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

Abstract

The invention discloses recombinant human II-type collagen as well as a preparation method and application thereof. The recombinant human II-type collagen provided by the invention is a truncated protein of a natural human II-type protein, and the amino acid sequence of the recombinant human II-type collagen is shown as SEQ ID No: 1. The recombinant human II-type collagen can be expressed in a commercial expression system such as a commercial pichia pastoris expression system, the expression yield is high, and the tested biological activity (promoting cell proliferation and promoting wound healing) of the recombinant human II-type collagen is obviously superior to that of natural human II-type collagen; and the collagen is also obviously superior to other-stage proteins of natural human II-type collagen.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a novel genetically engineered recombinant collagen, a preparation method thereof and uses 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. So far, at least 40 types of collagen chain coding genes have been discovered. These different collagen chains, combined in different ways, can form more than 20 different types of collagen molecules.

[0003] Among them, type II collagen is composed of three identical α1 chains, which are mainly distributed in cartilage and vitreous body, accounting for more than 90% of the total amount of collagen in human cartilage matrix. Biomaterials with type II collagen as the main raw material are widely used in the fields of tissue engineering and regenerative medicine. At present, different treatment methods and processes such as acid, alkali or protease have been developed mainly through animal tissue extraction to extract type II collagen from the cartilage of animals such as pigs, cattle, sheep, etc. However, collagen extracted from animal tissues has risks such as animal-derived diseases. At the same time, large-scale preparation will cause excessive demand for animals, and animal breeding on the supply side will cause huge pressure. On the other hand, those skilled in the art have tried many methods to try to recombinantly express human type II collagen in prokaryotic or eukaryotic expression systems, but unfortunately encountered the following problems: the recombinant expression of the full length of human type II collagen has not been successful; although some truncated proteins of human type II collagen can be recombinantly expressed, the yield is not high and / or the biological activity is much worse than the full length of human type II collagen, so it is not suitable for industrial-scale production.

[0004] It also has some unique biological functions, such as inhibiting inflammatory response, promoting wound healing, and promoting cell proliferation. Summary of the invention

[0005] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a recombinant human type II collagen with high yield in a commercial expression system and good cell proliferation promoting activity and wound healing promoting activity and its application.

[0006] In scientific research practice, the inventors screened and evaluated the efficacy of multiple truncated proteins of human type II collagen, and found a recombinant human type II collagen with high expression yield in a commercial expression system, easy separation and purification, and good activity in promoting the proliferation of human fibroblasts-HSF cells and inhibiting inflammatory responses to promote wound healing, thereby completing the present invention.

[0007] Specifically, the present invention includes:

[0008] 1. A recombinant human type II collagen, which is a truncated protein of human type II collagen, and its amino acid sequence is shown in SEQ ID No: 1.

[0009] 2. A nucleic acid encoding the recombinant human type II collagen described in any one of item 1.

[0010] 3. An expression vector comprising the nucleic acid described in item 2.

[0011] 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, and Bacillus licheniformis, eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli and Pichia pastoris, and more preferably Pichia pastoris.

[0012] 5. A method for producing the recombinant human type II collagen described in item 1, comprising: culturing the host cell described in item 4 to express the recombinant human type II collagen, and collecting the cell culture. 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.

[0013] 6. The method according to item 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation. Preferably, the combination of chromatography and membrane separation is used, and more preferably, the combination of ion exchange chromatography and membrane separation is used.

[0014] 7. A product comprising the recombinant human type II collagen described in item 1, wherein the product is a medicine, a cosmetic or a medical device.

[0015] 8. Use of the recombinant human type II collagen described in item 1 in the preparation of medicines, cosmetics or medical devices.

[0016] The beneficial effects of the present invention are that the recombinant human type II collagen of the present invention can be expressed in a commercial expression system such as a commercial Pichia pastoris expression system with a high expression yield, and its tested biological activities (promoting cell proliferation and promoting wound healing) are significantly better than natural human type II collagen, and are also significantly better than other stage proteins of natural human type II collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure shows the results of SDS-PAGE electrophoresis of recombinant human type II collagen.

[0018] Figure 2 This figure shows that recombinant human type II collagen has the effect of promoting cell proliferation.

[0019] Figure 3A The graph shows the inhibitory effect of recombinant human type II collagen on inflammatory factor TNF-α.

[0020] Figure 3B The graph shows the inhibitory effect of recombinant human type II collagen on the inflammatory factor IL-8. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below through specific embodiments. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the present invention.

[0022] Example 1: Preparation of recombinant collagen in yeast expression system

[0023] 1. Preparation of shuttle plasmid

[0024] According to the amino acid sequences shown in SEQ ID No: 1, 2, and 3 (corresponding to recombinant collagen II-1, II-2, and II-3, respectively), the codon optimization of the yeast expression system was performed to obtain the target gene sequence. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis, and the synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-II plasmid.

[0025] 2) Preparation of yeast expression strains

[0026] pPicZαA-II was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0027] 2. Induced expression of target protein

[0028] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0029] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C, 200 rpm for 24 h to serve as the seed for the tank;

[0030] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0031] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0032] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30%, the pH was about 5.0, the induction was carried out for 40 hours, the fermentation was terminated, the culture solution was centrifuged at 12000 rpm for 2 minutes, the supernatant was taken, and the protein yield was detected by BCA method. The results are shown in Table 1 below.

[0033] Table 1 Test results of each recombinant collagen after fermentation

[0034]

[0035] The results in Table 1 show that the yield of recombinant human type II collagen of SEQ ID No: 1 reaches more than 16 g / L, and its recombinant expression level is high and can meet the requirements of fermentation production. On the other hand, in the same expression system, the yields of recombinant human type II collagen of SEQ ID No: 2 and 3 are both less than 10 g / L, which does not meet the requirements of efficient expression production.

[0036] Example 2: Purification of recombinant human type II collagen

[0037] Collect the obtained engineered bacteria fermentation liquid, use a Thermo Fisher desktop centrifuge to separate the bacterial liquid and the bacterial body, and collect the supernatant. Prepare the buffer according to the characteristics of the protein: 20mM / L potassium phosphate buffer (liquid A, pH 6.0), and use 20mM / L potassium phosphate buffer + 1mol / L NaCl (liquid B, pH 6.0) as the eluent. The collected supernatant is adjusted to pH and filtered before being loaded on a hydrophobic cation exchange chromatography column. Before loading, the column is balanced with liquid A, and then washed with 20% liquid B after loading. Finally, it is eluted with liquid B. The eluted protein is the target protein. The purified recombinant human type II collagen corresponding to the designed sequences SEQ ID No: 1, 2, and 3 are represented by II-1, II-2, and II-3, respectively, and the corresponding SDS-PAGE electrophoresis diagrams are shown in the following figure. Figure 1 shown.

[0038] Example 3: Determination of the effect of recombinant human type II collagen on promoting cell proliferation

[0039] Human keratinocytes (HACAT) cells were grown to logarithmic phase and cultured at 8 × 10 4 Each well was inoculated with 0.1 mL per well in a 96-well plate at 37°C and 5% CO 2 Culture under culture conditions. When the cells grow to 50% to 60%, the experiment is started, the culture medium is discarded, and the cells are washed with PBS buffer. The experiment is divided into a control group and a sample group including a recombinant human type II collagen (II-1, II-2, II-3) group and a human type II collagen group. The human type II collagen was purchased from Sigma-Aldrich, item number CC052. General culture medium was added to the control group, and 10% of the sample was added to the culture medium in the sample group. Each group has 6 replicate wells at 37°C and 5% CO 2 Incubate for 24 h under culture conditions.

[0040] Add MTT reagent and place in a carbon dioxide incubator for 1 to 2 hours. Add dimethyl sulfoxide (DMSO) to dissolve the formazan in the cells and use a microplate reader to measure the absorbance at a wavelength of 490 nm. Use the following formula to calculate the cell proliferation rate:

[0041] Cell proliferation rate = (experimental group - blank group) / (control group - blank group) * 100%.

[0042] The proliferation rate of cells by collagen solution was calculated according to the formula, and IC50 was calculated by Graphpad Prism 5.0 software.

[0043] like Figure 2 As shown, the recombinant human type II collagen (II-1) of the present invention has the best effect in promoting cell proliferation, which is 10.9% higher than that of human recombinant collagen type II.

[0044] Example 4: Determination of the effect of recombinant human type II collagen on wound healing

[0045] Cell experiment on LPS-induced macrophage RAW264.7 inflammatory factor model (TNFα, IL-8).

[0046] 1) Press 1×10 5 Cells were seeded into 24-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0047] 2) Groups: blank control group (normal culture medium), model group (LPS-induced group), positive group (100 μg / mL dexamethasone), experimental groups: recombinant human type II collagen (II-1, II-2, II-3) (0.1 mg / mL) group and human type II collagen group (0.1 mg / mL).

[0048] 3) Induction and drug administration: When the cell plating rate in the 24-well plate reaches 40% to 60%, add 1 μg / mL LPS culture medium to the model group for modeling according to the experimental design. Add the test sample of corresponding concentration to the positive group and the experimental group on the basis of modeling. Each group has 3 replicate wells. After drug administration, place the cells in an incubator (37°C, 5% CO 2 )Continue culturing for 24 h.

[0049] 4) Sample collection: After the incubation is completed, the cell culture supernatant is collected in an EP tube (Note: the amount of collected samples is determined according to the detection indicators). After collection, the samples are placed in a -80°C refrigerator for freezing and storage.

[0050] 5) Detection of TNF-α content: Detection was performed according to the operating instructions of the Mouse TNF-α ELISA kit. Detection of IL-8 content: Detection was performed according to the operating instructions of the Mouse IL-8 ELISA kit.

[0051] The results showed that, as shown in Figure 3, compared with the model group, the positive control group (dexamethasone) had the best effect in inhibiting inflammatory factors (TNF-α, IL-8), followed by recombinant human type II collagen (II-1), then human type II collagen, and recombinant human type II collagen (II-2) and recombinant human type II collagen (II-3) had the worst effects.

[0052] Therefore, the protein recombinant human type II collagen (II-1) expressed by the designed sequence SEQ ID No: 1 has the best effects in promoting cell proliferation, inhibiting inflammatory response and promoting wound healing.

[0053] Sequence information

[0054] SEQ ID No 1:

[0055] gppgergapgnrgfpgqdglagpkgapgergpsglagpkgangdpgrpgepglpgargltgrpgdagpqgkvgpsgapgedgrpgppgpqgargqpgvmcfpgpkgangepgkagekglpgapglrglpgkdgetgaagppgpagpager

[0056] SEQ ID No 2:

[0057] gergppgpqgargfpgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagaagargndgqpgpagppgpvgpaggpgfpgapgakgeagptgargpegaqgprgepgtpgspgpagasgnp

[0058] SEQ ID No. 3:

[0059] gppgatgfpgaagrvgppgsngnpgppgppgpsgkdgpkgargdsgppgragepglqgpagppgekgepgddgpsgaegppgpqglagqrgivglpgqrgergfpglpgpsgepgkqgapgasgdrgppgpvgppgltgpa

Claims

1. A recombinant human type II collagen, which is a truncated protein of human type II collagen, and its amino acid sequence is shown in SEQ ID No:

1. 2 . The nucleic acid encoding the recombinant human type II collagen according to claim 1 .

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced, wherein the host cell is preferably Pichia pastoris.

5. A method for producing the recombinant human type II collagen according to claim 1, comprising: The step of culturing the host cell according to claim 4 to express the recombinant human type II collagen, and collecting the cell culture.

6. The method according to claim 5, further comprising the step of separating and purifying the cell culture, wherein the separation and purification adopts one or a combination of salting out, chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation.

7. A product comprising the recombinant human type II collagen according to claim 1, wherein the product is a medicine, a cosmetic or a medical device.

8. Use of the recombinant human type II collagen according to claim 1 in the preparation of medicines, cosmetics or medical devices.

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