Recombinant human III-type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant human III-type collagen as well as a preparation method and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant human III-type collagen is shown as SEQ ID No.1, and the nucleotide sequence of a gene for coding the recombinant human III-type collagen is shown as SEQ ID No.2; the invention also provides a method for expressing the recombinant human III type collagen in mammalian cells. The recombinant human III-type collagen provided by the invention is high in purity, has triple helix characteristic circular dichroism, has the molecular weight of 21.8 kDa, is far smaller than that of natural full-length collagen, has the effects of promoting cell proliferation and cell migration, and reduces obstacles for the collagen to penetrate through a skin barrier to exert biological functions.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and in particular relates to a recombinant human type III collagen and a preparation method and application thereof. Background Art

[0002] Collagen accounts for 25% to 30% of the total protein in the human body. Among the 28 types of collagen, type I, type II, and type III account for 90%. Human type III collagen is a fibrous protein formed by three alpha chain helices. Each protein chain of mature type III collagen contains 1068 amino acids, and the molecular weight of the three protein chains forming a triple helix is ​​close to 400kDa. Type III collagen contains multiple signal pathway recognition sites, such as binding to integrin recognition sites to activate collagen regeneration, binding to von Willebrand factor to participate in wound healing, binding to DDR2 to participate in cell proliferation, adhesion and bone cell growth, and binding to LAIR1 to inhibit the occurrence of inflammation. The amino acid sequence of type III collagen contains a sequence Gly-XY repeated 343 times. X and Y can be any amino acids, but X is usually proline (Pro), and Y is usually hydroxyproline (Hyp) and lysine (Hyl). Hydroxylated proline and lysine play an important role in maintaining the triple helical structure of collagen. The natural collagen rich in hydroxyl groups can combine with water molecules to produce hydration, which has the effects of promoting tissue growth and healing, retaining moisture, whitening, and nourishing the skin. However, when used as an ingredient in skin care products, the natural micelle structure and high molecular weight of collagen make it difficult to penetrate the skin barrier to reach the dermis and exert its effects.

[0003] The main production methods of collagen on the market include animal-derived collagen extraction, recombinant expression in E. coli, and recombinant expression in yeast cells. Animal-derived collagen has the risk of immunogenicity, and the recombinant collagen expressed in E. coli and yeast cells has a low hydroxylation level, is not easy to form a triple helix structure or the triple helix structure is unstable, and cannot play its due function. Summary of the invention

[0004] In view of this, the first purpose of the present invention is to provide a recombinant human type III collagen. The recombinant human type III collagen provided by the present invention has high purity, a characteristic triple-helix circular dichroism spectrum, and a molecular weight of 21.8 kDa, which is much smaller than the natural full-length collagen. It has the effects of promoting cell proliferation and cell migration, and reduces obstacles for collagen to pass through the skin barrier to exert its biological functions.

[0005] The second object of the present invention is to provide a method for expressing the recombinant human type III collagen in mammalian cells.

[0006] The third object of the present invention is to provide the use of the recombinant human type III collagen in the preparation of skin care products.

[0007] The present invention provides a recombinant human type III collagen, the amino acid sequence of which is shown in SEQ ID No.1.

[0008] The present invention provides a gene encoding the recombinant human type III collagen, and the nucleotide sequence of the gene is shown as SEQ ID No.2.

[0009] The invention provides a recombinant vector for expressing the recombinant human type III collagen, comprising an initial vector and the gene.

[0010] Preferably, the initial vector comprises pcDNA3.1 or pcDNA3.4 vector.

[0011] The present invention provides a recombinant cell expressing the recombinant human type III collagen, wherein the recombinant vector is transfected into the recombinant cell.

[0012] Preferably, the initial cells are mammalian cells.

[0013] The present invention provides a method for preparing recombinant human type III collagen, comprising the following steps:

[0014] 1) Recombining the gene into the initial vector pcDNA3.1 to obtain the recombinant vector;

[0015] 2) The gene encoding proline hydroxylase PBCV was recombined into the initial vector pcDNA3.4 to obtain the first auxiliary vector, and the gene encoding lysine hydroxylase L230 was recombined into the initial vector pcDNA3.4 to obtain the second auxiliary vector;

[0016] 3) co-transfecting the recombinant vector described in step 1), the first auxiliary vector described in step 2), and the second auxiliary vector described in step 2) into the initial cells to obtain recombinant cells;

[0017] 4) After culturing the recombinant cells, centrifuging and collecting the supernatant;

[0018] 5) Separating and obtaining recombinant human type III collagen from the supernatant.

[0019] Preferably, the separation in step 5) comprises nickel column affinity purification, ion exchange chromatography and dialysis performed sequentially.

[0020] The present invention provides the recombinant human type III collagen and the use of the recombinant human type III collagen prepared by the preparation method in the preparation of skin care products and cartilage filling materials.

[0021] The present invention provides the recombinant human type III collagen and the use of the recombinant human type III collagen prepared by the preparation method in promoting cell growth and healing.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention analyzes the functional sites in the collagen sequence, and extracts the region that has a stabilizing effect on triple helix formation, the integrin binding site, the LAIR1 binding site, the alanine-rich region that improves protein elasticity, the trimer signal region that helps triple helix formation, and the secretion signal peptide to obtain recombinant human type III collagen. The molecular weight is 21.8 kDa, which is much smaller than the natural full-length collagen. Circular dichroism detection has a typical triple helix characteristic peak shape, has the effects of promoting cell proliferation and cell migration, and reduces obstacles for collagen to pass through the skin barrier and exert its biological function.

[0024] The present invention recombines the gene encoding the recombinant human type III collagen into a pcDNA3.1 vector, and co-transfects the gene with a proline hydroxylase recombinant plasmid and a lysine hydroxylase recombinant plasmid into mammalian cells to obtain recombinant cells. The recombinant human type III collagen secreted into the supernatant is collected by culturing the recombinant cells. The recombinant human type III collagen derived from mammalian cells overcomes the disadvantages of immunogenicity risk or structural instability existing in animal-derived collagen extraction and recombinant proteins derived from engineered bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the SDS-PAGE image of recombinant human type III collagen;

[0026] Figure 2 This is the circular dichroism spectrum of recombinant human type III collagen;

[0027] Figure 3 The circular dichroism spectra of recombinant human type III collagen after heat treatment at different temperatures for 30 minutes;

[0028] Figure 4 The circular dichroism spectra of recombinant human type III collagen after heat treatment at 90℃ for different time periods;

[0029] Figure 5 For the cell proliferation experiment of recombinant human type III collagen;

[0030] Figure 6 Cell scratch test for recombinant human type III collagen;

[0031] Figure 7 The confluence of cells was measured by the recombinant human type III collagen scratch assay. DETAILED DESCRIPTION

[0032] The present invention provides a recombinant human type III collagen, the amino acid sequence of which is shown in SEQ ID No. 1, and is specifically as follows (hereinafter referred to as JY11):

[0033] GKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGDAGAPGE RGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGAPGPQGPRGDKGETGERCCGGVGAAAGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH*.

[0034] The present invention provides a gene encoding the recombinant human type III collagen, the nucleotide sequence of which is shown in SEQ ID No. 2, specifically as follows (hereinafter referred to as JY-2):

[0035] .

[0036] The invention provides a recombinant vector for expressing the recombinant human type III collagen, comprising an initial vector and the gene.

[0037] In the present invention, the initial vector includes a pcDNA3.1 or pcDNA3.4 vector. The present invention does not specifically limit the preparation method of the recombinant vector, and the conventional preparation method of the recombinant vector in the art can be used. In the specific implementation of the present invention, the gene is preferably recombined onto the initial vector by homologous recombination. In the present invention, the gene JY-2 is preferably synthesized by a biotechnology company. In the present invention, the pcDNA3.1 vector contains a trimer signal region and a secretion signal peptide that help the formation of a triple helix.

[0038] The present invention also provides a recombinant cell expressing the recombinant human type III collagen, wherein the recombinant vector is transfected into the recombinant cell. In the present invention, the initial cell is preferably a mammalian cell, and more preferably a HEK293F cell (donated by Shenzhen Bowansen Biotechnology Co., Ltd.).

[0039] The present invention provides a method for preparing recombinant human type III collagen, comprising the following steps:

[0040] 1) Recombining the gene into the initial vector pcDNA3.1 to obtain the recombinant vector;

[0041] 2) The gene encoding proline hydroxylase PBCV was recombined into the initial vector pcDNA3.4 to obtain the first auxiliary vector, and the gene encoding lysine hydroxylase L230 was recombined into the initial vector pcDNA3.4 to obtain the second auxiliary vector;

[0042] 3) co-transfecting the recombinant vector described in step 1), the first auxiliary vector described in step 2), and the second auxiliary vector described in step 2) into the initial cells to obtain recombinant cells;

[0043] 4) After culturing the recombinant cells, centrifuging and collecting the supernatant;

[0044] 5) Separating and obtaining recombinant human type III collagen from the supernatant.

[0045] The present invention has no special limitation on the preparation method of the first auxiliary carrier and the second auxiliary carrier, and they can be prepared by conventional preparation methods in the art.

[0046] In the present invention, the recombinant vector, the first auxiliary vector and the second auxiliary vector are co-transfected into the initial cells at a ratio of 75:15:10 to obtain recombinant cells. In the present invention, the initial cell density is preferably 2 to 3×10 6 / mL, cell viability was greater than 98%.

[0047] In the present invention, the conditions for culturing the recombinant cells are preferably 37°C, 5% CO2, 120rpm; the time for culturing the recombinant cells is preferably 4 to 6 days, and more preferably 5 days. After the culturing is completed, the cell culture fluid is centrifuged; the speed of the centrifugation is preferably 2000g, and the time of the centrifugation is preferably 30 minutes. After the centrifugation, the supernatant is collected, and the supernatant is preferably filtered through a membrane for subsequent operations, and the pore size of the filter membrane is preferably 0.45 μm.

[0048] In the present invention, the separation preferably includes nickel column affinity purification, ion exchange chromatography and dialysis performed in sequence. The present invention has no special limitation on the separation and purification method, and conventional protein separation and purification methods in the art can be used; the preferred technical scheme is described in the examples.

[0049] The present invention also provides the recombinant human type III collagen. The recombinant human type III collagen prepared by the preparation method has a typical triple helix circular dichroism spectral peak shape.

[0050] The present invention also provides the recombinant human type III collagen and the use of the recombinant human type III collagen prepared by the preparation method in the preparation of skin care products.

[0051] The present invention also provides the recombinant human type III collagen and the use of the recombinant human type III collagen prepared by the preparation method in promoting cell growth and healing.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] By analyzing the functional sites in the collagen sequence, amino acids and nucleic acid sequences that stabilize triple helix formation, integrin binding sites, LAIR1 binding sites, and alanine-rich regions that improve protein elasticity were intercepted, and the nucleic acid fragment JY11 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. JY-11 was fused to the pCDNA3.1 vector by homologous recombination, which contains a trimer signal region and a secretion signal peptide that helps triple helix formation. The DNA sequences containing proline hydroxylase PBCV and lysine hydroxylase L230 were constructed into the expression vector pCDNA3.4 respectively. The plasmid bacteria that were successfully constructed by sequencing were cultured in large quantities and the plasmids were extracted, and filtered with a 0.22μm filter membrane before transfecting the cells.

[0055] Prepare a 125ml cell culture flask and preheat SMM-293-TI culture medium. Take out a tube of frozen HEK293F cells from the liquid nitrogen storage tank and quickly shake to thaw the cells in a 37°C water bath. Take a 15ml centrifuge tube, add 5ml culture medium, use a pipette to pipette the cells into the culture medium, blow gently to mix, and centrifuge at 1000rpm for 5min. Use a pipette to remove the supernatant, and then add 3ml culture medium to resuspend the cell pellet. Pipette 20μl of cell suspension and 20μl of 0.04% trypan blue into the EP tube and blow to mix. Pipette 20μl into the small hole of the cell counting plate and place it on the cell counter. Record data such as cell density and cell viability. After counting, transfer to a 125ml cell culture flask and add a certain volume of culture medium to make the cell density reach 3-5×10 5 / mL, cultured in a shaker at 37°C, 5% CO2, 120rpm. Cells were cultured and subcultured continuously until the volume reached the required cell volume and the cell density was 2-3×10 6 Plasmid transfection can be performed when the viability is greater than 98%.

[0056] Collagen protein plasmids and plasmids containing proline hydroxylase PBCV and lysine hydroxylase L230 were transfected into 1L HEK293F cells and cultured continuously for 5 days in a shaker at 120 rpm, 37°C, and 5% CO2. On the fifth day, the cells were removed, centrifuged at 2000g for 30 minutes, and the supernatant was collected and filtered through 0.45 μm.

[0057] Add the balanced NI-Beads filler to the gravity column, and use the peristaltic pump to draw the cell supernatant into the filler to enrich the protein. Wash the impurities with low imidazole buffer, and wash away the impurities with about 90 filler volumes. Elute the target protein with high imidazole buffer.

[0058] The target protein eluted by nickel column affinity was subjected to cation exchange chromatography. Observe the SDS-PAGE protein bands after ion exchange chromatography, collect the target protein with higher purity into the same tube, cut a dialysis bag of appropriate length, soak it in the dialysate 50mMK2HPO4·KH2PO4 (pH=8.0) for 2-3min, transfer the eluted protein solution into the dialysis bag, put the dialysis bag containing the eluted protein solution into a 3L50mMK2HPO4·KH2PO4 measuring bucket, put a magnetic stirrer in the measuring bucket, put it on the magnetic stirrer and dialyze for more than 16 hours, and replace the dialysate once in the middle. The purified protein was named JY11LP, and the purity of the purified protein was analyzed by SDS-PAGE as follows Figure 1 shown.

[0059] The concentration of the dialyzed collagen was determined using the BCA protein concentration assay kit (enhanced). Two replicate wells were used for each protein, and the dialysate was used as the protein diluent. The protein concentration was calculated based on the BSA standard curve. The protein concentration was diluted to 0.05 mg / ml using the diluent. The secondary structure of the protein (e.g., Figure 2 ). The purity of the purified recombinant collagen is higher than 90%. The circular dichroism spectrum shows a weak positive absorption peak near 225nm and a large negative absorption peak near 197nm, which is consistent with the triple helix characteristic absorption. Comparative sample 1 and comparative sample 2 have no positive absorption peak near 225nm.

[0060] In order to detect the structural stability of recombinant collagen JY11LP after heat treatment, the purified recombinant collagen was placed at 45°C for 30 minutes, 60°C for 30 minutes, 70°C for 30 minutes, 90°C for 30 minutes, 90°C for 1 hour, 90°C for 2 hours and 90°C for 3 hours, and then restored to 4 degrees. After overnight, the protein structure was determined using circular dichroism spectrometry. The results showed that the 3 helical structural characteristics of the recombinant collagen were retained after heat treatment at different temperatures for 30 minutes and restored to 4 degrees for storage, and there was no obvious change (such as Figure 3 ); After heat treatment at 90℃ for more than 1 hour, the positive peak near 225nm was slightly weakened, the negative peak near 197nm was slightly shifted to the right, and the structure of recombinant collagen changed slightly, but the change was not obvious (such as Figure 4 ).

[0061] Example 2

[0062] Performance test of recombinant human type Ⅲ collagen JY11LP

[0063] Cell proliferation assay

[0064] After counting the mouse embryonic fibroblast ALB / 3T3 (Starfish Bio, Cat: TCM-C714), the cells were inoculated into a 96-well cell culture plate at 2000 cells / well and placed in a 37°C, 5% CO2 cell culture incubator for 24 hours. DMEM serum-free medium was replaced and cultured for 12 hours. 0.1 μg of purified recombinant human collagen solution (JY11LP) or ordinary recombinant collagen solution (comparison sample 1, comparison sample 2) and PBS (blank control) were added to the cell culture plate and cultured for 24 hours. 10 μL of CCK-8 reagent was added to each well and incubated in a 37°C, 5% CO2 cell culture incubator for 1 hour before taking out. Use an enzyme reader to read the absorbance value of the 96-well plate at 450nm and record the measurement results (such as Figure 5 ).

[0065] In the cell proliferation experiment, after adding 0.1 μg JY11LP recombinant human collagen to the experimental group cells and culturing for 24 hours, the cell proliferation rate was significantly faster than that of other recombinant collagen treatment groups (comparative sample 1, comparative sample 2) and the blank control group (PBS), indicating that JY11LP recombinant human collagen peptide has proliferative activity on BALB / 3T3 cells.

[0066] Cell migration assay

[0067] After counting BALB / 3T3 cells, 1×10 5 Cells / well were inoculated in a 6-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. When the cell density reached 90%, a line was drawn on the 6-well plate with a yellow pipette tip, and the initial scratch position was recorded under a microscope. 2 μg of purified recombinant human collagen JY11LP solution or ordinary collagen solution (comparative sample 1, comparative sample 2) and PBS (blank control) were added to each well of the 6-well plate, and culture was continued for 24 hours at 37°C, 5% CO2 incubator.

[0068] Cells at the same scratch position were photographed under a microscope. Image J software was used to calculate the scratch area of ​​cells at the beginning and after 24 hours of treatment, and the confluence rate was calculated. Confluence rate = (0 hour scratch area - 24 hour scratch area) / 24 hour scratch area.

[0069] The results are as follows Figure 6 and Figure 7 As shown in the figure, after adding 2μg JY11LP recombinant human collagen to the experimental group cells and culturing for 24h, the degree of scratch recovery was significantly faster than that of other recombinant collagen treatment groups and the blank control group. The scratch area was calculated by Image J software, and the results showed that the scratch recovery area of ​​cells in the JY11LP recombinant human collagen treatment group was 42.40%, the scratch recovery area of ​​cells in the comparison sample 1 collagen treatment group was 32.18%, the scratch recovery area of ​​cells in the comparison sample 2 collagen treatment group was 32.86%, and the scratch recovery area of ​​cells in the PBS treatment group was 28.17%. This shows that JY11LP recombinant human collagen has a migration-promoting activity on BALB / 3T3 cells.

[0070] It can be seen that the recombinant collagen JY11LP provided by the present invention has the effect of promoting cell proliferation and cell migration.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant human type III collagen, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No.

1.

2. The gene encoding the recombinant human type III collagen according to claim 1, characterized in that: The nucleotide sequence of the gene is as shown in SEQ ID No.2 or the nucleotide sequence of the gene has a homology of more than 90% with the sequence shown in SEQ ID No.

2.

3. The recombinant vector expressing the recombinant human type III collagen according to claim 1, characterized in that: It comprises an initial vector and the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The initial vector includes pcDNA3.1 vector or pcDNA3.

4.

5. The recombinant cell expressing the recombinant human type III collagen according to claim 1, characterized in that: The recombinant cell is transfected with the recombinant vector according to claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that The starting cells are mammalian cells.

7. The method for preparing recombinant human type III collagen according to claim 1, characterized in that: The following steps are involved: 1) Recombining the gene described in claim 2 into the initial vector pcDNA3.1 to obtain the recombinant vector described in claim 3; 2) The gene encoding proline hydroxylase PBCV was recombined into the initial vector pcDNA3.4 to obtain the first auxiliary vector, and the gene encoding lysine hydroxylase L230 was recombined into the initial vector pcDNA3.4 to obtain the second auxiliary vector; 3) co-transfecting the recombinant vector described in step 1), the first auxiliary vector described in step 2), and the second auxiliary vector described in step 2) into the initial cells to obtain recombinant cells; 4) After culturing the recombinant cells, centrifuging and collecting the supernatant; 5) Separating and obtaining recombinant human type III collagen from the supernatant.

8. The preparation method according to claim 7, characterized in that: The separation in step 5) comprises nickel column affinity purification, ion exchange chromatography and dialysis performed sequentially.

9. Use of the recombinant human type III collagen according to claim 1 or the recombinant human type III collagen prepared by the preparation method according to claim 7 or 8 in the preparation of skin care products and cartilage filling materials.

10. Use of the recombinant human type III collagen according to claim 1 or the recombinant human type III collagen prepared by the preparation method according to claim 7 or 8 in promoting cell growth and healing.

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