Recombinant human IV-type collagen as well as production method and application thereof

Abstract

The invention discloses a recombinant human IV type collagen as well as a production method and application thereof. The amino acid sequence of the recombinant human IV type collagen is shown as SEQ ID No: 1. The recombinant human IV-type collagen is a truncated protein of a human IV-type collagen alpha2 chain, special expression elements and means are not needed, the yield of the recombinant human IV-type collagen can reach up to 17g / L only through extracellular secretion expression of a commercial pichia pastoris expression system, the yield is obviously higher than the high yield level of recombinant collagen considered in the technical field, and the recombinant human IV-type collagen has a good application prospect. And the total length of the alpha 2 chain of the human IV-type collagen or other truncated proteins is obviously higher than that of the human IV-type collagen. In addition, the safety and biological activity of the recombinant human IV-type collagen are superior to the full length of the alpha 2 chain of the human IV-type collagen.

Description

[0001] This patent application claims priority to application number 202411658862.3 (application date: 2024-11-19; invention title: A recombinant human type IV collagen, its production method and application). Technical Field

[0002] This invention belongs to the field of synthetic biology technology, specifically relating to a high-yield genetically engineered recombinant human type IV collagen, its production method, and its uses. Background Technology

[0003] 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%–30% of total protein. There are many types of collagen, with common types being Type I, Type II, Type III, Type V, and Type XI. Due to its excellent biocompatibility, biodegradability, and bioactivity, collagen is widely used in food, medicine, tissue engineering, cosmetics, and other fields.

[0004] Currently, recombinant gene expression is one of the important methods for producing collagen. Recombinant human collagen, in particular, has an amino acid composition identical to that of the human body and is considered the closest to naturally synthesized collagen, exhibiting good biocompatibility and high safety. Commercially available recombinant human collagen products are widely used in medical aesthetics, cosmetics, medical devices, and health supplements.

[0005] Currently, the main problems in the production of recombinant collagen are as follows: 1) Easily degraded: Because recombinant collagen cannot form a stable triple helix structure like natural collagen under existing expression systems, it is easily degraded by proteases in the production system; 2) Difficult to purify: The easy degradation of collagen means that in addition to host protein interference, a large amount of degrading proteins need to be removed during the purification process of the prepared recombinant collagen, increasing the difficulty of purification and making it difficult to prepare high-purity target proteins; 3) Low yield: Due to the existence of degradation problems, the yield of recombinant collagen is low, far from meeting expectations. It is generally believed that, using commercial expression hosts and expression vectors, a yield of 10 g / L of recombinant collagen with a molecular weight of 70,000 or higher is already considered quite high. Therefore, the limited variety of mass-produced recombinant collagen severely restricts its further application.

[0006] As the market acceptance of recombinant collagen gradually increases, the market demand for it is also growing. Therefore, mass-producible recombinant collagen is urgently needed. Mass-producible recombinant collagen must possess characteristics such as high batch yield, good protein stability, and ease of purification. Thus, high-yield, stable, and easily purified recombinant collagen is currently in high demand in the market.

[0007] Recombinant collagen can be obtained by constructing truncated proteins or peptides of natural collagen. The uncertainty in the location and length of truncated proteins within natural collagen theoretically results in tens of millions of different types of recombinant collagen. However, there has been a lack of effective theoretical guidance on which of these recombinant collagens can achieve higher secretion yields. Extensive experimental verification and screening are necessary to determine the best results, which is the primary reason for the limited variety of recombinant collagen currently produced in large quantities. Summary of the Invention

[0008] In view of the technical problems existing in the prior art, the purpose of the present invention is to provide a recombinant human type IV collagen with high secretion yield, good safety and strong biological activity.

[0009] This invention includes: 1. A recombinant human type IV collagen, the amino acid sequence of which is shown in SEQ ID No: 1.

[0010] SEQ ID No: 1 1 GPPGERGLPG EVLGAQPGPR GDAGVPGQPG LKGLPGDRGP PGFRGSQGMP GMPGLKGQPG 61 LPGPSGQPGL YGPPGLHGFP GAPGQEGPLG LPGIPGREGL PGDRGDPGDT GAPGPVGMKG 121 LSGDRGDAGF TGEQGHPGSP GFKGIDGMPG TPGLKGDRGS PGMDGFQGMP GLKGRPGFPG 181 SKGEAGFFGI PGLKGLAGEP GFKGSRGDPG PPGPPPVILP GMKDIKGEKG DEGPMGLKGY 241 LGAKGIQGMP GIPGLSGIPG LPGRPGHIKG VKGDIGVPGI PGLPGFPGVA GPPGITGFPG 301 FIGSRGDKGA PGRAGLYGEI GATDGFGDIG DTINLPGRPG LKGERGTTGI PGLKGFFGEK 361 GTEGDIGFPG ITGVTGVQGP PGLKGQTGFP GLTGPPGSQG ELGRIGLPGG KGDDGWPGAP 421 GLPGFPGLRG IRGLHGLPGT KGFPGSPGSD IHGDPGFPGP PGERGDPGEA NTLPGPVGVP 481 GQKGDQGAPG ERGPPGSPGL QGFPGITPPS NISGAPGDKG APGIFGLKGY RGPPGPPGSA 541 ALPGSKGDTG NPGAPGTPGT KGWAGDSGPQ GRPGVFGLPG EKGPRGEQGF MGNTGPTGAV 601 GDRGPKGPKG DPGFPGAPGT VGAPGIAGIP QKIAVQPGTV GPQGRRRGPPG APGEMGPQGP 661 PGEPGFRGAP GKAGPQGRGG VSAVPGFRGD E 2. The nucleic acid encoding the recombinant human type IV collagen described in item 1. The nucleotide sequence of the nucleic acid may be, for example, as shown in SEQ ID No: 2.

[0011] 3. An expression vector comprising the nucleic acid described in item 2.

[0012] 4. A host cell in which the expression vector described in item 3 has been introduced. The host cell is preferably a eukaryotic cell, such as Pichia pastoris.

[0013] 5. A method for producing the recombinant human type IV collagen of item 1, comprising culturing the host cells of item 4 to induce them to secrete and express the recombinant human type IV collagen, and then separating and purifying them.

[0014] 6. The method according to item 5, wherein the separation and purification employs one or a combination of several selected from salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation. Preferably, a combination of chromatographic chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation, is used.

[0015] 7. Use of the recombinant human type IV collagen as described in item 1 in the preparation of cosmetics or medical devices.

[0016] 8. The use according to item 8, wherein the cosmetic or medical device is used to improve skin redness and repair sensitive skin.

[0017] 9. The use according to item 8, wherein the cosmetic or medical device is formulated in a form suitable for application to the skin, such as a face mask, face cream, spray, essence, etc.

[0018] The beneficial effects of this invention are as follows: 1. High Yield: In this technical field, it is generally considered that a yield of 10 g / L for recombinant collagen with a molecular weight of 70,000 or higher is already considered quite high when using commercially available expression hosts and vectors. This invention provides a novel recombinant human type IV collagen with a novel amino acid sequence composition. This recombinant human type IV collagen is a truncated protein of the human type IV collagen α2 chain. Without the need for special expression elements and methods, the yield of this recombinant human type IV collagen can reach as high as 17 g / L through extracellular secretion expression using a commercial Pichia pastoris expression system. This is significantly higher than the high yield level of recombinant collagen considered in this technical field, and also significantly higher than the full-length human type IV collagen α2 chain or other truncated proteins. Extracellular protein SDS-PAGE analysis shows that the target protein accounts for more than 70%, and the purity can reach 80% after simple ultrafiltration decolorization.

[0019] 2. The cytotoxicity is significantly less than that of the full-length α2 chain of human type IV collagen (see Example 3).

[0020] 3. Its bioactivity is significantly superior to that of the full-length α2 chain of human type IV collagen (see Examples 3 and 4). Attached Figure Description

[0021] Figure 1 This is an SDS-PAGE protein electrophoresis image of purified recombinant human type IV collagen-α2.

[0022] Figure 2 This is an SDS-PAGE protein electrophoresis image of the purified sequences II-1 and II-2.

[0023] Figure 3 A graph showing the effect of different concentrations of recombinant human type IV collagen-α2 solution on cell viability.

[0024] Figure 4 A graph showing the effect of different concentrations of full-length human type IV collagen α2 chain solutions on cell viability.

[0025] Figure 5 These are fluorescence micrographs of the cells before and after centrifugation.

[0026] Figure 6 Figure showing the effect of recombinant human type IV collagen-α2 on AhR gene expression in HaCat cells.

[0027] Figure 7 Figure showing the effect of recombinant human type IV collagen-α2 on Cyp1a1 gene expression in HaCat cells.

[0028] Figure 8A figure showing the effect of recombinant human type IV collagen-α2 on FLG gene expression in HaCat cells. Detailed Implementation

[0029] Example 1: Preparation of recombinant human type IV collagen-α2 in a yeast expression system I. Experimental Methods 1. Preparation of shuttle plasmids According to the amino acid sequence shown in SEQ ID No:1 (named recombinant human type IV collagen-α2), codon optimization was performed on the yeast expression system to obtain the type IV target gene sequence as shown in SEQ ID No:2. The obtained target gene sequence was then synthesized by Qingke Biotechnology Co., Ltd., and the synthesized gene was ligated into... p From the PicZαA plasmid, the pPicZαA-I-α2 plasmid was obtained.

[0030] SEQ ID No:2 2) Preparation of yeast expression strains pPicZαA-I-α2 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.

[0031] 2. Induced expression of the target protein (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 for activation; (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. (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. (4) Cultivate to OD 600 Start 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. (5) During the induction process, the dissolved oxygen should be controlled to be no less than 30% and the pH should be around 5.0. After induction for 40 hours, the fermentation was stopped. The culture medium was centrifuged at 12,000 rpm for 2 minutes. The supernatant was collected and the protein yield and purity were detected by BCA method and SDS-PAGE method.

[0032] 3. Collagen purification The collected supernatant was passed through a 30KD ultrafiltration membrane, then through a hollow fiber column and freeze-dried to obtain recombinant human type IV collagen-α2.

[0033] II. Results The recombinant human type IV collagen-α2 prepared by the above method achieved a fermenter yield as high as 17 g / L according to BCA analysis. SDS-PAGE protein electrophoresis results showed that the target protein content was over 70%, and the protein purity after ultrafiltration through a hollow fiber column reached over 80%. The purified recombinant human type IV collagen-α2 SDS-PAGE protein electrophoresis results are as follows... Figure 1 As shown.

[0034] Comparative Example 1: Preparation of other recombinant human type IV collagen (control protein) and full-length type IV collagen α2 chain in a yeast expression system. Using the same design approach as recombinant human type IV collagen-α2, protein sequences with similar molecular weights were designed. After codon optimization, the target gene was obtained. The target gene was synthesized and expressed using the same shuttle plasmid and host bacteria. The results of sequence design and expression are shown in Table 1. The SDS-PAGE electrophoresis images of the purified sequences II-1 and II-2 are shown below. Figure 2 Since the recombinant expression of full-length type IV collagen α2 chain protein using the above expression system was unsuccessful, full-length type IV collagen α2 chain protein (catalog number: ZY144Hu014) was purchased from Shanghai Zeye Biotechnology Co., Ltd. for comparative evaluation in subsequent bioactivity experiments.

[0035] Table 1. Different amino acid sequences and expression results

[0036] As can be seen from the results of Example 1 and Comparative Example 1, the yield of recombinant human type IV collagen-α2 of the present invention is much higher than that of sequences of different positions and sizes extracted in a similar design. Therefore, the recombinant human type IV collagen-α2 of the present invention is easier to prepare on a large scale and has greater potential for mass production.

[0037] For a long time, there has been a lack of effective theoretical guidance regarding which recombinant collagens can achieve higher secretion yields. Extensive experimental screening and verification are necessary, making high-yield recombinant collagens extremely rare. In this technical field, it is generally considered that a yield of 10 g / L for recombinant collagen with a molecular weight of 70,000 or higher is already considered quite high, using commercially available expression hosts and vectors without adding additional special expression elements or methods. However, the recombinant human type IV collagen of this invention can achieve a yield as high as approximately 17 g / L, significantly exceeding the high-yield levels considered typical in this technical field.

[0038] Example 3 Evaluation of the biological activity of recombinant human type IV collagen-α2 1. Cytotoxicity assay: HeLa cells grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and cultured in complete medium to a cell density of 1×10⁻⁶ cells / year. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity. After 24 h of cell culture, the complete culture medium was aspirated. Different concentrations of recombinant human type IV collagen-α2 solution (0.001-0.5 mg / ml, 4 replicates per concentration) and different concentrations of full-length human type IV collagen-α2 chain solution (0.001-0.5 mg / ml, 4 replicates per concentration) diluted in high-glucose DMEM medium were added to the experimental groups. The control group contained cells cultured in DMEM medium, and the blank group contained cell-free DMEM medium. Cells were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent was added to each group, and the cells were incubated for 2 h. The absorbance (OD) of each well was measured at 450 nm using an ELISA reader. Cell viability was calculated based on the mean absorbance of each group using the following formula: Experimental results: Figure 3 As shown, the cell viability of each concentration of recombinant type IV collagen solution in the experimental groups was above 90%, indicating that the different concentrations of recombinant human type IV collagen-α2 prepared in this invention have no cytotoxicity and good safety. Figure 4 As shown, the cell viability of the full-length human type IV collagen-α2 chain solution experimental group gradually decreased with increasing concentration. In particular, after the concentration exceeded 0.1 mg / mL, the cell morphology began to change and the viability decreased significantly, indicating that high concentrations of full-length human type IV collagen-α2 chain solution have potential cytotoxicity.

[0039] 2. Cell Adhesion Assay: NIH / 3T3 cells were cultured in a 37 ℃, 5% CO2 cell culture incubator. Cell density and status were observed daily under an inverted microscope. When cells reached 80%–90% confluence in the culture flask, cell passage or seeding was performed. Following the procedure in Appendix B (Normative) of the People's Republic of China Pharmaceutical Industry Standard YY / T 1849-2022 "Recombinant Collagen"—Cell Adhesion Assay—Centrifugation Method—cells were diluted to 5 × 10⁻⁶ cells using complete culture medium premixed with Hoechst 33342 fluorescent thermochromic agent (10%). 4100 μL of cell suspension was added to a 96-well plate, covered with aluminum foil, and incubated at 37 °C and 5% CO2 for 1 h. Prepared D-PBS solution, recombinant human type IV collagen-α2 solution (0.05 mg / mL), and full-length human type IV collagen-α2 chain solution (0.05 mg / mL) were added to separate wells of the 96-well plate, with four wells prepared for each sample. The plates were incubated at 37 °C and 5% CO2 for 2 h, and fluorescence tiling images were captured using an inverted fluorescence microscope. The wells were then filled with D-PBS, air bubbles were removed, and the plates were sealed with a sealing film. The plates were centrifuged at 350 g for 5 min. After centrifugation, the sealing film was discarded, and the supernatant was collected from the wells. The plates were washed once with D-PBS, and 100 μL of D-PBS was added. Fluorescence tiling images were captured again using an inverted fluorescence microscope. Finally, the number of fluorescently labeled cells was counted using ImageJ software, and the data were processed and analyzed.

[0040] An automated cell counting program was used to count the number of fluorescently labeled cell nuclei, and the cell count was determined before and after centrifugation. The adhesion percentage was calculated using the following formula: V = Nt / Nc × 100% In the formula: V --- adhesion percentage; Nt --- number of cells after centrifugation; Nc --- number of cells before centrifugation.

[0041] Using D-PBS as the blank control sample, recombinant human type IV collagen-α2 solution (0.05 mg / mL) and full-length human type IV collagen-α2 chain solution (0.05 mg / mL) were used in the experiment. Cell adhesion was measured under the same centrifugation conditions to compare and evaluate the effects of the two type IV collagens on cell adhesion. The results are shown in the figures. Under the experimental conditions, the adhesion percentage (%) of recombinant human type IV collagen-α2 solution was 36.551 ± 5.638, significantly better than that of full-length human type IV collagen-α2 chain solution (23.830 ± 4.096) (P < 0.05). Specific experimental results are as follows: Figure 5 As shown in Table 2.

[0042] Table 2 Adhesion percentage of different experimental groups

[0043] Example 4: The repair effect of recombinant human type IV collagen-α2 on the skin microbial barrier The following samples were prepared: recombinant type IV collagen-α2 solution (concentration of 0.1 mg / mL, aqueous solution), full-length human type IV collagen-α2 chain solution (0.1 mg / mL), and positive control: 100 μg / mL D-panthenol solution.

[0044] Sample Intervention: Hacat cells were placed in 6-well plates (5*10⁴ / well), with 2 mL added to each well. The plates were incubated at 37℃ in a 5% CO₂ incubator. Three parallel wells were set up for each group: normal control, model, positive control, and sample groups. After 24 h of culture, except for the normal control group, all other groups were treated with 55 μg / mL SLS (sodium dodecyl sulfate) for 24 h to construct an in vitro model of skin irritation / microbial barrier damage. After modeling, the model group was replaced with fresh culture medium, while the positive control and sample groups were replaced with fresh culture medium containing the samples for intervention, with 2 mL added to each well. Total mRNA was extracted after another 24 h of culture. The RNA concentration was measured using a micro spectrophotometer. A concentration greater than 20 ng / μL and an A260 / A280 value greater than 1.8 were considered acceptable. cDNA was synthesized using the RevertAid™ first-strand cDNA synthesis kit. SYBR Green qPCR Mix, cDNA template, primers (sequences shown in Table 3), and RNase-free ddH2O were dissolved at 4°C, and the Real-Time PCR reaction system was prepared on ice. A two-step PCR reaction was performed, with the following reaction program: Step 1: 95°C pre-denaturation for 300 s; Step 2: 95°C denaturation for 20 s, annealing at 55°C for 20 s, extension at 72°C for 20 s, for 40 reaction cycles. The mRNA expression level of each target relative to β-Actin mRNA was determined using the 2-ΔΔCt method. The expression levels of keratinocyte aromatic hydrocarbon receptor (AhR), cytochrome P450 enzyme family gene (Cyp1a1), and filaggrin (FLG) are shown in the table below. Figure 6 , Figure 7 , Figure 8 As shown.

[0045] Table 3 Primer sequence listing

[0046] Experimental results: Figure 6 , Figure 7 , Figure 8 It was found that recombinant type IV collagen-α2 significantly promoted the expression levels of AhR, Cyp1a1, and FLG genes, superior to that of the full-length human type IV collagen-α2 chain, and comparable to that of the commonly used positive control drug D-panthenol. This indicates that it has the effects of promoting keratinocyte differentiation and repairing the skin microbial barrier.

Claims

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

1.

2. A nucleic acid encoding the recombinant human type IV collagen of claim 1; the nucleotide sequence of the nucleic acid may be, for example, as shown in SEQ ID No:

2.

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

4. A host cell in which the expression vector of claim 3 is introduced; the host cell is preferably a eukaryotic cell, such as Pichia pastoris.

5. A method for producing the recombinant human type IV collagen of claim 1, comprising culturing the host cells of claim 4 to induce them to secrete and express the recombinant human type IV collagen, and then separating and purifying them.

6. The method according to claim 5, wherein, The separation and purification process employs one or a combination of several of the following methods: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation; preferably, a combination of chromatographic chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation.

7. The use of the recombinant human type IV collagen according to claim 1 in the preparation of cosmetics, topical medicines or medical devices.

8. The use according to claim 8, wherein, The cosmetics, topical medicines, or medical devices mentioned are used to improve skin redness and repair sensitive skin.

9. The use according to claim 8, wherein, The cosmetics, topical medicines, or medical devices are formulated for application to the skin. Examples include face masks, creams, sprays, and serums.

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