Preparation method and application of recombinant VI type collagen

Abstract

The invention discloses a preparation method and application of recombinant type VI collagen, amino acids from the 1489th site to the 1518th site of a human type VI collagen alpha5 chain are adopted as target proteins, after codon optimization, the target proteins are transformed to E.coli BL 21, expression of the target proteins is detected, high-yield strains are screened, and the recombinant type VI collagen is obtained. Systematically optimizing expression conditions such as a culture medium, an inducer, culture temperature, culture time and the like to obtain an engineering strain with stable expression; the recombinant collagen is purified through affinity chromatography, and the recombinant collagen which has antioxidant activity, has protection and repair effects on ultraviolet-induced skin cell photoaging and has influence effects on skin cell proliferation, adhesion and migration is obtained. The recombinant collagen can obviously reduce the mRNA expression quantity of inflammatory factors IL-1beta, IL-8 and TNF-alpha and matrix metalloproteinase MMP1, so that the degree of inflammatory reaction in photoaging HaCaT cells is reduced, and a theoretical basis is provided for application of the recombinant collagen in the fields of food nutrition, health care and beauty treatment, clinical treatment, medical beauty treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and in particular relates to the new technical field of a biosynthetic method of inositol. Background Art

[0002] Ultraviolet (UV) radiation accelerates skin cell aging through direct effects such as DNA damage and indirect effects such as oxidative stress. This can lead to skin edema, erythema, sunburn, hyperplasia, telangiectasia, and the development of skin cancer. The skin performs several important physiological functions and significantly impacts interpersonal relationships, psychological well-being, and body image. However, with aging and cumulative exposure to external factors, the skin undergoes degenerative changes. This inevitably leads to skin problems, which severely impact quality of life and place a significant burden on healthcare systems. Skin problems rank fourth among the leading causes of non-fatal disease burden. Anti-aging is widely considered a promising approach to prevent skin problems and a key research area in the dermatology and health care industries. There are two main types of skin aging: intrinsic factors, such as genetic susceptibility and hormonal changes, play a significant role in the protective zone. The primary cause of aging in exposed areas is attributed to external factors, with UV radiation from sunlight contributing up to 80% to this process, commonly referred to as photoaging. While skin aging is an inevitable physiological process, it can be delayed and mitigated to some extent. Therefore, preventing and delaying skin aging has long been a major concern in academia and the beauty industry.

[0003] Collagen is a general term for a class of biological macromolecular proteins. It is one of the main components of animal connective tissue and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein content. Collagen is widely present in skin, bones, joints, and hair, and plays a supporting, repairing, and anti-aging role. Type VI collagen was first discovered by Chung in 1976 from pepsin hydrolysates of human aortas. Its notable characteristics are its large molecular weight and high cysteine ​​content. Furuto and Miller isolated type VI collagen from placental tissue and provided a preliminary description of its structure. Under an electron microscope, type VI collagen is dumbbell-shaped, with a central rod approximately 150 nm in size and two spherical wings.

[0004] Collagen possesses strong biological activity and functions, participating in cell migration, differentiation, and proliferation, and providing mechanical strength to bones, tendons, cartilage, and skin. Collagen also promotes cell growth, acts as a hemostatic agent, and possesses excellent biocompatibility and biodegradability. These properties have led to its widespread use in a wide range of medical and health fields, including treatments for burns, trauma, corneal diseases, cosmetic procedures, orthopedics, hard tissue repair, and wound hemostasis. Summary of the Invention

[0005] Aiming at the technical problem that there are few reports on the technical solution of using prokaryotic expression system to efficiently prepare recombinant type VI human collagen with the protection and repair effect of human epidermal cell photoaging, this application uses the amino acids 1489 to 1518 of the α5 chain of human type VI collagen as the target protein, and after codon optimization, it is converted into E. coli BL 21, a stably expressed engineered strain was obtained; recombinant collagen was purified by affinity chromatography to obtain recombinant collagen with antioxidant activity, protective and repair effects on ultraviolet-induced photoaging of skin cells, and effects on skin cell proliferation, adhesion and migration, providing a theoretical basis for its application in food nutrition, health care and beauty, clinical treatment and medical cosmetology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: The present application provides a method for preparing recombinant type VI collagen, wherein the recombinant type VI collagen uses the amino acids 1489 to 1518 of the α5 chain of human type VI collagen as the target protein, is synthesized into the expression vector pET-28a after codon optimization to obtain the recombinant expression vector pET-28a-col6a5, and is transformed into E. coli BL 21, the nucleotide sequence of the human type VI collagen α5 chain is shown in SEQ ID NO: 1, and the amino acid sequence of the human type VI collagen α5 chain is shown in SEQ ID NO: 2.

[0007] In the preparation method, E. coli BL 21 was inoculated onto LB solid medium containing 50 mg / mL kanamycin and cultured at 37°C overnight. Positive clones were selected and inoculated into LB liquid medium containing 50 mg / mL kanamycin at a ratio of 1:100. The cells were cultured at 180 rpm and 37°C until the OD600 reached 0.6-0.8. IPTG was added at a concentration of 0.4-1.0 mmol / L and induced for 3-8 h at 16-37°C. After induction, the cells were centrifuged at 8000 rpm and 4°C for 15 min. The pellets were collected, resuspended and washed twice with PBS buffer (pH 7.4), and ultrasonically disrupted at 200 W for 3 s on and 6 s off. After disruption, the supernatant and pellet were collected separately.

[0008] In the preparation method, 3 ml of His-tag Purification Resin gel medium is added to an empty column tube, and 20% ethanol, deionized water, and PBS buffer (pH 7.4) are added in sequence to balance the buffer. The supernatant and crude protein extract in the precipitate collected after ultrasonic disruption are purified. 30 ml of the disrupted supernatant is added to the chromatography column, and the column is mixed at low temperature and low speed for 2 hours. After the liquid in the chromatography column is completely drained, the column is washed and eluted using PBS imidazole buffer of different concentrations. After mixing at low temperature and low speed for 15 minutes at each concentration, the drained contents are collected. After affinity chromatography, the eluted target protein is dialyzed with PBS buffer at a volume of 1:100 to remove imidazole. The dialysate is changed every 4 hours, the dialysis time is 2 days, and the dialysis temperature is 4°C.

[0009] Preferably, the concentration of added IPTG is 0.4 mmol / L.

[0010] Preferably, the induction expression temperature is 37°C.

[0011] Preferably, the induction expression time is 6 hours.

[0012] Furthermore, the present application also provides the use of the recombinant type VI collagen prepared by the above-mentioned method for preparing recombinant type VI collagen in the preparation of preparations with antioxidant function.

[0013] Furthermore, the present application also provides the use of the recombinant type VI collagen prepared by the above-mentioned method for preparing recombinant type VI collagen in the preparation of preparations related to the protection of epidermal cells damaged by UVB.

[0014] In the application of the preparation of preparations related to protecting epidermal cells after UVB damage, the concentration of recombinant type VI collagen is 0.1-1 mg / mL.

[0015] Furthermore, the present application also provides the use of the recombinant type VI collagen prepared by the above-mentioned method for preparing recombinant type VI collagen in the preparation of preparations related to stimulating collagen formation.

[0016] Through the above technical solutions, this application achieves the following technical effects: (1) The technical solution provided by this application selects the amino acids 1489 to 1518 of the α5 chain of human type VI collagen as the target protein, and after codon optimization, converts it into E. coliBL 21, obtained a stably expressed engineered strain; through affinity chromatography, recombinant collagen was purified to obtain recombinant collagen with antioxidant activity, protective and repair effects on ultraviolet-induced photoaging of skin cells, and effects on skin cell proliferation, adhesion and migration. Its mechanism of action in delaying skin aging was deeply explored, providing a theoretical basis for its application in food nutrition, health care and beauty, clinical treatment and medical cosmetology.

[0017] (2) The recombinant type VI collagen prepared by the technical solution provided in this application has a good scavenging ability for DPPH free radicals. The recombinant collagen with a concentration of 5 mg / mL has the strongest scavenging ability for DPPH free radicals, and its half maximal inhibitory concentration (IC50) is 0.98 mg / mL. + There is a positive correlation between the free radical scavenging rate and the ABTS scavenging rate at 5 mg / mL increased to 88%. + IC of free radicals 50 The value is 0.496 mg / mL.

[0018] (3) The recombinant type VI collagen prepared by the technical solution provided in this application has a certain proliferative effect on cell growth, is non-cytotoxic, and has good biocompatibility. The concentration of the obtained recombinant type VI collagen is 0.1 mg / mL. Compared with the control group, the number of cells adhered to the recombinant collagen was significantly increased, the cell adhesion activity was enhanced, and it was conducive to cell growth and reproduction.

[0019] (4) The recombinant type VI collagen prepared by the technical solution provided in this application has a certain protective effect on HaCaT cells before photoaging damage, and also has a certain repair effect on HaCaT cells after UVB irradiation. The damaged cells have obvious recovery and improvement. The cell survival rate of the recombinant collagen-treated group is significantly increased compared with the control group and has a certain dose-dependency. Recombinant collagen at a concentration of 0.75-1 mg / mL has a significant inhibitory effect on UVB-induced photoaging of HaCaT cells.

[0020] (5) The recombinant type VI collagen prepared by the technical solution provided in this application was used to detect the effect of recombinant collagen on the migration rate of cells after UVB irradiation through a cell scratch test. The results showed that compared with the control group, treatment with recombinant collagen significantly promoted the migration of NIH-3T3 cells, which is beneficial for the cells to migrate to the aged skin and secrete cytokines to stimulate collagen formation, reduce wrinkles and restore the skin to a younger appearance.

[0021] (6) The recombinant type VI collagen prepared by the technical solution provided in this application significantly reduced the ROS level in HaCaT cells after UVB irradiation, effectively removed the excess ROS produced in photoaged cells, and reduced the oxidative stress level of the cells. The results showed that the recombinant collagen significantly reduced the fluorescence intensity of ROS. The fluorescence intensity of 0.1 mg / mL recombinant collagen decreased by 37.03%, and the fluorescence intensity of 1.0 mg / mL recombinant collagen decreased by 59.22%.

[0022] (7) The recombinant type VI collagen prepared by the technical solution provided in this application can significantly reduce the mRNA expression of inflammatory factors IL-1β, IL-8, TNF-α and matrix metalloproteinase MMP1, thereby reducing the degree of inflammatory response in photoaged HaCaT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] picture Figure 1 shows the effect of different expression conditions on the expression level of recombinant type VI collagen.

[0024] Figure A shows the effect of different culture times on the expression level of recombinant protein, M is the protein molecular weight standard, 1 is before recombinant type VI collagen induction, 2-7 are after 3h, 4h, 5h, 6h, 7h, and 8h of induction of recombinant type VI collagen; Figure B shows the effect of different culture temperatures on the expression level of recombinant protein, M is the protein molecular weight standard; 1 is before recombinant type VI collagen induction; 2-4 are after recombinant type VI collagen induction at 25℃, 30℃, and 37℃; Figure C shows the effect of different IPTG concentrations on the expression level of recombinant protein, M is the protein molecular weight standard; 1 is before recombinant type VI collagen induction; 2-5 are after recombinant type VI collagen induction with final concentrations of 0.4, 0.6, 0.8, and 1.0 mmol / L IPTG.

[0025] Figure 2 Shown are the results of induced expression, purification and Western blotting detection of recombinant type VI collagen COL6A5.

[0026] Figure A shows the results of induced expression of the recombinant protein, where M is a protein molecular weight standard, 1 is recombinant type VI collagen before induction, 2 is recombinant type VI collagen after induction, 3 is the supernatant after recombinant type VI collagen fragmentation, and 4 is the precipitate after recombinant type VI collagen fragmentation; Figure B shows the results of purification of the recombinant protein, where M is a protein molecular weight standard, 1 is the flow-through, 2 is the 10 mM imidazole eluent, 3 is the 20 mM imidazole eluent, 4 is the 50 mM imidazole eluent, 5 is the 100 mM imidazole eluent, 6 is the 150 mM imidazole eluent, 7 is the 200 mM imidazole eluent, and 8 is the 500 mM imidazole eluent; Figure C shows the results of Western blotting detection; M is a protein molecular weight standard, and 1 is recombinant human-like type VI collagen.

[0027] Figure 3 Shown are the results of the scavenging activity of recombinant collagen and Vc on DPPH free radicals.

[0028] Figure A shows the results of the scavenging activity of recombinant collagen on DPPH free radicals; Figure B shows the results of the scavenging activity of Vc on DPPH free radicals.

[0029] Figure 4 Shown are ABTS of recombinant collagen and GSH + Figure 2. Cationic radical scavenging ability of the cations.

[0030] Figure 5 Shown is the effect of different concentrations of recombinant collagen on the growth of NIH-3T3 cells.

[0031] Figure 6 Shown is the effect of different concentrations of recombinant collagen on the proliferation of NIIH-3T3 cells.

[0032] Figure 7 Shown are images of NIH-3T3 cell adhesion (100X).

[0033] Figure 8 Shown is the adhesion of NIH-3T3 cells.

[0034] Figure 9 Shown is the effect of different doses of UVB on HaCaT cells.

[0035] Figure 10 Shown are the protective and repair effects of recombinant collagen on HaCaT cell damage, as well as the results of the protective and repair effects.

[0036] Figure A shows the protective effect of recombinant collagen on HaCaT cell damage; Figure B shows the repair effect of recombinant collagen on HaCaT cell damage; Figure C shows the protective and repair effects of recombinant collagen on HaCaT cell damage.

[0037] Figure 11 Shown are the results of the effect of recombinant collagen on the migration of NIH-3T3 cells after UVB irradiation.

[0038] Figure 12 Shown is the cell migration in the presence of 0.01% Col VI.

[0039] Figure 13 Shown is the ROS intensity under an inverted fluorescence microscope (×100).

[0040] Figure A shows the blank control group; Figure B shows the UVB model group; Figure C shows the UVB+0.1 mg / mL collagen group; Figure D shows the UVB+1.0 mg / mL collagen group; and Figure E shows the 0.01 mg / mL Vc positive control group.

[0041] Figure 14 Shown are the relative ROS levels in HaCaT cells (n = 3).

[0042] Figure 15 Shown are the changes in mRNA expression levels of IL-8, TNF-α, IL-1β, and MMP-1 in HaCaT cells.

[0043] Figure A shows the changes in IL-8 mRNA expression; Figure B shows the changes in TNF-α mRNA expression; Figure C shows the changes in IL-1β mRNA expression; and Figure D shows the changes in MMP-1 mRNA expression. DETAILED DESCRIPTION

[0044] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0045] The reagents used in this study were: tryptone, yeast extract, isopropyl β-D-thiogalactoside, tris(hydroxymethyl)aminomethane, sodium dodecylsulfonate, acrylamide, ammonium persulfate, ethylenediaminetetraacetic acid, bovine serum albumin, reduced glutathione, kanamycin (Kana), RealBand prestained protein marker (10-180 kDa), DMEM high-glucose medium, fetal bovine serum, CCK-8 cell counting kit, and DCFH-DA reactive oxygen species fluorescent probe, all purchased from Shanghai Sangon Biotechnology Co., Ltd. Penicillin-streptomycin solution, 0.25% trypsin digestion buffer, RNA extraction kit, cDNA synthesis premix, and SYBR Green qPCR Mix were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Mouse fibroblast cell line (NIH-3T3) and human immortalized epidermal cell line (HaCaT) were provided by the cell bank of Zigong Fifth People's Hospital. All other reagents not specified were domestically produced and of analytical grade.

[0046] The instruments and equipment used in this application are: intelligent electric constant temperature incubator (DHP-9160B) purchased from Shanghai Yiheng Scientific Instrument Co., Ltd.; ultrapure water manufacturing system (UPH-Ⅱ-20T) purchased from Sichuan Youpu Ultrapure Technology Co., Ltd.; constant temperature culture shaker (HZ150L) purchased from Shanghai Jingsheng Scientific Instrument Co., Ltd.; electrophoresis system (Trans-blot SD Cell, SDS-PAGE) purchased from Bio-Rad; ultrasonic experimental equipment (JY92-IIDN) purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; medical refrigerated centrifuge (TGL-16) purchased from Sichuan Shuke Instrument Co., Ltd.; desktop centrifuge (TGL-16M) purchased from Shanghai Lu Xiangyi; full wavelength microplate reader (ReadMax1200) purchased from Shanghai Shanpu Biotechnology Co., Ltd.; fluorescence inverted microscope purchased from Germany OSRAM Company; cell culture incubator (Celstar185) purchased from Shanghai Rundu Biotechnology Co., Ltd.

[0047] The Escherichia coli used in this application E. coli BL 21 (DE3) is a common strain that can be purchased by the general public from any biological company.

[0048] Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0049] Example 1: A method for preparing recombinant type VI collagen The present application provides a method for preparing recombinant type VI collagen, wherein the recombinant type VI collagen uses the amino acids 1489 to 1518 of the α5 chain of human type VI collagen as the target protein, is synthesized into the expression vector pET-28a after codon optimization to obtain the recombinant expression vector pET-28a-col6a5, and is transformed into E. coli BL 21, the nucleotide sequence of the human type VI collagen α5 chain is shown in SEQ ID NO: 1, and the amino acid sequence of the human type VI collagen α5 chain is shown in SEQ ID NO: 2.

[0050] In the preparation method, E. coli BL 21 was inoculated onto LB solid medium containing 50 mg / mL kanamycin and cultured at 37°C overnight. Positive clones were selected and inoculated into LB liquid medium containing 50 mg / mL kanamycin. The cells were cultured at 180 rpm and 37°C until the OD600 reached 0.6-0.8. IPTG was added at a concentration of 0.4-1.0 mmol / L and induced for 3-8 h at 16-37°C. After induction, the cells were centrifuged at 8000 rpm and 4°C for 15 min. The pellets were collected, resuspended and washed twice with PBS buffer (pH 7.4), and ultrasonically disrupted at 200 W for 3 s on and 6 s off. After disruption, the supernatant and pellet were collected separately.

[0051] In the preparation method, 3 ml of His-tag Purification Resin gel medium is added to an empty column tube, and 20% ethanol, deionized water, and PBS buffer (pH 7.4) are added in sequence to balance the buffer. The supernatant and crude protein extract in the precipitate collected after ultrasonic disruption are purified. 30 ml of the disrupted supernatant is added to the chromatography column, and the column is mixed at low temperature and low speed for 2 hours. After the liquid in the chromatography column is completely drained, the column is washed and eluted using PBS imidazole buffer of different concentrations. After mixing at low temperature and low speed for 15 minutes at each concentration, the drained contents are collected. After affinity chromatography, the eluted target protein is dialyzed with PBS buffer at a volume of 1:100 to remove imidazole. The dialysate is changed every 4 hours, the dialysis time is 2 days, and the dialysis temperature is 4°C.

[0052] Preferably, the concentration of added IPTG is 0.4 mmol / L.

[0053] Preferably, the induction expression temperature is 37°C.

[0054] Preferably, the induction expression time is 6 hours.

[0055] Example 2: Application of recombinant type VI collagen Furthermore, the present application also provides a method for preparing recombinant type VI collagen and the use of the recombinant type VI collagen prepared in the preparation of preparations with antioxidant function.

[0056] Furthermore, the present application also provides a method for preparing recombinant type VI collagen and the use of the recombinant type VI collagen prepared in the preparation of preparations related to protecting epidermal cells damaged by UVB.

[0057] In the application of the preparation of preparations related to protecting epidermal cells after UVB damage, the concentration of recombinant type VI collagen is 0.1-1 mg / mL.

[0058] Furthermore, the present application also provides a method for preparing recombinant type VI collagen and the use of the recombinant type VI collagen prepared in the preparation of preparations related to stimulating collagen formation.

[0059] Example 3: Construction of recombinant protein 1. Test methods (1) Prokaryotic expression The nucleotide sequence of the human type VI collagen α5 chain as shown in SEQ ID NO: 1 and the amino acid sequence of the human type VI collagen α5 chain as shown in SEQ ID NO: 2 were used with the pET-28a vector to construct a recombinant expression vector pET-28a-col6a5. The recombinant expression vector pET-28a-col6a5 preserved in the laboratory was extracted and transformed into Escherichia coli. E. coli BL 21 (DE3) competent cells were inoculated onto LB solid medium containing 50 mg / mL kanamycin and cultured overnight at 37°C. Positive clones were selected and inoculated into LB liquid medium containing 50 mg / mL kanamycin. The cells were cultured at 180 rpm and 37°C until the OD600 reached 0.6-0.8. IPTG was added to a final concentration of 80 μM and induced at 37°C for 6 h. After induction, the cells were centrifuged at 8000 rpm and 4°C for 15 min. The pellets were collected, resuspended, and washed twice with PBS buffer (pH 7.4). The cells were then ultrasonically disrupted (power 200 W, supersonication for 3 s on, 6 s off). The supernatant and pellet were collected, and protein expression was analyzed by SDS-PAGE.

[0060] (2) Protein purification Add 3 ml of His-tag Purification Resin gel medium to an empty column tube, and add 20% ethanol, deionized water, and PBS buffer (pH 7.4) equilibration buffer in sequence to purify the crude protein extract. Add 30 ml of the crushed supernatant to the chromatography column and mix at low temperature and low speed for 2 hours. After the liquid in the chromatography column is exhausted, wash and elute with PBS imidazole buffer of different concentrations. After mixing at low temperature and low speed for 15 minutes at each concentration, collect the exhausted contents and take samples for SDS-PAGE detection of protein elution. After affinity chromatography, the eluted target protein is dialyzed with PBS buffer at a volume of 1:100 to remove imidazole. The dialysate is changed every 4 hours, the dialysis time is 2 days, and the dialysis temperature is 4°C.

[0061] (3) Western blot detection of target protein The dialyzed target protein was detected by SDS-PAGE. The protein gel was placed in transfer buffer for equilibration for 20 minutes. The transfer electrophoresis tank was placed in an ice-water bath and transferred at a constant current of 200 mA in the transfer tank for 70 minutes. The membrane was blocked with blocking buffer on a shaker at room temperature for 1.5 hours. The primary antibody (Anti×His-Tag mouse polyclonal antibody) was diluted 1:3000 with blocking buffer and incubated overnight at 4°C. The membrane was washed three times with TBST buffer for 5 minutes each time. The secondary antibody (HRP-conjugated Goat Anti-Mouse IgG) was diluted 1:10000 and incubated at 37°C for 1 hour. The membrane was washed three times with TBST buffer and stained with ECL staining solution in the dark for 1 minute. The results were visualized using a Bio-Rad gel imaging system after exposure for 15 seconds.

[0062] 2. Experimental Results See attached Figure 1 As shown in the figure, after IPTG induction, recombinant type VI human collagen was expressed. A clear band appeared at 25-35 kDa, with a relative molecular mass of approximately 28 kDa, consistent with the size of recombinant type VI collagen. A clear target band was found in the supernatant sample after fragmentation, indicating that the protein was soluble. Ni-NTA His-Bind resin was used to purify the recombinant protein containing 6×HisTag. Since the engineered bacteria expressed a soluble recombinant protein ( Figure 1 -A), so the treated supernatant was directly purified under native conditions. Figure 1-B analysis showed that the target protein with high purity was found in the elution solution with 200 mM imidazole, with the purity reaching over 90%. The soluble recombinant protein gel detected by SDS-PAGE was electrotransferred to a PVDF membrane again for Western-blot immunoblotting detection. Figure 1 -C test results showed that the recombinant human type VI collagen obtained by purification was the expected protein.

[0063] Example 4: Optimization of the induction expression conditions of recombinant collagen 1. Experimental Methods The cultured bacteria were inoculated into LB medium at a ratio of 1:100, and cultured at 37°C and a final IPTG concentration of 1.0 mM for 3, 4, 5, 6, 7, and 8 h, respectively. The optimal induction time was determined by SDS-PAGE electrophoresis. At a final concentration of 1.0 mM IPTG and the optimal induction time, the cultures were cultured at 16, 25, and 37°C, respectively, to determine the optimal induction temperature. At the optimal induction time and optimal induction temperature, 0.4, 0.6, 0.8, and 1.0 mM IPTG were added, respectively, to determine the optimal induction concentration.

[0064] 2. Experimental results: In order to obtain a stable expression engineered strain, the expression conditions such as culture time, culture temperature, inducer concentration, etc. were systematically optimized, and samples were taken in sequence for SDS-PAGE electrophoresis analysis. The results are as follows Figure 2 As shown in Figure 1-A, the culture time has some influence on the expression of recombinant protein. When cultured for 6 h, the expression level is higher. Figure 2 As shown in Figure 2-B, temperature has a great influence on the expression of recombinant protein. The higher the temperature, the higher the expression level. Therefore, we chose 37℃ for culture. Figure 2 As shown in Figure 2-C, the IPTG concentration affected the expression rate of the recombinant protein. Considering its toxicity and cost, we selected 0.4 mmol / L. Therefore, the subsequent expansion culture conditions were selected at 37°C and 0.4 mmol / L IPTG for 6 h.

[0065] Example 5: Antioxidant activity detection 1. Test methods (1) DPPH free radical scavenging ability determination Following the method of Liu Yang et al., 4 mg of DPPH was dissolved in 5 mL of anhydrous ethanol and then diluted to 100 mL with 50% (v / v) ethanol-water solution to prepare a 0.1 mmol / L DPPH solution. Five 1.5 mL centrifuge tubes were filled with 100 μL of the 0.1 mmol / L DPPH solution and 100 μL of five sample solutions of varying concentrations (1, 2, 3, 4, and 5 mg / mL). The mixture was incubated in the dark for 30 minutes, and the absorbance was measured at 517 nm. The result was designated as A1. Another 100 μL of the 0.1 mmol / L DPPH solution was added to 100 μL of 50% (v / v) ethanol-water solution. The mixture was incubated in the dark for 30 minutes, and the absorbance was measured at 517 nm. The result was designated as A3. Take 100 μL of 50% ethanol aqueous solution by volume, add 100 μL of the sample solution as above, react in the dark for 30 minutes, measure the absorbance at 517 nm, and set the result as A2.

[0066] DPPH free radical scavenging rate calculation formula: [A3-(A1-A2)] / A3×100% Vc was used as a positive control and BSA as a negative control. Three parallel experiments were performed for each concentration of sample, and the average value was taken to calculate the clearance rate.

[0067] (2) Determination of ABTS cationic radical scavenging ability Following the method of Zhang Yixuan et al., 5 mL of 7 mmol / L ABTS solution was mixed with 88 mL of 2.45 mmol / L potassium persulfate solution, stored in the dark at room temperature for 12 h, and diluted with anhydrous ethanol to an absorbance of 0.70 ± 0.02 at 734 nm to prepare the ABTS reagent. 0.4 mL of recombinant collagen solution of varying concentrations was mixed with 3.6 mL of ABTS reagent and reacted at room temperature in the dark for 6 min. The absorbance value (A1) was measured at 734 nm. The corresponding absorbance value (A2) was determined by replacing the ABTS reagent with an equal amount of ethanol.

[0068] ABTS cation radical scavenging rate calculation formula: (A2-A1) / A2×100% GSH was used as a positive control and BSA as a negative control. Three parallel experiments were performed for each concentration of sample, and the average value was taken to calculate the clearance rate.

[0069] 2. Experimental Results (1) DPPH free radical scavenging ability DPPH free radicals are converted into DPPH-H (non-free radicals) in the presence of antioxidants or hydrogen donors, thereby reducing the color intensity of the solution. The scavenging effect of different concentrations of recombinant collagen on DPPH free radicals is shown in the attached Figure 3As shown in the figure, several concentrations of recombinant collagen have a certain scavenging effect on DPPH free radicals, and show a good dose-effect relationship within the experimental concentration range. With the increase of concentration, its scavenging ability gradually increases, but the scavenging ability is not as good as that of the positive control V c Overall, the recombinant collagen with a concentration of 5 mg / mL had the strongest DPPH free radical scavenging ability, with a half-maximal inhibitory concentration (IC50) of 0.98 mg / mL.

[0070] (2) ABTS cationic radical scavenging ability ABTS + The method is also a more accurate method for determining the antioxidant activity in vitro, which is similar to the DPPH method. Under appropriate conditions, ABTS is oxidized to a stable blue-green free radical cation (ABTS + ), antioxidants and ABTS + The reaction of ABTS with antioxidants was calculated by measuring the change of its absorbance at 734 nm. + scavenging capacity to evaluate its antioxidant effect.

[0071] See attached Figure 4 The clearance curve of recombinant collagen showed that + There is a positive correlation between the free radical scavenging rate and the free radical scavenging rate. At 5 mg / mL, the ABTS scavenging rate increased to 88%; however, it is still insufficient compared with the positive control GSH. + IC of free radicals 50 The value is 0.496 mg / mL.

[0072] Example 6: Cell test 1. Test methods (1) Cytotoxicity NIH-3T3 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin solution in a 5% CO2, 37°C incubator. The cells were collected after digestion with 0.25% trypsin, resuspended, counted using a hemocytometer, and diluted to 1×10 4A single-cell suspension of 100 μL / well was seeded into a 96-well cell culture plate. After the cells reached the logarithmic growth phase, recombinant collagen (0.01, 0.1, and 0.5 mg / mL) was added and incubated in an incubator for 24 hours. Five replicates were set for each concentration, and a control group without protein and a blank group were established. After incubation, the original culture medium containing the different drug concentrations was removed, and serum-free culture medium containing 10% CCK-8 solution was added to each well. The cells were cultured in an incubator for an additional 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0073] The formula for calculating cell survival rate is: (experimental group-blank group) / (control group-blank group)×100% (2) Cell proliferation The cell culture method was the same as step 1.6 above. The cells were diluted to 5000 cells / well and inoculated into a 96-well plate. Recombinant collagen containing different concentrations was added and the cell proliferation rate was measured at different time points (24 h, 48 h, and 72 h).

[0074] The formula for calculating cell proliferation rate is: (experimental group-control group) / control group×100% (3) Cell adhesion The 96-well plates were coated with recombinant collagen at different concentrations, and 1×10 4 Cells were inoculated at 100 μL / 100 μL. After 3 h, non-adherent cells were washed with PBS and washed three times. The cells were fixed with 4% paraformaldehyde for 20 min and washed three times with PBS. 100 μL of 0.1% crystal violet was added and stained for 15 min. The cells were washed three times with PBS. The images were observed with an inverted fluorescence microscope, and the percentage of cell adhesion area was calculated using the analysis software Image J.

[0075] 2. Experimental Results (1) Cytotoxicity evaluation and cell proliferation ability determination of recombinant type VI collagen Through CCK-8 experiments, it was proved that different concentrations (0.001%, 0.005%, 0.01%, 0.05%) of recombinant collagen at different time points (24 h, 48 h, 72 h) had a certain proliferative effect on cell growth, without cytotoxicity, and had good biocompatibility. Figure 5 , Attachment Figure 6 shown.

[0076] (2) Cell adhesion ability Cells adhere to each other or to the extracellular matrix (ECM) through cell surface proteins called cell adhesion molecules (CAMs). Cell adhesion molecules are a class of membrane surface glycoproteins that function through "ligand-receptor" specific binding. They can mediate adhesion between cells and between cells and ECM, participate in various activities such as cell recognition, cell activation, and signal transduction, and play an important biological role.

[0077] The recombinant collagen was used to detect the adhesion activity of NIH-3T3 cells. The control group was PBS and the experimental group was recombinant collagen at a concentration of 0.1 mg / mL. Figure 7 , Attachment Figure 8 It can be seen that the number of cells adhered to the recombinant collagen increased significantly compared with the control group, which enhanced the cell adhesion activity and contributed to cell growth and reproduction.

[0078] Example 7: Establishment of UVB-induced photoaging model of HaCaT cells 1. Test methods (1) Establishment of UVB-induced photoaging model of HaCaT cells In order to screen the optimal dose of UVB irradiation for the photoaging model of HaCaT cells, 0, 15, 30, 45, and 60 mJ / cm 2 Irradiate cells and calculate IC 50 This is the dose for subsequent modeling.

[0079] The trypsin-digested HaCaT cell suspension was inoculated into a 96-well plate. After the cells grew to 70-80%, they were irradiated with different doses of UVB. During irradiation, the original culture medium was aspirated and 100 μL PBS was added. After irradiation, the culture medium was replaced with serum-free culture medium and cultured for 24 h. The cell morphology was observed under a fluorescence inverted microscope, and CCK-8 solution was added to determine the cell viability.

[0080] Radiation dose (mJ / cm 2 ) = radiation intensity (mw / cm 2 )×radiation time (2) Detection of the protective effect of recombinant human-like collagen on cells after UVB damage The digested HaCaT cell suspension was inoculated into 96-well plates. When the cells grew to 70-80%, the blank group was not treated with light or recombinant collagen. The culture medium containing recombinant collagen was replaced with an equal amount of PBS. The UVB damage group and the experimental group were exposed to 20 mJ / cm 2 The cells were treated with light and then cultured without serum for 24 h. Each group was repeated 5 times.

[0081] Different concentrations of recombinant collagen (0.1, 0.25, 0.5, 0.75, and 1 mg / mL) were added before UVB irradiation, and serum-free medium was replaced after irradiation to continue culture for 24 h.

[0082] (3) Detection of the repair effect of recombinant human-like collagen on cells after UVB damage After UVB irradiation, different concentrations of recombinant collagen (0.1, 0.25, 0.5, 0.75, and 1 mg / mL) were added, and the culture medium was replaced with an equal amount of PBS during irradiation. After irradiation, the cells were cultured for another 24 h using culture medium containing different concentrations of recombinant collagen.

[0083] (4) Detection of the protective and repair effects of recombinant human collagen on cells after UVB damage Different concentrations of recombinant collagen (0.1, 0.25, 0.5, 0.75, and 1 mg / mL) were added before UVB irradiation, and the cells were cultured for another 24 h using culture medium containing different concentrations of recombinant collagen after irradiation.

[0084] 2. Test results A UVB-irradiated cell photoaging model was established. A series of irradiation doses were selected to irradiate HaCaT cells. The irradiation time was calculated based on the irradiation power, and the cell morphology after irradiation was observed. The half inhibitory dose IC was calculated based on the logarithm of the UVB irradiation dose. 50 The value is 19.61 mJ / cm 2 Therefore, 20 mJ / cm 2 The dose for subsequent modeling is shown in the attached Figure 9 shown.

[0085] By the attached Figure 10 The results showed that recombinant collagen had a protective effect on HaCaT cells before photoaging damage and also had a certain repair effect on HaCaT cells after UVB irradiation, with damaged cells showing significant recovery and improvement. The cell survival rate in the recombinant collagen-treated group was significantly increased compared with the control group in a dose-dependent manner. This indicates that recombinant collagen at a concentration of 0.75-1 mg / mL has a significant inhibitory effect on UVB-induced photoaging of HaCaT cells.

[0086] Example 8: Cell Migration 1. Test methods NIH-3T3 cell suspension digested with 0.25% trypsin was taken and 5×10 5Cells were seeded into six-well plates at 100 μL per well and incubated for 24 hours until the cell confluence reached 90%. A scratch was made with the tip of a 20 μL pipette, and the culture medium was aspirated. The cells adhered to the plate and washed three times with PBS to remove residual cell debris. The cells were then divided into three groups: a blank group was not irradiated with UVB; a control group was irradiated with UVB without the addition of drug; and an experimental group was irradiated with UVB containing collagen. After irradiation, fresh culture medium was replaced. The experimental group was treated with recombinant collagen, while the blank and control groups were replaced with an equal volume of PBS. Cell status was observed and recorded at 0 h. The cells were then cultured in a 37°C, 5% CO2 incubator. Cell migration was photographed and recorded at 12 h, 24 h, and 36 h. Cell migration rates were calculated using Image J.

[0087] Cell migration rate = (initial scratch area - final scratch area) / initial scratch area × 100% 2. Test results The effect of recombinant collagen on the migration rate of cells irradiated by UVB was detected by cell scratch test. Figure 11 , see attached Figure 12 The results showed that compared with the control group, treatment with recombinant collagen significantly promoted the migration of NIH-3T3 cells, which was beneficial for the cells to migrate to aging skin and secrete cytokines to stimulate collagen formation, reduce wrinkles and restore skin to youthfulness.

[0088] Example 9: Detection of Reactive Oxygen Species (ROS) 1. Test methods ROS, also known as reactive oxygen species, are generated by ultraviolet radiation. Excessive free radicals can damage biomacromolecules in cells, leading to cell degeneration. When ROS levels exceed the body's antioxidant defenses, they cause oxidative stress in the skin and accelerate skin aging. The DCFH-DA fluorescent probe method is used to detect intracellular ROS. DCFH-DA is a non-fluorescent, indicative probe. Intracellular ROS interact with DCFH and oxidize to DCF, which emits a green fluorescent signal. The green fluorescent signal is proportional to the accumulation of intracellular ROS. The fluorescent signal is detected using a fluorescence microscope at a maximum excitation wavelength of 480 nm and a maximum emission wavelength of 525 nm.

[0089] HaCaT cells were seeded in 96-well plates and cultured in a 37°C, 5% CO2 incubator until the cell density reached 60-70%. The DCFH-DA probe was diluted 1:1000 in PBS to a final concentration of 10 μmol / L. The original cell culture medium was removed, and 100 mL of diluted DCFH-DA (protected from light) was added. Drug-treated cells were then added and irradiated with 20 mJ / cm2 UVB. After irradiation, the cells were incubated in a 37°C cell culture incubator for 30 min. The cells were washed three times with PBS to fully remove any DCFH-DA that had not entered the cells. 50 μL of PBS was then added, and images were taken using an inverted fluorescence microscope (excitation wavelength: 488 nm, emission wavelength: 525 nm). Fluorescence intensity was analyzed using Image J software.

[0090] The mice were divided into three groups: a control group without any treatment, a UVB damage group, and an experimental group in which recombinant type VI collagen was added during UVB irradiation.

[0091] 2. Experimental Results See attached Figure 13 , Attachment Figure 14 As shown in the results, it can be seen that recombinant collagen significantly reduced the level of ROS in HaCaT cells after UVB irradiation, and can effectively remove the excessive ROS produced in photoaged cells and reduce the oxidative stress level of cells. Figure 14 The results showed that recombinant collagen significantly reduced the fluorescence intensity of ROS. 0.1 mg / mL recombinant collagen reduced the fluorescence intensity by 37.03%, and 1.0 mg / mL recombinant collagen reduced the fluorescence intensity by 59.22%.

[0092] Example 10: Effects of recombinant collagen on the expression of related pro-inflammatory factors and MMPs genes in UVB-induced HaCaT cells 1. Test methods Cell plating and drug administration procedures were the same as before. After UVB irradiation, the culture medium was aspirated and 1 mL of cell lysis buffer was added to each well. Total RNA was extracted according to the instructions of the Biyuntian Total RNA Extraction Kit. Complementary deoxyribonucleic acid (cDNA) was obtained by reverse transcription using a 5× cDNA synthesis premix. The cDNA stock solution was serially diluted and used as a template for a real-time quantitative polymerase chain reaction (qPCR) system for each gene using SYBR Green qPCR Mix. qPCR reaction conditions included a pre-denaturation at 94°C for 20 s followed by a three-step amplification cycle at 94°C for 10 s, 60°C for 10 s, and 72°C for 10 s (40 cycles). Specific primers for the target gene and the internal reference gene (PP1A) are shown in Table 1. The relative expression level of each gene was calculated.

[0093] Table 1: Fluorescence quantitative PCR primer sequences

[0094] 2. Test results See attached Figure 15 As shown, the results showed that recombinant collagen could significantly reduce the mRNA expression of inflammatory factors IL-1β, IL-8, TNF-α and matrix metalloproteinase MMP1, thereby reducing the degree of inflammatory response in photoaged HaCaT cells.

[0095] This application successfully prepared and purified recombinant human-like type VI collagen (rhCOL6) using a prokaryotic expression system. The results also preliminarily confirmed that recombinant human-like type VI collagen has a significant inhibitory effect on UVB-induced photoaging in HaCaT cells. The results reduced the level of MMP1 in UVB-photoaged HaCaT cells, thereby reducing extracellular matrix degradation, and decreased the levels of inflammatory factors IL-1β, IL8, and TNF-α, thereby alleviating the inflammatory response. The authors also conducted preliminary exploration of its functions and mechanisms related to anti-photoaging, providing a theoretical basis for the development of functional skin care products or medical dressings based on type VI collagen. With the advancement of synthetic biology, the type specificity and functional diversity of recombinant collagen will become an important research direction in the anti-aging field.

[0096] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Its purpose is to enable people who need this technology to understand the content of the present invention and implement it. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing recombinant type VI collagen, characterized in that: The recombinant type VI collagen protein uses the amino acids 1489 to 1518 of the α5 chain of human type VI collagen as the target protein, which is synthesized into the expression vector pET-28a after codon optimization to obtain the recombinant expression vector pET-28a-col6a5, and is transformed into E. coli BL 21, the nucleotide sequence of the human type VI collagen α5 chain is shown in SEQ ID NO: 1, and the amino acid sequence of the human type VI collagen α5 chain is shown in SEQ ID NO:

2.

2. The method for preparing recombinant type VI collagen according to claim 1, wherein In the preparation method, E. coli BL 21 was inoculated onto LB solid medium containing 50 mg / mL kanamycin and cultured at 37°C overnight. Positive clones were selected and inoculated into LB liquid medium containing 50 mg / mL kanamycin at a ratio of 1:

100. The cells were cultured at 180 rpm and 37°C until the OD600 reached 0.6-0.

8. IPTG was added at a concentration of 0.4-1.0 mmol / L and induced for 3-8 h at 16-37°C. After induction, the cells were centrifuged at 8000 rpm and 4°C for 15 min. The pellets were collected, resuspended and washed twice with PBS buffer (pH 7.4), and ultrasonically disrupted at 200 W for 3 s on and 6 s off. After disruption, the supernatant and pellet were collected separately.

3. The method for preparing recombinant type VI collagen according to claim 1, wherein: In the preparation method, 3 ml of His-tag Purification Resin gel medium is added to an empty column tube, and 20% ethanol, deionized water, and PBS buffer (pH 7.4) are added in sequence to balance the buffer. The supernatant and crude protein extract in the precipitate collected after ultrasonic disruption are purified. 30 ml of the disrupted supernatant is added to the chromatography column, and the column is mixed at low temperature and low speed for 2 hours. After the liquid in the chromatography column is completely drained, the column is washed and eluted using PBS imidazole buffer of different concentrations. After mixing at low temperature and low speed for 15 minutes at each concentration, the drained contents are collected. After affinity chromatography, the eluted target protein is dialyzed with PBS buffer at a volume of 1:100 to remove imidazole. The dialysate is changed every 4 hours, the dialysis time is 2 days, and the dialysis temperature is 4°C.

4. The method for preparing recombinant type VI collagen according to claim 2, wherein: The concentration of added IPTG is 0.4 mmol / L.

5. The method for preparing recombinant type VI collagen according to claim 2, wherein: The induction expression temperature is 37°C.

6. The method for preparing recombinant type VI collagen according to claim 2, wherein: The induction expression time is 6 hours.

7. Use of the recombinant type VI collagen prepared by the method for preparing recombinant type VI collagen according to any one of claims 1 to 6 in preparing preparations having antioxidant function.

8. Use of the recombinant type VI collagen prepared by the method for preparing recombinant type VI collagen according to any one of claims 1 to 6 in preparing preparations for protecting epidermal cells damaged by UVB.

9. Use of the recombinant type VI collagen according to claim 8 in the preparation of a preparation for protecting epidermal cells damaged by UVB, characterized in that: The concentration of recombinant type VI collagen in the application is 0.1-1 mg / mL.

10. Use of the recombinant type VI collagen prepared by the method for preparing recombinant type VI collagen according to any one of claims 1 to 6 in preparing preparations related to stimulating collagen formation.

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