A recombinant human VI+XVII collagen and its preparation method

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a recombinant human VI+XVII collagen and a preparation method thereof, wherein the recombinant human VI+XVII collagen is composed of a water-soluble fragment of recombinant human type VI collagen 84aa and a recombinant human type XVII collagen 149aa connected end to end. The recombinant plasmid obtained by construction can stably and massively express recombinant human VI+XVII collagen in an Escherichia coli expression system. The sequences of type VI and type XVII collagen selected by the present invention are both natural human amino acid sequences, and will not produce immune rejection reactions when applied to the human body. Recombinant human VI+XVII collagen has excellent biological activities of cell adhesion, cell migration and cell proliferation, enhances the repair effect on skin tissue and hair follicles, and provides new ideas and methods for its application in the fields of skin care products, hair care, skin repair dressings and medical cosmetology.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant human VI+XVII collagen and a preparation method thereof. Background Art

[0002] Collagen is the main protein in the human extracellular matrix, accounting for one-third of human proteins and three-quarters of the dry weight of the skin. It has the function of maintaining the structure and normal physiological function of tissues and organs such as skin, cartilage, tendons, ligaments and internal organs. In addition to its structural role, collagen can also interact with integrins, fibronectin, heparin and matrix metalloproteinases (MMPs), and is very important in regulating tissue regeneration. 29 different collagens with 48 different chains have been identified. As a scaffold for the attachment of cells and matrix proteins, it has excellent physical and chemical properties, biological efficacy, biocompatibility, biodegradability, etc., and is an ideal biomedical and beauty skin care material.

[0003] At present, the recombinant human collagen that has been studied more is mainly type I, II, and III, and there is little research on other types of collagen. Type XVII collagen is a unique transmembrane protein and a key component of the hemidesmosomes that connect epithelial cells and basement membrane zones. It can improve the state of the basement membrane, anchor epidermal cells, achieve skin anti-aging effects, and allow the basement membrane to better exchange information and substances. It achieves basement membrane adhesion, tissue regeneration, and maintains the activity of hair follicle stem cells and epidermal stem cells, which plays an important role in cell aging and skin differentiation.

[0004] At present, due to the low content of human type XVII collagen in the human body and its complex molecular structure, it is very difficult to directly extract and mass produce. As a major method for producing collagen in recent years, genetic engineering has solved the immunogenicity and viral risks of animal protein compared to collagen obtained by traditional extraction, and has low rejection reaction. At the same time, it has the advantages of processability and stable quality, making it more suitable for application in the biomedical field.

[0005] However, in the prior art, prokaryotic expression systems are often used to express type XVII collagen in the form of inclusion bodies, which makes expression difficult. Human type XVII collagen is a transmembrane collagen with intracellular, transmembrane and extracellular regions. When transmembrane proteins are expressed in cells, they are not secreted outside the cells but are fixed on the cell membrane. In addition, the amino acid sequence of human type XVII collagen is very long (1497 amino acids) and the protein molecular weight is large (180 kDa). It is difficult to effectively secrete it outside the cells and is easily degraded, resulting in the complexity of the preparation process and high preparation costs.

[0006] The existing production method of type XVII collagen is difficult to achieve low-cost large-scale production, and the single-sequence type XVII collagen has poor stability, which limits its use conditions and effects. The current type XVII collagen production is difficult to match the increasingly strong market demand. Summary of the invention

[0007] In view of the above problems, the present invention provides a method for preparing recombinant human VI+XVII collagen.

[0008] In order to achieve the above-mentioned object, the technical scheme adopted by the present invention is: a recombinant human VI+XVII collagen, whose amino acid sequence is as follows: MGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQ GPKGDKGDPGEMGHKGEKGDKGDQGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPL GHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQGPKGDKGDPGEMGHKGEKGDKGDQ.

[0009] A method for preparing recombinant human VI+XVII collagen comprises the following steps:

[0010] Step 1. Protein sequence design: The recombinant human VI+XVII collagen is characterized by being composed of a recombinant human type VI collagen fragment and a recombinant human type XVII collagen fragment connected at the first position;

[0011] Step 2: construct an expression vector for recombinant VI+XVⅡ collagen: design and synthesize a gene fragment encoding VI+XVⅡ collagen, clone it into an expression vector, use Escherichia coli as an expression host, perform codon optimization, and determine the gene sequence of recombinant VI+XVⅡ collagen; transfer the synthetic gene sequence into an expression plasmid by double enzyme digestion to obtain a recombinant expression plasmid;

[0012] Step 3: Prokaryotic expression: The constructed recombinant expression plasmid is transformed into competent E. coli cells by a one-step chemical method, transformants containing the recombinant expression plasmid are screened out, and single colonies are selected for induction culture;

[0013] Step 4, preparation of recombinant VI+XVII collagen: the collected cell lysis supernatant containing recombinant VI+XVII collagen is subjected to membrane filtration, and a recombinant VI+XVII collagen solution is prepared by a cation exchange chromatography method, and the buffer is replaced by ultrafiltration and concentrated, and freeze-dried to collect collagen freeze-dried powder;

[0014] Step 5. Efficacy verification: Through specific experiments, it is verified that the recombinant collagen has the biological activity of cell adhesion, cell migration promotion and cell proliferation promotion.

[0015] Preferably, the recombinant type VI+XVII collagen in step 1 uses a short peptide connected end to end as a repeating unit, which is repeated at least twice to obtain a repeating short peptide.

[0016] Preferably, the method for constructing the expression vector of recombinant VI+XVII collagen in step 2 is: the gene fragment of the synthesized recombinant human VI+XVII collagen is connected to the pET30a(+) plasmid by double restriction of NdeI and Hind Ⅲ to obtain pET30a(+)-VI+XVⅡ.

[0017] Preferably, the expression method of the expression strain in step 3 is: transform the successfully constructed expression plasmid pET30a (+) -VI + XVII into the competent Escherichia coli; culture in a 37 ° C incubator for about 20 hours until clearly visible colonies grow; pick a single clone colony from the transformed LB plate and culture it in 10 ml of LB medium containing 50 μg / ml kanamycin for 12h-16h, and then transfer it to 12g / L peptone, The culture was expanded in TB medium containing 24 g / L yeast extract and 0.4% glycerol; the culture was carried out at 37°C and 220 rpm until the OD600 of the bacterial solution was 0.8-1.0, IPTG with a final concentration of 0.5 mM was added to induce expression, the bacteria were collected by centrifugation, ultrasonically disrupted, and the cell lysis supernatant and precipitate were separated by centrifugation. The cells were lysed, and the supernatant was stored in an environment of -20°C and purified. The obtained recombinant VI+XVII collagen was detected by SDS-PAGE.

[0018] Preferably, the Escherichia coli is BL21 (DE3), OrigamiB (DE3), BL21 Star Plyss (DE3), Rosetta gami B (DE3) or Arctic Express (DE3).

[0019] Preferably, the induction conditions are culturing at 10°C and 180 rpm for 20 h.

[0020] Preferably, the ultrafiltration replacement buffer composition of step 4 is: 25mM Tris-HCl and 1M NaCl, pH 8.0.

[0021] Preferably, the efficacy verification includes cell adhesion activity detection, cell migration promotion experiment and cell proliferation promotion experiment. Compared with the prior art, the advantages and positive effects of the present invention are:

[0022] (1) The present invention screens and obtains a functional region amino acid fragment of human type VI collagen with high water solubility and good stability, and fuses it with a functional region amino acid fragment of human type XVII collagen to express it, thereby improving the stability and water solubility of type XVII collagen molecules;

[0023] (2) Recombinant humanized VI+XVII collagen with excellent biological activity is obtained by repeating this fusion peptide, achieving efficient and safe expression of recombinant human VI+XVII collagen; the E. coli expression system is used, which is suitable for large-scale amplification, with a short fermentation cycle of only a few hours and very low production cost. Since the target gene sequence is screened, constructed and designed, and E. coli codons are optimized, the target protein expression level is high;

[0024] (3) By fusion-expressing type VI+XVII collagen, higher activity in promoting cell migration, cell proliferation and cell adhesion is achieved. The excellent biological activity of cell adhesion, cell migration and cell proliferation verified by experiments enhances the repair effect on skin tissue and hair follicles, providing new ideas and methods for its application in skin care products, hair care, skin repair dressings and medical cosmetology.

[0025] (4) The recombinant collagen involved in the present invention is not designed with an affinity tag, which simplifies the two-step purification process and only uses one-step ion exchange chromatography to produce recombinant collagen as a biomaterial, providing feasibility for its application in medical devices, skin care product raw materials, and biopharmaceuticals.

[0026] (5) The present invention uses an engineered strain containing a molecular chaperone to express the target protein, thereby reducing the number of bases of the exogenous sequence inserted into the recombinant plasmid, helping to improve the stability of the recombinant plasmid and reducing the uncontrollable factors caused by the instability of the plasmid in large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following is a brief introduction to the drawings required for describing the embodiment:

[0028] Figure 1This is a schematic diagram of the recombinant plasmid of recombinant human VI+XVII collagen.

[0029] Figure 2 This is the protein expression diagram of recombinant human VI+XVⅡ collagen.

[0030] Figure 3 This is the electrophoresis diagram of the purification results of recombinant human VI+XVⅡ collagen.

[0031] Figure 4 The results of the cell adhesion experiment are shown in Figure 2.

[0032] Figure 5 The results of the cell migration experiment are shown in Figure 2.

[0033] Figure 6 This is a graph showing the results of a cell proliferation promotion experiment. DETAILED DESCRIPTION

[0034] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below with reference to the accompanying drawings and embodiments.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0036] Example 1

[0037] The following is combined with Figure 1-Figure 6To further describe the present invention, a recombinant human VI+XVII collagen protein has an amino acid sequence as follows: MGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQGPKGD KGDPGEMGHKGEKGDKGDQGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPLGH PGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQGPKGDKGDPGEMGHKGEKGDKGDQ.

[0038] A method for preparing recombinant human VI+XVII collagen,

[0039] Step 1: protein sequence design: the recombinant human VI+XVII collagen is characterized by being composed of a recombinant human type VI collagen fragment and a recombinant human type XVII collagen fragment connected at the first position, and the amino acid sequence is designed;

[0040] The hydrophobicity and protein stability of the amino acid sequence of human type XVⅡ α1 chain collagen and the functional region of human type VI α1 chain collagen were analyzed respectively. According to the results of hydrophobicity analysis, a 149 amino acid short peptide fragment of type XVⅡ collagen and an 84 amino acid short peptide fragment of human type VI collagen were selected. These two short peptide fragments were connected end to end into a 233aa short peptide as a repeating unit. The repeated short peptide was repeated twice to obtain the recombinant human VI+XVⅡ collagen amino acid sequence.

[0041] The amino acid sequences with advantages such as high water solubility and cell adhesion were screened from the functional region of type VI collagen, and fused with the amino acid sequences selected from the extracellular region of type XVII collagen to improve the stability and water solubility of the recombinant protein. Expasy, a professional protein analysis platform of the Swiss Institute of Bioinformatics at SIB, was used to predict the stability of the recombinant protein, the total amino acid hydrophobicity, and the hydrophilicity of the protein. The hydrophobicity of all amino acids in the recombinant protein was evaluated to be less than 0.5, and the hydrophilicity (GRAVY) was -1.249, indicating that the recombinant protein had good water solubility.

[0042] Step 2: Construct an expression vector for recombinant VI+XVⅡ collagen: Design and synthesize a gene fragment encoding VI+XVⅡ collagen, clone it into an expression vector, use E. coli as the expression host, perform codon optimization, and determine the gene sequence of recombinant VI+XVⅡ collagen; transfer the synthetic gene sequence into an expression plasmid by double enzyme digestion to obtain a recombinant expression plasmid, such as Figure 1 shown.

[0043] Step 3, prokaryotic expression: The constructed expression plasmid pET30a (+) - Ⅵ + XVII was transformed into competent E. coli; the E. coli was BL21 (DE3) strain, cultured in a 37°C incubator for about 20 hours until clearly visible colonies grew; a single clone colony was picked from the transformed LB plate and cultured in 10 ml of LB medium containing 50 μg / ml kanamycin for 12h-16h, and then transferred to TB medium containing 12g / L peptone, 24g / L yeast extract, and 0.4% glycerol at a ratio of 1:100 for expansion culture; cultured at 37°C, 220rpm until the bacterial solution OD600 was 0.8-1.0, and a final concentration of 0.5mM IPTG was used to induce expression. The induction conditions were 10°C and 180 rpm for 20 h. Then the cells were collected by centrifugation, ultrasonically broken, and the cell lysis supernatant and precipitate were separated by centrifugation. The cells were lysed and the supernatant was stored at -20°C. After purification, the recombinant VI+XVII collagen obtained was tested by SDS-PAGE. The results are as follows. Figure 2 As shown in the figure: M is protein Marker, 1 is uninduced whole cells, 2 is cell lysis supernatant induced at 10°C for 20 hours, 3 is cell lysis supernatant induced at 10°C for 25 hours, 4 is cell lysis precipitate induced at 10°C for 20 hours, and 5 is cell lysis precipitate induced at 10°C for 25 hours.

[0044] Step 4, preparing recombinant protein; filtering the collected cell lysis supernatant, preparing recombinant VI+XVⅡ collagen solution by cation exchange chromatography, replacing the buffer by ultrafiltration and concentrating, freeze drying, collecting collagen lyophilized powder, purifying, and electrophoresing the purified recombinant VI+XVⅡ collagen. The results are as follows: Figure 3 As shown in the figure: M is protein marker, 1 is the pretreated protein solution, 2 is the flow-through sample, 3 is the 0.5M NaCl elution result, and 4 is the 0.75M NaCl elution result.

[0045] Step 5: Efficacy verification:

[0046] Cell adhesion activity assay:

[0047] Normally culture NIH / 3T3 cells. Recombinant VI+XVII collagen purified lyophilized powder, reference substance type XVII collagen, and bovine serum albumin ultrapure water were dissolved in PBS with a pH of 7.4 and diluted to 0.5 mg / mL. 100 μL of various protein solutions and blank PBS solution were added to the 96-well cell culture plate and allowed to stand at room temperature for 60 min. Then 105 3T3 cells in good culture state were added to each well and incubated at 37°C and 5% CO2 for 60 min. The cells in the wells were washed 4 times with PBS. The absorbance value at OD492nm was detected using the LDH detection kit. The absorbance at OD492nm can characterize the cell adhesion activity of the collagen sample: the higher the absorbance value, the more cells the protein adheres to, the higher the adhesion activity, and the more collagen can help cells adhere to the wall or the extracellular matrix in a short time, which is more conducive to building a better extracellular environment.

[0048] The results are as follows Figure 4 As shown, the recombinant human type VI+XVII collagen has better cell adhesion activity than the existing human XVII collagen.

[0049] Cell migration assay:

[0050] Scratch is defined as when cells grow to a fused monolayer state, a blank area is artificially created on the fused monolayer cells. Cells at the edge of the scratch will gradually enter the blank area to heal the "scratch". During the cell migration process, images are captured regularly at the starting point and during the process, and the cell migration rate is determined by calculation.

[0051] Take the purified lyophilized powder of recombinant VI+XVⅡ collagen, the reference substance type XVII collagen and bovine serum albumin and dissolve them in ultrapure water, and dilute the samples to 0.5 mg / mL with DMEM serum-free medium, pH7.0-7.4. Culture and passage NIH / 3T3 cells normally. Inoculate cells in good condition into 6-well plates, inoculate 2 mL of cell suspension in each well at a density of 20,000 cells / mL, and culture for 36 hours. Prepare scratches with a 200μL gun tip, wash the cells 3 times with PBS, and remove the scratched cells. Add the protein solution diluted with DMEM serum-free medium to the wells, continue to culture in a 37℃, 5% CO2 incubator, take samples at 0h and 24h, and take pictures. Use Image J software to obtain the initial scratch area and the area of ​​the cell-free blank area, and calculate the migration rate, migration rate = (1-cell-free blank area / initial scratch area) * 100%. Cell migration activity is a more effective indicator of the biological activity of collagen. The higher the migration rate and the faster the speed, the better the biological activity of collagen. The experimental results are as follows Figure 5 shown.

[0052] Cell proliferation assay:

[0053] The MTT method was used to detect the ability of recombinant VI+XVⅡ collagen, reference XVⅡ collagen and BSA to promote cell proliferation using NIH / 3T3 cells as experimental cells. Figure 6 As shown, recombinant type VI + XVⅡ collagen showed better ability to promote cell proliferation than BSA and XVⅡ collagen, and recombinant type VI + XVⅡ collagen had the highest ability to promote cell proliferation.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A recombinant human VI+XVII collagen, characterized in that: The amino acid sequence is: MGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPLGHPGP QGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQGPKGDKGDPGEMGHKGEKGD KGDQGLPGCKGSPGFDGIQGPPGPKGDPGAFGLKGEKGAPGERGGPGERGPRGTPGTRGPRGDPGEAGARGAPGPAGPPGDPGLMGERGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKG QKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPKGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGPPGPPGPQGPKGDKGDPGEMGHKGEKGDKGDQ.

2. A method for preparing the recombinant human VI+XVII collagen according to claim 1, characterized in that: The following steps are involved: Step 1. Protein sequence design: The recombinant human VI+XVII collagen is characterized by being composed of a recombinant human type VI collagen fragment and a recombinant human type XVII collagen fragment connected at the first position; Step 2: construct an expression vector for recombinant VI+XVII collagen: design and synthesize a gene fragment encoding VI+XVII collagen, clone it into an expression vector, use Escherichia coli as an expression host, perform codon optimization, and determine the gene sequence of recombinant VI+XVII collagen; transfer the synthetic gene sequence into an expression plasmid by double enzyme digestion to obtain a recombinant expression plasmid; Step 3: Prokaryotic expression: The constructed recombinant expression plasmid is transformed into competent E. coli cells by a one-step chemical method, transformants containing the recombinant expression plasmid are screened out, and single colonies are selected for induction culture; Step 4, preparation of recombinant VI+XVII collagen: the collected cell lysis supernatant containing recombinant VI+XVII collagen is subjected to membrane filtration, and a recombinant VI+XVII collagen solution is prepared by a cation exchange chromatography method, and the buffer is replaced by ultrafiltration and concentrated, and freeze-dried to collect collagen freeze-dried powder; Step 5. Efficacy verification: Through specific experiments, it is verified that the recombinant collagen has the biological activity of cell adhesion, cell migration promotion and cell proliferation promotion.

3. The method for preparing recombinant human VI+XVII collagen according to claim 2, characterized in that: The recombinant type VI+XVII collagen in step 1 uses a short peptide connected end to end as a repeating unit, which is repeated twice to obtain a repeating short peptide.

4. The method for preparing recombinant human VI+XVII collagen according to claim 2, characterized in that: The method for constructing the expression vector of recombinant VI+XVII collagen in step 2 is: the gene fragment of the synthesized recombinant human VI+XVII collagen is connected to the pET30a(+) plasmid by double restriction of NdeI and Hind Ⅲ to obtain pET30a(+)-VI+XVII.

5. The method for preparing recombinant human VI+XVII collagen according to claim 2, characterized in that: In the step 3, the successfully constructed expression plasmid pET30a (+) -VI + XVII is transformed into competent Escherichia coli; cultured in a 37°C incubator for 20 hours until clearly visible colonies grow; a single clone colony is picked from the transformed LB plate and cultured in 10 ml of LB medium containing 50 μg / ml kanamycin for 12 hours to 16 hours, and then transferred to TB medium containing 12g / L peptone, 24g / L yeast extract, and 0.4% glycerol at a ratio of 1:100 for expansion culture; cultured at 37°C and 220rpm until the OD600 of the bacterial solution is 0.8-1.0, IPTG with a final concentration of 0.5mM is added to induce expression, the bacteria are collected by centrifugation, ultrasonically broken, and the cell lysis supernatant and precipitate are separated by centrifugation, the cells are lysed, the supernatant is taken and stored in a -20°C environment, purified, and the obtained recombinant VI + XVII collagen is detected by SDS-PAGE.

6. The method for preparing recombinant human VI+XVII collagen according to claim 5, characterized in that The Escherichia coli is BL21 (DE3), OrigamiB (DE3), BL21 Star (DE3) pLysS, Rosetta-gamiB (DE3) or ArcticExpress (DE3).

7. The method for preparing recombinant human VI+XVII collagen according to claim 5, characterized in that: The induction conditions are 10° C. and 180 rpm for 20 h.

8. The method for preparing recombinant human VI+XVII collagen according to claim 2, characterized in that: The ultrafiltration replacement buffer composition of step 4 is: 25mM Tris-HCl and 1M NaCl, pH 8.

0.

9. The method for preparing recombinant human VI+XVII collagen according to claim 2, characterized in that: The efficacy verification includes cell adhesion activity detection, cell migration promotion experiment and cell proliferation promotion experiment.

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