Recombinant human collagen type ii, method for producing the same, and use thereof

Abstract

This patent application discloses a recombinant human type II collagen, its preparation method, and its uses. The recombinant human type II collagen of this patent application is a truncated protein of natural human type II collagen, and its amino acid sequence is shown in SEQ ID No: 1. The recombinant human type II collagen of this invention can be expressed in commercial expression systems such as the commercial Pichia pastoris expression system with high expression yields. Furthermore, its tested biological activities (promoting cell proliferation and wound healing) are significantly superior to those of natural human type II collagen, and also significantly superior to other stages of natural human type II collagen.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a novel genetically engineered recombinant collagen, its preparation method, and its uses. Background Technology

[0002] Collagen is a biological macromolecule, a major component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein. To date, at least 40 genes encoding collagen chains have been discovered. These different collagen chains combine in different ways to form more than 20 different types of collagen molecules.

[0003] Type II collagen, composed of three identical α1 chains, is mainly distributed in cartilage and vitreous humor, accounting for over 90% of the total collagen in human cartilage matrix. Biomaterials using type II collagen as the primary raw material are widely used in tissue engineering and regenerative medicine. Currently, type II collagen is mainly extracted from animal tissues using various treatment methods and processes, such as acid, alkali, or protease extraction, from the cartilage of animals like pigs, cattle, and sheep. However, collagen extracted from animal tissues carries risks such as animal-derived diseases, and large-scale production would lead to excessive demand from animals, placing enormous pressure on animal husbandry. On the other hand, those skilled in the art have attempted many methods to recombinantly express human type II collagen in prokaryotic or eukaryotic expression systems, but unfortunately, the following problems have been encountered: recombinant expression of the full-length human type II collagen has not been successful; while some truncated versions of type II collagen can be recombinantly expressed, the yield is low and / or the biological activity is significantly lower than that of the full-length human type II collagen, making them unsuitable for industrial-scale production.

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

[0005] In view of the above-mentioned technical problems existing in the prior art, the purpose 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 and wound healing activities, and its application.

[0006] In their scientific research practice, the inventors screened and evaluated the efficacy of several truncated proteins of human type II collagen. As a result, they discovered a recombinant human type II collagen that exhibits high expression yield, is easy to isolate and purify, and has good activity in promoting the proliferation of human fibroblast-HSF cells and inhibiting inflammatory response and promoting wound healing in a commercial expression system, thus completing this 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, the amino acid sequence of which is shown in SEQ ID No: 1.

[0009] 2. The nucleic acid of recombinant human type II collagen as described in any one of coding items 1.

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

[0011] 4. A host cell in which the expression vector described in item 3 is introduced. The host cell can 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, more preferably Pichia pastoris.

[0012] 5. A method for producing the recombinant human type II collagen as described in item 1, comprising the steps of: culturing the host cells described in item 4 to express the recombinant human type II collagen, and collecting the cell culture. The expression may be one or a combination of constitutive expression and induced expression. The inducer for induced expression may be an inducer such as IPTG, β-galactoside, methanol, or ethanol.

[0013] 6. The method according to claim 5 further includes a step of separating and purifying the cell culture, wherein the separation and purification employs one or a combination of several of the following: salting out, chromatographic chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation. A combination of chromatographic chromatography and membrane separation is preferred, and a combination of ion exchange chromatography and membrane separation is more preferred.

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

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

[0016] The beneficial effects of the present invention are as follows: the recombinant human type II collagen of the present invention can be expressed in a commercial expression system such as the commercial Pichia pastoris expression system with high expression yield, and its tested biological activities (promoting cell proliferation and wound healing) are significantly better than those of natural human type II collagen, and also significantly better than other stages of natural human type II collagen. Attached Figure Description

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

[0018] Figure 2 A diagram illustrating the cell proliferation-promoting effect of recombinant human type II collagen.

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

[0020] Figure 3B A figure illustrating the effect of recombinant human type II collagen in inhibiting the inflammatory factor IL-8. Detailed Implementation

[0021] The present invention will now be described in detail through specific embodiments. It should be noted that these descriptions are merely exemplary and do not constitute a limitation on the scope of the present invention.

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

[0023] 1. Preparation of shuttle plasmids

[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 codons of the yeast expression system were optimized to obtain the target gene sequence. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was ligated into 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. Transformants were screened using bleomycin resistance as a selection marker to obtain the yeast expression strain.

[0027] 2. Induced expression of the target protein

[0028] (1) Select 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 incubate at 30℃ and 200 rpm for 48 h to activate it;

[0029] (2) Transfer 1% of the inoculum to a 500ml Erlenmeyer flask (containing 200ml of YPD culture medium), and incubate at 30℃ and 200rpm for 24h to serve as the seed for the next flask.

[0030] (3) Prepare 3L of BSM medium and add it to a 5L fermenter. Sterilize at 121℃ for 20min. After cooling to 30℃, adjust the pH to 5.0. Add the seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture.

[0031] (4) Cultivate to OD 600 Begin adding 50% glycerin when the OD reaches 70, until... 600 Once the dissolved oxygen level reaches approximately 120, stop adding more methanol and wait for it to rebound to 100% before starting to add methanol for induction.

[0032] (5) During the induction process, the dissolved oxygen was controlled to be no less than 30% and the pH to be around 5.0. The induction was carried out for 40 hours. After the fermentation was stopped, the culture medium was centrifuged at 12,000 rpm for 2 minutes. The supernatant was collected and the protein yield was detected by BCA method. The results are shown in Table 1 below.

[0033] Table 1. Detection results of each recombinant collagen protein after fermentation.

[0034]

[0035] The results in Table 1 show that the recombinant human type II collagen yield of SEQ ID No: 1 reached over 16 g / L, indicating high recombinant expression levels that met the requirements for fermentation production. On the other hand, in the same expression system, the recombinant human type II collagen yields of SEQ ID No: 2 and 3 were both less than 10 g / L, failing to meet the requirements for efficient expression and production.

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

[0037] The collected fermentation broth of the engineered bacteria was separated from the bacterial culture and cells using a Thermo Fisher Scientific benchtop centrifuge, and the supernatant was collected. Based on the characteristics of the protein, a buffer solution was prepared: 20 mM / L potassium phosphate buffer (Solution A, pH 6.0), with 20 mM / L potassium phosphate buffer + 1 mol / L NaCl (Solution B, pH 6.0) as the elution buffer. The collected supernatant was adjusted to pH, filtered, and then loaded onto a hydrophobic cation exchange chromatography column. Before loading, the column was equilibrated with Solution A; after loading, impurities were washed with 20% Solution B; and finally, elution was performed with Solution B. The eluted protein was the target protein. The purified recombinant human type II collagen corresponding to the designed sequences SEQ ID No: 1, 2, and 3 are represented as II-1, II-2, and II-3, respectively. The corresponding SDS-PAGE electrophoresis images are shown below. Figure 1 As shown.

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

[0039] Human keratinocytes-HACAT cells were brought to the logarithmic phase at a concentration of 8 × 10⁻⁶. 4 Cells were seeded at 0.1 mL / mL in 96-well plates and cultured at 37°C with 5% CO2. When the cells reached 50%–60% confluence, the culture medium was discarded, and the cells were washed with PBS buffer. The experiment consisted of a control group and a sample group, including recombinant human type II collagen (II-1, II-2, II-3) and human type II collagen. The human type II collagen was purchased from Sigma-Aldrich (catalog number CC052). The control group was cultured in standard medium, while the sample group was cultured with 10% of the sample added to the medium. Each group was divided into six replicates and cultured at 37°C with 5% CO2 for 24 hours.

[0040] After adding MTT reagent and reacting in a CO2 incubator for 1-2 hours, dimethyl sulfoxide (DMSO) was added to dissolve formazan in the cells. The absorbance was measured at 490 nm using a microplate reader. The cell proliferation rate was calculated using the following formula:

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

[0042] The cell proliferation rate of collagen solution was calculated according to the formula, and IC50 was calculated using 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 cell proliferation-promoting effect, which is 10.9% higher than that of human type II recombinant collagen.

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

[0045] Cellular experiments using LPS-induced RAW264.7 macrophage inflammatory factor model (TNFα, IL-8).

[0046] 1) Press 1×10 5 Cells were seeded at a density of cells / well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0047] 2) Groups: blank control group (normal culture medium), model group (LPS-induced group), positive group (100 μg / mL dexamethasone), experimental group: 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 deposition rate in the 24-well plates reached 40%–60%, LPS medium containing 1 μg / mL was added to the model group to establish the cell model, according to the experimental design. The positive control group and experimental group were treated with the corresponding concentration of experimental sample. Each group had three replicates. After drug administration, the cells were placed in an incubator (37℃, 5% CO2) and cultured for another 24 hours.

[0049] 4) Sample collection: After incubation, collect the cell culture supernatant into EP tubes (Note: Determine the amount of sample to be collected according to the detection indicators). After collection, freeze the sample in a -80℃ freezer.

[0050] 5) TNF-α level detection: Perform the detection according to the instructions of the Mouse TNF-α ELISA kit. IL-8 level detection: Perform the detection according to the instructions of the Mouse IL-8 ELISA kit.

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

[0052] Therefore, the recombinant human type II collagen (II-1) protein expressed by the designed sequence SEQ ID No:1 has the best efficacy 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] gergenpppgpqgargfpgtpglpgvkghrgypgldgakgeagapgvkgesgspgengspgpmgprglpgergrtgpagaagargndgqpgpagppgpvgpaggpgfpgapgakgeagptgargpegaqgprgepgtpgspgpagasgnp

[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, the amino acid sequence of which is shown in SEQ ID No:

1.

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

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

4. The host cell into which the expression vector of claim 3 is introduced.

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

6. A product comprising the recombinant human type II collagen of claim 1, wherein the product is a pharmaceutical, cosmetic, or medical device.

7. The use of the recombinant human type II collagen according to claim 1 in the preparation of pharmaceuticals, cosmetics, or medical devices, wherein, The medicine is used to promote wound healing.

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