCATTGATTAAGGTACC.

[0053] (2) Gene synthesis:

[0054] According to the nucleic acid sequence shown above, a nucleic acid fragment encoding the recombinant human collagen type VI fragment was synthesized by Kings River Biotech Co., Ltd.

[0055] Example 2: Construction of expression vector pET30a(+)-HC122

[0056] The nucleic acid fragment of SEQ ID NO. 2 obtained in Example 1 was ligated to the pET30a(+) plasmid through the multiple cloning sites Ndel and Kpnl to obtain the recombinant plasmid pET30a(+)-HC122.

[0057] Example 3: Construction of expression strain BL21(DE3) / pET30a(+)-HC122

[0058] The construction of the expression strain was performed according to the method described in "Molecular Cloning: A Laboratory Manual (3rd Edition)" (J. Sambrook et al.), and the specific steps were as follows:

[0059] An E. coli BL21 (DE3) single colony was picked into a test tube containing LB liquid medium, and after inoculation, it was cultured at 37°C overnight with shaking. 0.5 mL of the overnight LB culture was added to a 50 mL LB liquid medium in a triangular flask, and the bacteria were cultured at 37°C with vigorous shaking for about 2 hours to grow to the early logarithmic phase. The bacteria were transferred to a 50 mL polypropylene tube pre-cooled with ice under sterile conditions, and placed on ice for 10 minutes. Centrifugation was performed at 4°C and 4000 rpm, and the supernatant was poured out. The tube was inverted to allow the residual liquid to flow out as much as possible. 6 mL of 0.1 mol / L CaCl2 pre-cooled with ice was added to resuspend the precipitate, and placed on ice for 30 minutes. Centrifugation was performed at 4°C and 3000 rpm, and the supernatant was poured out. The tube was inverted to allow the residual liquid to flow out as much as possible. 1.2 mL of 0.1 mol / L CaCl2 pre-cooled with ice was added to resuspend the precipitate (if you want to prepare -70°C stored for standby competent cells, add 0.1 mol / L CaCl2 containing 20% glycerol to suspend the bacteria), and placed at 4°C for 5-24 hours. 200 μL of competent cell suspension was taken, and the recombinant plasmid pET30a(+)-HC122 prepared in Example 2 (volume <10 μL, DNA <50 ng) was added, mixed gently, and placed on ice for 30 minutes. 42°C water bath was used for static heat shock for 90 seconds, and then immediately placed on ice for cooling. 500 μL of liquid LB medium was added, mixed well, and placed in a 37°C shaking incubator for recovery for 45 minutes (LB can also be added directly to a 37°C water bath for recovery for 1 hour, and the tube was shaken in the middle to suspend the cells). The transformed cells were taken and plated on a plate containing 50 μg / mL of antibiotic (kanamycin), and placed in a 37°C incubator for inverted culture. The colonies that grew were the expression strain BL21(DE3) / pET30a(+)-HC122.

[0060] Example 4: Induced expression of expression strain BL21(DE3) / pET30a(+)-HC122

[0061] A single colony of the expression strain BL21(DE3) / pET30a(+)-HC122 prepared in Example 3 was inoculated into LB liquid medium containing 50 μg / mL Kan (kanamycin) and incubated at 37°C, 200 r / min overnight to become activated seed, which was then inoculated into a 5L fermenter containing 3L complete medium at a 3% inoculation amount for incubation. The fermentation process was controlled at a temperature of 37°C, a dissolved oxygen of 30%, and a pH of 7.0. When the OD 600 reached 60, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM for induction of expression, and incubation was continued for 12 hours. The bacterial cells were collected by centrifugation.

[0062] Example 5: Purification of Recombinant Human Collagen Type VI HC122

[0063] The specific purification steps of HC122 are as follows:

[0064] 1. Cell disruption:

[0065] The bacterial cells prepared in Example 4 were resuspended with an appropriate amount of cell disruption solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) at a ratio (m / v) of 1:5 to 1:20, and a ratio of 1:10 was selected in this example. The bacterial cells were disrupted using a high-pressure homogenizer. The disrupted bacterial solution was centrifuged at 12000 g for 1 h, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane to obtain a supernatant.

[0066] 2. Affinity chromatography:

[0067] (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.

[0068] (2) Sample loading: The supernatant obtained after filtration in step 1 was loaded onto the chromatography column, and the column retention time was controlled to be no less than 5 min. The flow-through was detected by electrophoresis.

[0069] (3) Column washing: The chromatography column was washed with buffer A until the baseline was stable, and the volume of buffer A used was no less than 5 column volumes.

[0070] (4) Elution of target protein: Elution was performed using 80% buffer A and 20% buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole), and the eluate was collected in a new centrifuge tube to obtain a solution containing recombinant human collagen type VI HC122.

[0071] 3. Desalting:

[0072] The solution containing the recombinant human collagen type VI HC122 obtained in step 2 was subjected to volume replacement with purified water using an ultrafiltration membrane with a molecular weight cut-off of 5 kDa to remove salts from the solution, and the solution was filtered through a 0.22 μm filter and lyophilized to obtain a recombinant collagen lyophilisate, with a yield of 8 g / L of fermentation broth.

[0073] Test Example 1: SDS-PAGE protein electrophoresis detection

[0074] Sample treatment: The bacteria prepared in Example 4 and the whole bacteria, supernatant, flow-through and eluate in Example 5 were collected, mixed with loading buffer, boiled in a water bath for 10 minutes, and naturally cooled for use. The Kindsure SurePAGE™ precast gel (4-12%) was selected for loading, and electrophoresis was performed at a voltage of 140 V for 45-55 minutes until the bromophenol blue band reached the bottom of the gel.

[0075] Microwave staining of Coomassie Brilliant Blue R-250: (1) Prepare the staining solution: dissolve Coomassie Brilliant Blue R-250 in a 40% ethanol and 10% acetic acid solution to a final concentration of 0.1% (W / V). (2) Prepare the destaining solution: dissolve 10% (V / V) ethanol and 7.5% (V / V) acetic acid together. (3) After electrophoresis, pry open the gel plate and remove the gel, then place it in a staining container containing 100 mL of the staining solution. (4) Cover the container and place it in a microwave oven at high heat for 8 minutes. To avoid danger, please do not let the solution boil. (5) Remove the staining container from the microwave oven and place it on a destaining shaker at room temperature for 5 minutes. (6) Pour off the staining solution and carefully wash the gel with deionized water. (7) Pour off the deionized water and add 100 mL of the destaining solution. (8) Cover the container and place it in a microwave oven at high heat for 8 minutes. (9) Pour off the destaining solution and add new destaining solution, and repeat step 8. (10) Remove from the microwave oven and place on a destaining shaker at room temperature for gentle shaking until the background is clear.

[0076] As Figure 1 shown, after one-step chromatography, the recombinant collagen type VI has almost no impurity bands in electrophoresis, indicating that the recombinant human collagen type VI obtained by the present application has high purity.

[0077] Test Example 2: HPLC detection

[0078] Take the recombinant collagen freeze-dried product of 10 mg obtained in Example 5, dissolve and dilute to 1 mg / mL with the mobile phase (0.1M phosphate buffer PB+0.2M NaCl). According to the “molecular exclusion chromatography” of “chromatography” in the “People's Republic of China Pharmacopoeia” (2020 edition), the detection conditions are: chromatographic column: TSKgel G3000SWxl, flow rate: 0.5 mL / min, column temperature: 25℃, mobile phase: phosphate buffer solution, detector wavelength: 220 nm.

[0079] The mobile phase (phosphate buffer solution PBS) is prepared as follows:

[0080] 0.5M NaH2PO4·2H2O solution: weigh 78g, add 800mL purified water, magnetically stir to dissolve, add purified water to constant volume to 1000mL;

[0081] 0.5M Na2HPO4·12H2O solution: weigh 179.07g, add 800mL purified water, magnetically stir to dissolve, add purified water to constant volume to 1000mL;

[0082] Take 0.5M Na2HPO4·12H2O solution 61mL, NaH2PO4·2H2O solution 39mL, NaCl 5.84g, constant volume to 500mL with purified water, filter with 0.45µm microporous filter membrane, then ultrasonic degassing, to obtain (0.1M PB+0.2M NaCl) solution with pH=6.85.

[0083] As shown in Figure 2 , the sample purity is calculated by area normalization method, and it is calculated that the recombinant human collagen VI obtained by the application has a purity higher than 98%, reaching the purity ≥95% specified in “YY / T 1888-2023 Recombinant human collagen”.

[0084] Effect example 1: recombinant human collagen VI HC122 promotes cell migration test

[0085] Marker pen is used to draw lines on the back of a 6-well culture plate, with three equal parts in the horizontal and vertical directions of each well. About 10×10 5 L929 mouse fibroblasts are plated in each well. When the cells reach 95% confluence, 10µL of the gun head is used to draw a line to form a cell scratch. The cells are rinsed with PBS for 3 times to remove the scratched cells. The intersection of the horizontal and vertical lines is taken as the core, and a photo is taken under a microscope to record the scratch area at this time, which is recorded as the 0h scratch area.

[0086] The blank control group is added with 100μL of culture medium, and the experimental group is added with 100μL of sample liquid. The blank control group and the experimental group are continuously cultured under the same conditions. After 24h and 48h of culture, a photo is taken under a microscope.

[0087] The sample liquid of the experimental group is: the HC122 lyophilized product obtained in Example 5 is dissolved with complete culture medium, filtered with a 0.22 μm filter membrane to remove bacteria, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively.

[0088] As shown in Table 1, the recombinant human collagen type VI HC122 obtained by the application has good cell adhesion promoting effect at the microgram level. Figure 3 As shown in Table 1, the recombinant human collagen type VI HC122 obtained by the application has good cell adhesion promoting effect at the microgram level.

[0089] Effect Example 2: Recombinant human collagen type VI HC122 promotes cell proliferation test

[0090] After trypsin digestion, HaCaT cells are inoculated at 8×10 3 After inoculating cells in the 96-well culture plate, the plate is placed in a 37°C, 5% CO2 incubator and cultured for 24 h. The culture solution in the hole is discarded, 100 μL of culture medium is added to each hole of the blank control group, and 100 μL of sample liquid is added to each hole of the experimental group. The 96-well culture plate is placed in a 37°C, 5% CO2 incubator and cultured for 24 h. 10 μL of CCK8 reagent is added to each hole, and the plate is incubated in the incubator for 2 h. After incubation, the absorbance value of each hole at a wavelength of 450 nm is detected using an enzyme marker. The absorbance value of the blank control group is set to 100%, and the absorbance ratio of each group to the blank control group is multiplied by 100% to obtain the cell viability of each group.

[0091] The sample liquid of the experimental group is: the HC122 lyophilized product obtained in Example 5 is dissolved with complete culture medium, filtered with a 0.22 μm filter membrane to remove bacteria, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively.

[0092] As shown in Table 1, the recombinant human collagen type VI HC122 obtained by the application has good cell adhesion promoting effect at the microgram level. Figure 4 As shown in Table 1, the recombinant human collagen type VI HC122 obtained by the application has good cell adhesion promoting effect at the microgram level.

[0093] Effect Example 3: Recombinant human collagen type VI HC122 promotes dermal hair papilla cell adhesion test

[0094] Human dermal papilla cells HDPC are digested and counted, and 1×10 5100 μL / well. The medium was aspirated and washed 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 liquid, and the positive control group was added with 100 μL of basic medium (sample maximum enzyme activity control group, PC), and was incubated for 24 h under the condition of 5% CO2, 37°C and >90% humidity.

[0095] One hour before detection, the positive control group was added with LDH release reagent in an amount of 10% of the volume of the culture solution (10 μL / well). After adding the LDH release reagent, it was mixed by repeatedly blowing and was incubated for 1 h. Before detection, the cell culture plate was centrifuged at 400g for 5 min by using a multi-well centrifuge. 120 μL of supernatant was taken in a new 96-well plate; 60 μL of LDH detection liquid was added to each well, mixed, and incubated at room temperature for 30 min in the dark; and the absorbance value was measured at 490 nm, and the ratio of the absorbance value of each group to that of the blank control group was the LDH release ratio.

[0096] Among them, the sample liquid of the experimental group was: the HC122 lyophilisate obtained in Example 5 was dissolved with complete culture medium, filtered with a 0.22 μm filter membrane to remove bacteria, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively. The LDH release reagent and the LDH detection liquid were from an LDH detection kit (Biyun Tian, C0016).

[0097] The results are shown in Table 1. Figure 5 Compared with the control group, the recombinant human collagen type VI HC122 obtained in the application can improve 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, and HC122 has a significant effect at a concentration of 200 μg / mL.

[0098] Example 4: Antioxidant performance of recombinant collagen type VI HC122

[0099] The density of human dermal papilla cells HDPC was adjusted to 5×10 5 The density of human dermal papilla cells HDPC was adjusted to 5×10

[0100] The medium was aspirated, and 500 μL of sample or complete culture medium was added to each well, and was incubated for 24 h (5% CO2, 37°C, >90% humidity).

[0101] Dilute DCFH-DA (2',7'-dichlorofluorescin diacetate) with serum-free medium at 1:1000 to make a final concentration of 10 μM. Remove the medium and add an appropriate volume of the diluted DCFH-DA working solution to cover the cells, and incubate at 37°C for 20-30 min. Wash the cells with serum-free medium for 1-2 times to remove the DCFH-DA that has not entered the cells. Take a picture directly in the fluorescence 1 channel (excitation wavelength 488 nm, emission wavelength 525 nm / FITC).

[0102] The blank control group sample is complete culture medium;

[0103] The experimental group sample is the HC122 lyophilisate obtained in Example 5, which is dissolved in complete culture medium, filtered with a 0.22 μm filter to remove bacteria, and then diluted to 10 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL and 800 μg / mL, respectively;

[0104] DCFH-DA itself has no fluorescence, but can freely pass through the cell membrane, enter the cells, and be hydrolyzed by intracellular esterase to generate DCFH (dichlorofluorescin), and DCFH cannot permeate the cell membrane, so the probe can be easily loaded into the cells. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to generate fluorescent DCF (2',7'-dichlorofluorescin), and the fluorescence of DCF can be detected to know the level of intracellular reactive oxygen species.

[0105] The results are shown in Table 1. Figure 6 As shown in Table 1, the fluorescence intensity of the experimental group to which the recombinant human collagen type VI HC122 is added is obviously smaller than that of the negative control group to which the recombinant human collagen type VI HC122 is not added, and the fluorescence intensity gradually weakens as the content of the added recombinant human collagen type VI increases, proving that the recombinant human collagen type VI obtained in the application has an antioxidant stress ability.

[0106] Although the application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions recorded in the foregoing embodiments or make equivalent replacements to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application.

Claims

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

1.

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

2.

3. An expression vector, characterized in that Containing the nucleic acid according to claim 2.

4. A host cell, characterized in that Containing the nucleic acid 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 designing a coding nucleic acid sequence based on the amino acid sequence obtained in step S1, and performing codon optimization to obtain a nucleotide sequence encoding recombinant human type VI collagen, followed by gene synthesis to obtain a nucleic acid fragment encoding the recombinant human type VI collagen fragment; S3. Construction of expression vector: ligating 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 transformed into competent host cells to obtain expression strains; S5, inducing expression: inducing expression of the expression strain obtained in step S4, and collecting the bacterial solution; 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 is pET30a(+).

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

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 is carried out by adding inducer IPTG to induce the strain to express.

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