Recombinant human I-type collagen and application thereof

Abstract

The invention discloses recombinant human I-type collagen and application thereof. The amino acid sequence of the recombinant human I-type collagen is as shown in SEQ ID No: 2. The recombinant human I-type collagen has good cell proliferation promoting activity, cell migration promoting activity and cell adhesion promoting activity (superior to full-length recombinant human I-type collagen or commercially available human collagen) and is easy to produce. The recombinant human I-type collagen can be expected to have the effects of whitening, moisturizing, relieving and repairing, and can be applied to the fields of cosmetics, external medicines, skin care products, health care products, tissue engineering materials, medical instruments and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant human type I collagen and a use thereof. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.

[0003] The sources of natural human collagen are limited. The natural collagen currently used in industry is mainly extracted from animal skin or bones by acid, alkali or enzyme methods, and its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases, and large-scale preparation puts tremendous pressure on animal breeding on the supply side.

[0004] With the large-scale application of genetic engineering technology, genetic engineering recombinant expression of collagen has successfully solved the bottleneck of large-scale preparation of collagen by using patterned expression hosts to express exogenous proteins. Compared with traditional animal collagen extraction, genetic engineering recombinant preparation of collagen has the following advantages: 1. A wide variety of collagen is produced, which can be collagen from common sources such as cattle, pigs, and fish, and human collagen can also be produced, which has better safety and immune efficacy in the fields of medical devices. 2. It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Its cell pathogens cannot be transmitted to each other due to the huge difference in cell structure with humans. 3. The production cycle is short, cost-saving, can be quickly amplified, and is suitable for industrial large-scale production. Compared with the long animal culture of 3-5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Large-scale culture can be carried out by providing simple C source and N source, which is easy to prepare at industrial level. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot spot in collagen production research. Summary of the invention

[0005] The purpose of the present invention is to provide a new recombinant human type I collagen protein that has good cell proliferation promoting activity, cell migration promoting activity, cell adhesion promoting activity and is easy to express, and its production method and use. The recombinant human type I collagen protein is expected to have whitening, moisturizing, soothing and repairing effects, and can be applied to cosmetics, external medicines, skin care products, health products, tissue engineering materials, medical devices and other fields.

[0006] That is, the technical solution of the present invention includes:

[0007] 1. A recombinant human type I collagen, the amino acid sequence of which is shown in SEQ ID No: 2.

[0008] SEQ ID No:2

[0009]

[0010]

[0011] The recombinant human type I collagen also has a tag for easy purification, which can be, for example, 6*His, GST, MBP or SUMO.

[0012] 2. A nucleic acid encoding the recombinant human type I collagen described in item 1. The nucleotide sequence of the nucleic acid may be, for example, as shown in SEQ ID No: 1.

[0013] SEQ ID No:1

[0014]

[0015] 3. An expression vector comprising the nucleic acid according to item 2. The expression vector may be, for example, pPIC9k, pPICZαA, pGAPZA, pHIL-S1 or pYAM75P.

[0016] 4. A host cell into which the expression vector described in item 3 is introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris, such as Pichia pastoris.

[0017] 5. A method for producing the recombinant human type I collagen described in item 1, comprising the steps of culturing the host cell described in item 4 to express the recombinant human type I collagen described in item 1, and collecting the culture containing the recombinant human type I collagen. The expression can be constitutive expression, induced expression, or a combination of the two. The inducer for inducing expression can be an inducer such as IPTG, β-galactoside, methanol, ethanol, etc.

[0018] 6. The production method according to item 5 further comprises the step of separating and purifying the culture to obtain purified recombinant human type I collagen.

[0019] 7. The production method according to item 6, wherein the separation and purification adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation. The affinity chromatography can adopt Ni-nickel affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography, or SUMO affinity chromatography.

[0020] 8. Use of the recombinant human type I collagen described in item 1 in the preparation of cosmetics, external medicines, skin care products, health care products, tissue engineering materials, and medical devices.

[0021] 9. The use according to item 8, wherein the tissue engineering material is selected from hemostatic sponges, subcutaneous fillers, artificial bones, artificial skin, oral absorbable biomembranes, bone implants, and bone repair scaffolds.

[0022] Effects of the Invention

[0023] The recombinant human type I collagen has good cell proliferation promoting activity, cell migration promoting activity, cell adhesion promoting activity (all better than full-length recombinant human type I collagen or commercially available human collagen) and is easy to produce. The recombinant human type I collagen is expected to have whitening, moisturizing, soothing, and repairing effects, and can be used in cosmetics, topical medicines, skin care products, health products, tissue engineering materials, medical devices and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the Pichia pastoris expression vector and the recombinant human type I collagen expression vector.

[0025] Figure 2 This is a schematic diagram of the expression and purification of the obtained recombinant human type I collagen, wherein M is the protein marker, 1 is the expression of recombinant human type I collagen, 2 is the empty load expression, 3 is the flow-through, 4 is the NTA-10mM wash, 5 is the NTA-40mM wash, 6 is the NTA-200mM elution, and 7 is the protein after ultrafiltration concentration.

[0026] Figure 3 It is a schematic diagram showing the cell proliferation promoting effects of the recombinant human type I collagen of the present invention, the full-length recombinant type I collagen and commercially available collagen.

[0027] Figure 4 It is a schematic diagram showing the cell adhesion promoting effects of the recombinant human type I collagen of the present invention, the full-length recombinant type I collagen and commercially available collagen.

[0028] Figure 5 It is a schematic diagram showing the cell migration promoting effects of the recombinant human type I collagen of the present invention, the full-length recombinant type I collagen and commercially available collagen. DETAILED DESCRIPTION

[0029] Example 1 Preparation of recombinant human type Ⅰ collagen

[0030] The nucleotide sequence of recombinant human type I collagen was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. (the nucleotide sequence is shown in SEQ ID No: 1, and the amino acid sequence is shown in SEQ ID No: 2). The nucleotide sequence fragment was used as a template to design upstream and downstream primers containing restriction sites for PCR reaction. Among them, the upstream primer was added with a 6*His tag to facilitate subsequent purification.

[0031] The sequence of the upstream primer P1 is shown in SEQ ID No: 3, and its nucleotide sequence is: 5'-CGGAATTCATCAAGGGTGATCG-3', which contains an EcoRI restriction endonuclease cleavage site;

[0032] The sequence of the downstream primer P2 is shown in SEQ ID No: 4, and its nucleotide sequence is: 5'-TTGCGGCCGCTTTGTCACCT-3', which contains a NotI restriction endonuclease cleavage site.

[0033] The reaction conditions of PCR were as follows: 98°C pre-denaturation for 1 min, 98°C denaturation for 10 s, 60°C annealing for 10 s, 72°C extension for 10 s, 30 cycles and then 72°C extension for 10 min. The reaction system is shown in Table 1:

[0034] Table 1 PCR reaction system

[0035]

[0036]

[0037] The recovered PCR product and expression vector pPIC9K (purchased from Invitrogen) were digested with restriction endonucleases EcoRI and Not I. The digestion reaction system is shown in Table 2:

[0038] Table 2 Enzyme digestion reaction of expression vector and recombinant human type Ⅰ collagen PCR product

[0039] Reagents Dosage pPIC9K vector or PCR product 10μL (≤1μg) Restriction enzymes 0.5μL 10×buffer 2μL <![CDATA[ddH2O]]> 7.5μL

[0040] The digested products were recovered by gel recovery kit and connected with T4 DNA ligase (purchased from Thermo Fisher Scientific (China) Co., Ltd.) at a temperature of 16°C and a connection time of 30 min. The connection reaction is shown in Table 3:

[0041] Table 3 Ligation reaction between expression vector and recombinant human type Ⅰ collagen

[0042]

[0043] The ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Thermo Fisher Scientific (China) Co., Ltd.), mixed, ice bathed for 30 minutes, 42°C water bathed for 60 seconds (no shaking), and then ice bathed for 1-2 minutes; 500μL LB medium was added and incubated at 37°C for 1 hour; the bacterial solution was shaken, 100μL was spread on an ampicillin agar plate (LB / AP+), and inverted at 37°C for 12 to 16 hours. A single colony was picked from the agar plate as a template, and then PCR was performed with the upstream primer P1 (SEQ ID No: 3) and the downstream primer P2 (SEQ ID No: 4). The positive clones screened by PCR were further identified by restriction enzyme digestion reaction using restriction enzymes EcoRI and NotI. The clones with both positive results were sequenced and analyzed. The sequencing primers are universal sequencing primers for plasmid pPIC9K. If the DNA sequencing results are consistent with the designed gene sequence, it is determined that the expression vector is successfully constructed. At the same time, a recombinant Escherichia coli transformed with the empty vector pPIC9K is constructed as a control.

[0044] 2. Expression and Purification of Recombinant Human Type Ⅰ Collagen

[0045] The transformants with correct DNA sequencing were inoculated into LB liquid medium containing 100 mg / mL Amp, and cultured overnight at 37°C and 220 rpm for large-scale extraction of pPIC9k-COL-S plasmid (using a plasmid extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the plasmid concentration was detected by agarose gel electrophoresis after gradient dilution. pPIC9k-COL-S was digested with SacI to linearize the vector. The digestion system is shown in Table 4:

[0046] Table 4 Linearization reaction of recombinant human type Ⅰ collagen

[0047] Reagents Dosage pPIC9k-COL-S 20μL Sal I 1μL 100×BSA 0.5μL 10×buffer H 5μL <![CDATA[ddH2O]]> 5μL

[0048] After the DNA digested with the above enzymes was purified, 10 μL of linearized DNA pPIC9k-COL-S was added to 80 μL of LGS115 yeast competent cells. The operation diagram is as follows: Figure 1As shown, and transfer to a pre-cooled 0.2cm electrode cup, ice bath for 5min. After carefully wiping the ice on the electrode cup, put it in the electrode slot and tighten it, put down the glass cover, adjust the voltage to 1500V, the resistance to 200Ω, and the capacitance to 50μF for conversion. Immediately after the electric shock, open the lid and add 1mL of 1M sorbitol pre-cooled on ice. After gently mixing, transfer all the liquid to a 1.5mL sterilized centrifuge tube and apply MD plate. Incubate the plate upside down in a 30℃ biochemical incubator for 2 to 5 days until a single colony appears. Use COL-S primers (upstream primer P1 and downstream primer P2) for colony PCR positive screening, and in the clean bench, the colonies that grow the target bands are spotted on YPD plates containing 0.25mg / mL~4.0mg / mL G418 in sequence for high copy screening.

[0049] Take a high-copy single colony and inoculate it in YPD medium at 30℃, 220rpm shaking culture overnight, take an appropriate amount of culture medium to transfer to BMGY medium at 30℃, 220rpm shaking overnight until OD600 reaches 2-8, take an appropriate amount of culture medium and transfer it to a 50mL sterilized centrifuge tube, centrifuge at 4000rpm for 3min at room temperature, discard the supernatant, resuspend the bacteria in BMMY medium until OD600 equals 1, and induce culture at 30℃, 250rpm. Yeast strain GS115 and yeast strain GS115 transformed with empty vector pPIC9k are induced and cultured in the same way. Add 250μL methanol every 24h. Take 1mL sample every 24h, centrifuge at 12000rpm for 1min, and store the supernatant and bacteria at -20℃ for subsequent detection. Stop the culture after 5 days of induction, centrifuge the fermentation broth, and save the supernatant and bacteria separately.

[0050] The fermentation supernatant was subjected to SDS-PAGE electrophoresis, in which the concentration of the concentrated gel was 5%, the concentration of the separation gel was 15%, and the supernatant loading volume was 15 μL. After running the gel at 60V for 30 minutes, it was adjusted to 100V and the electrophoresis was continued for about 1 to 2 hours until the bromophenol blue reached the bottom of the gel plate and the electrophoresis was stopped. After Coomassie Brilliant Blue R-250 staining for 30 minutes, it was destained with a decolorant for about 1 hour, and the decolorant was changed several times until the protein bands were clearly visible. The results showed that the empty vector pPIC9k did not express protein in the yeast strain, while the pPIC9k-COL-S recombinant human type I collagen was expressed in the yeast strain GS115, such as Figure 2 shown.

[0051] Recombinant human type I collagen was purified by Ni-nickel affinity chromatography. The fermentation supernatant was added to the Ni-nickel affinity chromatography medium and mixed to allow the target protein to fully bind to the Ni-nickel affinity chromatography gel. The binding flow rate was 0.6 mL / min. The baseline was washed with NTA-10mM imidazole buffer at a flow rate of 1 mL / min. Then, the impurities were washed with NTA-40mM imidazole buffer at a flow rate of 1 mL / min, and the target protein was eluted with NTA-200mM imidazole buffer at a flow rate of 1 mL / min. The target protein was found in the supernatant and the recovered liquid after NTA-200mM elution. The size was about 14KD, which was consistent with the expected result. After ultrafiltration and centrifugation of the purified protein, 20 μg was loaded, and there was basically no impurity band. The protein purity exceeded 95%, such as Figure 2 shown.

[0052] Experimental Example 2 Application of recombinant human type Ⅰ collagen in promoting cell proliferation

[0053] The recombinant human type I collagen described in Example 1 was used to act on HSF (human skin fibroblast) cells for cell proliferation experiments. The specific steps are as follows: human HSF cells (purchased from ATCC cell bank, product catalog number HTX2132) were inoculated in a sterile 96-well plate, 200 μL of culture medium per well, DMEM / F12 complete culture medium was used for cell culture, and 4 mg / mL of recombinant human type I collagen, full-length recombinant type I collagen, and commercially available collagen were added to each well. Six replicate wells were set for each group concentration, and an equal volume of culture medium was added to the blank control group, and the plates were placed in a 37°C incubator for 48 hours. The supernatant was discarded, 100 μL of culture medium and 50 μL of MTT solution were added to each well, shaken, and incubated in an incubator for 4 hours. After 4 hours, the mixture was aspirated, 150 μL of DMSO was added to dissolve the purple crystal formazan, shaken for 10 minutes, and placed in a microplate reader. The detection wavelength was 570 nm, and the absorbance value of each well was measured. The data of recombinant human type I collagen promoting HSF (human skin fibroblast) cell proliferation are shown in Table 5. The schematic diagram of its effect on the proliferation of human skin fibroblasts compared with full-length recombinant type I collagen and commercially available collagen (purchased from Abcam) is shown in Figure 3. Among them,

[0054] Proliferation rate = (OD of experimental group - OD of blank group) × 100% / blank group.

[0055] Table 5 Proliferation rate of skin fibroblasts by recombinant human type Ⅰ collagen, full-length recombinant type Ⅰ collagen and commercially available collagen

[0056]

[0057] From Table 5 and Figure 3It can be seen that the cell proliferation rate promoted by the recombinant human type I collagen described in the present invention is close to 24%, while the cell proliferation rate promoted by the full-length recombinant type I collagen and commercially available collagen is about 5%, indicating that the recombinant human type I collagen described in the present invention can significantly promote cell proliferation.

[0058] Experimental Example 3 Application of recombinant human type Ⅰ collagen in promoting cell adhesion

[0059] The recombinant human type I collagen described in Example 1 was used to act on HSF (human skin fibroblast) cells to promote cell adhesion experiments. The specific steps are as follows: human HSF cells were inoculated in a sterile 96-well plate, and 200 μL of 4 mg / mL recombinant human type I collagen, full-length recombinant type I collagen, and commercially available collagen solution prepared by complete culture medium were added to each well of the experimental group, and only culture medium was added to the blank control group. After being placed at 37°C for 2 hours, each group was washed 3 times with PBS to wash away non-adherent cells, 100 μL of culture solution and 50 μL of MTT solution were added to each well, shaken well, and placed in an incubator for incubation for 4 hours, and then the mixed solution was aspirated, 150 μL of DMSO was added to dissolve the purple crystal formazan, shaken for 10 minutes, placed in an ELISA reader, and the detection wavelength was 570 nm, and the absorbance value of each well was measured.

[0060] The data of recombinant human type I collagen promoting HSF (human skin fibroblast) cell adhesion are shown in Table 6, and the schematic diagram of its adhesion effect on human skin fibroblasts with commercially available collagen (purchased from Abcam) is shown in Figure 4, where:

[0061] Cell adhesion promotion rate = (OD of experimental group - OD of blank group) × 100% / blank group.

[0062] Table 6 Adhesion rate of recombinant human type I collagen, full-length type I collagen and commercially available collagen to skin fibroblasts

[0063]

[0064] From Table 6 and Figure 4 It can be seen that the cell adhesion promoting rate of the recombinant human type I collagen described in the present invention is 38%, while the cell adhesion promoting rates of the full-length recombinant type I collagen and the commercially available collagen are 33% and 29% respectively, indicating that the recombinant human type I collagen described in the present invention can significantly promote cell adhesion.

[0065] Example 4 Application of recombinant human type I collagen in promoting cell migration

[0066] The recombinant human type I collagen described in Example 1 was used to act on HSF (human skin fibroblast) cells to promote cell migration experiments. The specific steps are as follows: human HSF cells were inoculated in a sterile 6-well plate, with 2 mL of culture medium per well. After 24 hours, a 200 μL gun tip was used to align the well plate vertically with a ruler, and the longitudinal line was gently pushed down to form a scratch. The cells were rinsed 3 times with PBS to remove the scratched cells. The experimental group was added with 2 mL of serum-free culture medium, 4 mg / mL of recombinant human type I collagen, full-length recombinant type I collagen, and commercially available collagen solution, respectively, and the blank group was added with only serum-free culture medium. Cultured in a 37°C, 5% CO2 incubator, photographed under a microscope after 0h and 24h, the cell migration area was calculated, and the cell migration was analyzed.

[0067] The data of recombinant human type I collagen promoting HSF (human skin fibroblast) cell migration are shown in Table 7, and the schematic diagram of its effect on the migration of human skin fibroblasts with commercially available collagen (purchased from Abcam) is shown in Figure 5, where:

[0068] Cell migration promotion rate = (cell migration area of ​​experimental group - cell migration area of ​​blank group) × 100% / blank group.

[0069] Table 7 Migration rate of recombinant human type I collagen, full-length recombinant type I collagen and commercially available collagen on skin fibroblasts

[0070]

[0071] From Table 7 and Figure 5 It can be seen that the cell migration promoting rate of the recombinant human type I collagen described in the present invention is 41%, while the cell migration promoting rates of the full-length recombinant type I collagen and the commercially available collagen are 30% and 28% respectively, indicating that the recombinant human type I collagen described in the present invention can significantly promote cell migration.

[0072] In summary, the recombinant human type I collagen provided by the present invention can promote cell proliferation, adhesion and migration, so that the recombinant human type I collagen can be applied to multiple application fields.

Claims

1. A recombinant human type I collagen, the amino acid sequence of which is shown in SEQ ID No:

2.

2. The recombinant human type I collagen according to claim 1, further comprising a tag for easy purification; the tag for easy purification may be, for example, 6*His, GST, MBP or SUMO.

3. A nucleic acid encoding the recombinant human type I collagen according to claim 1.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell into which the expression vector according to claim 4 has been introduced; the host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris, such as Pichia pastoris.

6. A method for producing the recombinant human type I collagen according to claim 1, comprising the steps of culturing the host cell according to claim 5 to express the recombinant human type I collagen according to claim 1, and collecting the culture containing the recombinant human type I collagen.

7. The production method according to claim 6, further comprising the step of separating and purifying the culture to obtain purified recombinant human type I collagen.

8. The production method according to claim 7, wherein: The separation and purification adopts a combination of one or more of salting out, chromatography, affinity chromatography, acid-base precipitation, and membrane separation, preferably a combination of chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation; the affinity chromatography can adopt Ni-nickel affinity chromatography, reduced glutathione affinity chromatography, maltose affinity chromatography or SUMO affinity chromatography.

9. Use of the recombinant human type I collagen according to claim 1 in the preparation of cosmetics, external medicines, skin care products, health products, tissue engineering materials, and medical devices.

10. The use according to claim 9, wherein The tissue engineering material is selected from hemostatic sponge, subcutaneous filler, artificial bone, artificial skin, oral absorbable biomembrane, bone implant, and bone repair scaffold.

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