Recombinant XII type collagen as well as preparation method and application thereof

Abstract

The invention discloses a recombinant XII type collagen as well as a preparation method and application thereof. The amino acid sequence of the recombinant XII type collagen is any one of (A1)-(A3); (A1) is an amino acid sequence as shown in SEQ ID NO: 1; (A2) is an amino acid sequence which is obtained by substitution, deletion or insertion of one or more amino acid residues of the amino acid sequence as shown in SEQ ID NO: 1 and has the same functional protein; and (A3) is an amino acid sequence of a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of the sequence (A1) or (A2). The recombinant XII type collagen provided by the invention has good antioxidant activity, and has a repairing effect on the problems of skin laxity, wrinkle increase and the like caused by degradation of type I collagen fibers of aged skin.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a recombinant type XII collagen and a preparation method and application thereof. Background Art

[0002] Fibrillar-associated collagen with interrupted triple helix (FACIT) is a subfamily of the collagen family. FACIT plays an important role in the structuring of the extracellular matrix, participates in the regulation of collagen fiber size, and is also a link between different extracellular matrix components and different cells. Type XII collagen is a type of FACIT collagen, which interacts with type I collagen and affects the formation of type I collagen fibers in many tissues. Collagen XII helps maintain the structure of collagen and absorb stress. Mutations or deletions of collagen XII can lead to reduced muscle and bone strength and loose skin. Based on this, type XII collagen may be a potential drug for treating skin loosening caused by collagen fiber degradation after aging. Research on collagen has always focused on fibrillar collagen, while less research has been done on fibril-associated collagen. So far, there are no products related to type XII collagen.

[0003] Fibroblasts are the main cells in the dermis that can synthesize collagen, and the percentage of type I collagen they secrete can reach 90%. Type I collagen is assembled into collagen fibers in the ECM to maintain skin strength and provide skin elasticity. During the aging process of the skin, due to the reduction of collagen synthesis in cells, the cross-linking becomes worse, and the collagen fiber arrangement becomes chaotic, the skin atrophies and produces wrinkles.

[0004] The traditional approach to treating skin aging is to stimulate fibroblasts to promote collagen production and matrix deposition through natural extracts or traditional drugs (such as ascorbic acid and retinol). However, these molecules have multiple other negative effects in the body, such as abnormal angiogenesis and inflammatory response.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a recombinant type XII collagen and its preparation method and application in view of the above-mentioned deficiencies in the prior art. The recombinant type XII collagen has good antioxidant activity and has a repairing effect on skin sagging, increased wrinkles and other problems caused by the degradation of type I collagen fibers in aging skin.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a recombinant type XII collagen, characterized in that the amino acid sequence of the recombinant type XII collagen is any one of the following (A1) to (A3);

[0008] (A1) is the amino acid sequence shown in SEQ ID NO: 1;

[0009] (A2) is an amino acid sequence of a protein having the same function obtained by replacing, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1;

[0010] (A3) is the amino acid sequence of a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the sequence (A1) or (A2);

[0011] SEQ ID NO: 1:

[0012] GPPGPPGPAGGPGAKGPRGERGISGAIGPPGPRGDIGPPGPQGPPGPQGPNGLSIPGEQGRQGMKGDAGEPGLPGRTGTPGLPGPPGPMGPPGDRGFTGKDGAMGPRGPPGPPGSPGSPGVTGPSGKP GKPGDHGRPGPSGLKGEKGDRGDIGPPGPPGPPGSAGARGEPGPGGRPGFGTPGMQGPPGERGLPGEKGERGTGSSGPRGLPGPPGPQGESRTGPPGSTGSRGPPGPPGRPGNSGIRGPPGPPGYC.

[0013] A nucleic acid molecule encoding the above-mentioned recombinant type XII collagen, characterized in that the nucleic acid molecule is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2, or a DNA molecule having 80% or more identity with the nucleotide sequence as shown in SEQ ID NO: 2 and encoding the recombinant type XII collagen;

[0014] SEQ ID NO:2:

[0015] .

[0016] An expression cassette comprising the above nucleic acid molecule, a recombinant microorganism comprising the nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, a recombinant vector comprising the expression cassette, a recombinant microorganism comprising the expression cassette and a recombinant microorganism comprising the recombinant vector.

[0017] A method for preparing the above-mentioned recombinant type XII collagen, characterized in that it includes: inserting a nucleotide sequence encoding the amino acid sequence into an expression vector to obtain a recombinant plasmid vector, then introducing the recombinant plasmid vector into a host cell, then culturing the host cell introduced with the recombinant plasmid vector, collecting and separating and purifying the cell culture to obtain the recombinant type XII collagen.

[0018] The above method is characterized in that the host cell includes a prokaryotic cell or a eukaryotic cell; the prokaryotic cell includes Escherichia coli or Bacillus subtilis; the eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae or mammalian cells.

[0019] The above method is characterized in that the host cell is Pichia pastoris.

[0020] The above method is characterized in that the culture conditions of the recombinant cells are: the recombinant cells are inoculated into YPD medium, cultured for 20h to 25h at 25°C to 30°C and 160 to 240rpm until OD600=10 to 15 as seed liquid, and inoculated into a fermenter at an inoculation rate of 5% to 20%, the culture temperature is 25°C to 30°C, pH=4.5 to 5.5, the dissolved oxygen is controlled at 20% to 50%, and when glycerol is exhausted, induction expression is started. After the induction is completed, the fermentation liquid is collected and centrifuged to collect the supernatant.

[0021] The above method is characterized in that the purification includes: purifying the fermentation supernatant by using at least one of salting out, ultrafiltration, ion exchange chromatography, affinity chromatography and gel filtration chromatography.

[0022] A use of the above-mentioned recombinant type XII collagen, the above-mentioned nucleic acid molecule, or the above-mentioned expression box, recombinant microorganism or recombinant vector in the preparation of products for anti-oxidation and / or treatment of collagen degradation in aging skin.

[0023] The above application is characterized in that the product includes cosmetics, medicines and / or medical devices.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The recombinant type XII collagen of the present invention has good antioxidant activity and has a repairing effect on problems such as skin sagging and increased wrinkles caused by degradation of type I collagen fibers in aging skin. At the same time, the present invention preferably constructs Pichia pastoris engineered bacteria to express the recombinant type XII collagen in large quantities. The production of the recombinant type XII collagen using the Pichia pastoris engineered bacteria has the advantages of high yield and easy purification.

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the map of the recombinant plasmid pPiczαA-XII in Example 1.

[0028] Figure 2 This is the result of colony PCR identification after pPiczαA-XII in Example 2 was transformed into Pichia pastoris, where lanes 2, 5, and 6 are positive transformants; lane 7 is a plasmid positive control.

[0029] Figure 3 This is the SDS-PAGE protein electrophoresis detection diagram of the shake flask fermentation supernatant of the screened strain in Example 3.

[0030] Figure 4 This is a fermentation verification of the strain constructed in Example 4 in a 5L fermenter, and an SDS-PAGE protein electrophoresis detection diagram of the fermentation supernatant at different times after induction.

[0031] Figure 5 This is a diagram for detecting ROS content in Example 6.

[0032] Figure 6 This is the standard curve diagram of the Elisa experiment in Example 7. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0034] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0035] Example 1

[0036] This example is about the synthesis of recombinant type XII collagen gene sequence and the construction of expression strain, and the specific steps are as follows:

[0037] The nucleotide sequence of the recombinant type XII collagen gene was synthesized by BGI (the nucleotide sequence is shown in SEQ ID NO: 2). The nucleotide sequence has an XhoI restriction site introduced at its 5' end and an XbaI restriction site introduced at its 3' end.

[0038] The synthetic gene and pPiczαA expression vector were double-digested with restriction endonucleases XhoI and XbaI, the target fragment was purified by gel recovery, and then the digested fragments were connected with T4-DNA ligase, connected overnight at 4°C, transformed into E. coli DH5α competent cells, positive clones were screened on LB plates containing antibodies, single clones were randomly picked, recombinant plasmids were extracted, and positive clones were identified by enzyme digestion. Then, the recombinant plasmid was double-digested with XhoI and XbaI, the target fragment was recovered by gel recovery, and then connected to the pPiczαA vector fragment digested with the same enzymes to obtain the recombinant plasmid pPiczαA-XII (such as Figure 1 shown) is used for expression.

[0039] Example 2

[0040] This example is about Pichia pastoris strain transformation and plate screening, and the specific operations are as follows:

[0041] Recombinant plasmid pPiczαA-col-XII and empty plasmid pPiczαA were digested with restriction endonuclease SalI, and 2μg-10μg of plasmid was taken. The voltage was 1.5kV, the capacitance was 25μF, the resistance was 200Ω, and the discharge time was 5.0ms. The competent cells of Pichia pastoris X-33 were transformed by electroporation. Then, 1.0mL of ice-cold 1.0mol / L sorbitol solution was immediately added, and YPD plates were quickly coated. The plates were cultured at 28-30℃, and 100ug / mL bleomycin was used to select transformants. Hundreds of transformants were randomly selected, and colony PCR was performed with the original plasmid as a control. Positive transformants were selected (according to the designed primers, the PCR results showed that the positive transformants were those with bands at 750-1000bp, such as Figure 2 shown);

[0042] Example 3

[0043] This example is a fermentation screening of recombinant Pichia pastoris strains, and the specific operations are as follows:

[0044] Positive strains were randomly selected (blank plasmid transformed strains were selected as controls) for preliminary screening by shake flask fermentation. The screening method was as follows: single clones were randomly selected from YPD plates and cultured in YPD medium at 30°C and 200rpm overnight; 5% (v / v) bacterial solution was added to liquid BMGY medium and cultured at 30°C and 200rpm. When the bacterial cell density grew to OD600 of 10, the cells were collected by centrifugation at 4000rpm for 5min, and the precipitate was resuspended in liquid BMMY medium and then inoculated into BMMY medium (50mL / 500mL shake flask, pH=6.0) to make its OD about 1.0, cultured at 30°C and 220rpm, and 1% methanol (v / v) was added every 24h. After induction for 48h, the protein expression was detected by SDS-PAGE (such as Figure 3The strains with higher expression levels were screened out.

[0045] Example 4: Fermentation verification of recombinant Pichia pastoris strain in 5L fermenter

[0046] The strain correctly expressing recombinant type XII collagen in Example 3 was selected for plate activation, and the liquid seed culture medium was transferred. When OD600 = 10, the upper tank was inoculated (the substrate culture medium was BSM culture medium). After the substrate glycerol was completely consumed and the dissolved oxygen recovered, methanol was added for induction. The flow rate of methanol was adjusted according to data feedback. The fermentation was terminated after 40 hours of induction. Samples were taken every 20 hours during the process, and protein electrophoresis was performed. The detection was as follows: Figure 4 As shown, different lanes represent fermentation times of 20 h and 40 h.

[0047] Example 5

[0048] This example is the separation and purification of the recombinant type XII collagen obtained in Example 4, and the specific operations are as follows:

[0049] (1) A 10KDa pore size ultrafiltration membrane was first balanced with a phosphate buffer solution having a pH of 6.0 and a concentration of 20 mmol / L, and the inlet and outlet pressures were maintained at 1.5 bar and 0.5 bar, respectively. The fermentation supernatant in Example 4 was then concentrated. When the supernatant was concentrated to about 1 L, it was diluted with 2 times the volume of the above phosphate buffer solution, and then ultrafiltration was continued to about 1 L. The dilution was repeated 3 times, and the concentrate was collected for later use.

[0050] (2) The chromatography column was equilibrated with a 20 mmol / L phosphate equilibration buffer at a pH of 6.0 for 2 to 3 column volumes, and the concentrated solution after ultrafiltration was loaded onto the column for cation exchange chromatography.

[0051] The specific steps are:

[0052] 1) Equilibrate the chromatography column with ultrapure water for 3 column volumes, then wash with 20 mmol / L, pH 6.0 phosphate buffer for 3 column volumes. After the baseline is stable, linearly elute with 20 mmol / L, pH 6.0 phosphate buffer containing 0.5 M NaCl from 0-0.5 M NaCl for 10 column volumes, and collect the eluate.

[0053] 2) The eluate sample obtained by cation exchange chromatography is concentrated to a certain volume by ultrafiltration through an ultrafiltration tube with a molecular weight cutoff of 5 KDa, with the protein concentration not exceeding 50 mg / mL.

[0054] 3) A 1.6 cm x 80 cm Sephadex G-75 column was used for gel filtration chromatography. Two column volumes were first balanced with 20 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl buffer. The concentrated sample separated by cation exchange chromatography was then loaded and eluted at a flow rate of 0.5 mL / min. The collected solution was detected at a wavelength of 280 nm. The eluate curve had only one absorption peak. The peak eluate was collected and vacuum freeze-dried by a conventional method to obtain a solid protein powder.

[0055] Example 6

[0056] This example is an experiment to evaluate the antioxidant activity of the lyophilized protein sample of recombinant type XII collagen obtained in Example 5 by detecting the level of intracellular reactive oxygen species (ROS). The experimental method is as follows:

[0057] The experimental materials were prepared into a protein solution with a protein concentration of 1 mg / mL using dd H2O and filtered using a 0.22 μm sterile filter in an ultra-clean bench. HSFS cells were seeded in a 6-well plate with a volume of 2 mL per well, containing approximately 2.0×10 5 cells and incubated for 24 hours. Three groups were established, including a control group, a model group and a drug-treated group. 2 mL of culture medium was added to the control group and the model group, respectively, and 500 μg / mL of recombinant type XII collagen was added to the culture medium in the drug-treated group. The cells were then cultured for another 24 hours. Subsequently, other groups, excluding the control group, were incubated for 2 hours at a certain hydrogen peroxide concentration. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. After the cells were collected, they were suspended in the diluted DCFH-DA and incubated in a cell culture incubator at 37°C for 30 minutes. Invert and mix every 3 to 5 minutes to allow full contact between the probe and the cells. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Subsequently, an inverted fluorescence microscope was used for direct observation and photography. The test results are as follows Figure 5 As shown, the intracellular ROS level was the highest and the field of view was the brightest in the H2O2-induced aging model. Compared with the H2O2-induced aging model, the fluorescence intensity of the drug-treated group and the blank control group was dimmer and the intracellular ROS content was lower. The intracellular oxidative stress response caused by increased ROS is closely related to cell senescence, so these results indicate that the antioxidant properties of recombinant type XII collagen can alleviate cell senescence caused by H2O2-induced oxidative stress.

[0058] Example 7

[0059] This example is a test to verify the performance of recombinant type XII collagen in repairing wrinkles on aging skin. Type I collagen is assembled into collagen fibers in the ECM to maintain skin strength and provide skin elasticity. During skin aging, due to the reduction of type I collagen synthesis in cells, cross-linking becomes poor, and the collagen fiber arrangement becomes chaotic, the skin atrophies and produces wrinkles. Therefore, we used a human type I collagen Elisa kit to compare the ability of recombinant type XII collagen and commercial products to stimulate fibroblasts to secrete type I collagen. The standard curve drawn in the experiment is shown in the figure below. Figure 6 The experimental results are shown in Table 1. The conditions of the market product control group and the recombinant type XII collagen solution experimental group were consistent in terms of concentration, dosage, etc.

[0060] Table 1 Detection of type I collagen content

[0061] Grouping Type I collagen content (ng / mL) Blank control group 2.65 Aging Modeling Group 1.70 Recombinant Type XII Collagen 4.07 Products on the market 2.82

[0062] According to the experimental results in Table 1, it can be found that compared with the products on the market, the recombinant type XII collagen of the present invention can more effectively stimulate fibroblasts to synthesize type I collagen. This shows that compared with the products on the market, its effect in treating the problems of sagging skin and increased wrinkles caused by aging is more significant.

[0063] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant type XII collagen, characterized in that: The amino acid sequence of the recombinant type XII collagen is any one of the following (A1) to (A3); (A1) is the amino acid sequence shown in SEQ ID NO: 1; (A2) is an amino acid sequence of a protein having the same function obtained by replacing, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; (A3) is the amino acid sequence of a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the sequence (A1) or (A2).

2. A nucleic acid molecule encoding the recombinant type XII collagen as claimed in claim 1, characterized in that: The nucleic acid molecule is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2, or a DNA molecule having 80% or more identity with the nucleotide sequence as shown in SEQ ID NO: 2 and encoding the recombinant type XII collagen.

3. An expression cassette comprising a nucleic acid molecule as described in claim 2, a recombinant microorganism comprising the nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, a recombinant vector comprising the expression cassette, a recombinant microorganism comprising the expression cassette, and a recombinant microorganism comprising the recombinant vector.

4. A method for preparing the recombinant type XII collagen as claimed in claim 1, characterized in that: include: The nucleotide sequence encoding the amino acid sequence is inserted into an expression vector to obtain a recombinant plasmid vector, which is then introduced into a host cell. The host cell introduced with the recombinant plasmid vector is then cultured, and the cell culture is collected, separated and purified to obtain the recombinant type XII collagen.

5. The method according to claim 4, characterized in that The host cell includes a prokaryotic cell or a eukaryotic cell; the prokaryotic cell includes Escherichia coli or Bacillus subtilis; the eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae or a mammalian cell.

6. The method according to claim 5, characterized in that The host cell is Pichia pastoris.

7. The method according to claim 4, characterized in that The culture conditions of the recombinant cells are as follows: the recombinant cells are inoculated into a YPD medium, cultured for 20 h to 25 h at 25° C. to 30° C. and 160 to 240 rpm until OD600=10 to 15 as a seed solution, inoculated into a fermentation tank at an inoculation rate of 5% to 20%, the culture temperature is 25° C. to 30° C., the pH is 4.5 to 5.5, the dissolved oxygen is controlled at 20% to 50%, and the induction expression is started after the glycerol is exhausted. After the induction is completed, the fermentation liquid is collected and centrifuged, and the supernatant is collected.

8. The method according to claim 7, characterized in that The purification comprises: purifying the fermentation supernatant by using at least one of salting out, ultrafiltration, ion exchange chromatography, affinity chromatography and gel filtration chromatography.

9. Use of the recombinant type XII collagen as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, or the expression cassette, recombinant microorganism or recombinant vector as claimed in claim 3 in the preparation of a product for anti-oxidation and / or treatment of collagen degradation in aging skin.

10. The use according to claim 9, characterized in that: The products include cosmetics, pharmaceuticals and / or medical devices.

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