Recombinant human type iv collagen and preparation method therefor

Abstract

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

Description

Recombinant human type IV collagen and preparation method thereof Technical Field

[0001] The present invention relates to recombinant human type IV collagen and a preparation method thereof, belonging to the technical field of bioengineering. Background Art

[0002] Collagen is the primary extracellular matrix in the human body, comprising approximately 85% of the extracellular matrix. It is also the primary structural protein in human tissues and organs, accounting for approximately 30%-40% of the total human protein. Commonly found in the skin, blood vessels, tendons, fascia, and other areas, it performs numerous important biological functions. Twenty-eight different types of collagen have been discovered. Members of the collagen family possess a unique trimeric helical structure and are classified as either fibrillar or nonfibrillar collagen, depending on whether they can form collagen fibrils with periodic striations. Common fibrillar collagens include types I, II, III, V, and XI, while nonfibrillar collagens include types IV and X. Type IV collagen is the primary structural component of the basement membrane and is composed of six α-peptide chains: α1, α2, α3, α4, α5, and α6. The six types of α-peptide chains are composed of three different heterotrimers: α1α1α2, α3α4α5, and α5α5α6. Only α1α1α2 and α5α5α6 are expressed in the skin. Type IV collagen not only provides a scaffold for cell growth but also interacts with specific receptors on the cell surface, activating intracellular signal transduction pathways and participating in important physiological processes such as cell adhesion, migration, growth, proliferation, and differentiation.

[0003] Currently, the primary source of collagen is animal-derived collagen, mostly from terrestrial and marine animals. Terrestrial collagen is cross-linked and embedded in native tissue, requiring stringent extraction and purification techniques. Furthermore, pathogen contamination and allergic reactions are unavoidable issues with terrestrial collagen. Marine collagen effectively avoids the pathogen contamination and allergic reactions associated with terrestrial collagen, but it carries disadvantages such as difficulty in extraction and high purification costs. Type IV collagen is present in low concentrations in animals, making it difficult to extract a single component. With the advancement of genetic engineering technology, recombinant collagen has become the preferred alternative to animal-derived collagen in biomedicine and tissue engineering. Recombinant collagen is obtained by cloning the human collagen gene into a selected expression vector, transforming it into expressing cells, and then purifying it. Compared to collagen obtained through traditional extraction, recombinant collagen offers advantages such as processability, freedom from viral hazards, improved water solubility, batch stability, and low rejection.

[0004] Currently, many recombinant expression systems are used for the recombinant expression of human collagen, such as prokaryotes (Escherichia coli), yeast, animal cells, transgenic animals, and transgenic plants. However, there are relatively few studies and patent applications for the expression of human type IV collagen using recombinant expression systems. At present, the expression systems are mainly based on Escherichia coli and yeast. Escherichia coli is unable to perform corresponding post-translational modifications on recombinant collagen-based proteins, and the target protein is expressed intracellularly on a large scale. The large amount of impurity host proteins and naturally occurring endotoxins and peptidoglycans produced when the bacteria are lysed require a complex purification process to remove. Yeast, as a eukaryotic organism, is prone to glycosylation and phosphorylation of recombinant collagen. In addition, the existing results of Pichia pastoris expressing type IV collagen do not use sequences that are native to human type IV collagen, and their application to the human body may result in rejection reactions. Summary of the Invention

[0005] The purpose of the present invention is to overcome some technical problems existing in the prior art. The present invention provides a recombinant human type IV collagen and a preparation method thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The present invention first provides a recombinant human type IV collagen monomer, wherein the amino acid sequence of the recombinant human type IV collagen monomer is as shown in SEQ ID No: 1, or an amino acid sequence modified to a certain extent by amino acid substitution, insertion, replacement, addition, deletion, etc. based on SEQ ID No: 1, or an amino acid sequence having greater than 80% identity with the amino acid sequence shown in SEQ ID No: 1. The monomer sequence is a partial active amino acid sequence of the α2 chain of human type IV collagen.

[0008] The present invention also provides a recombinant human type IV collagen, the amino acid sequence of which comprises n monomer sequences, where n is an integer greater than or equal to 3 and less than or equal to 20; wherein each monomer sequence is identical or different and is repeated in series with the monomer as the basic unit.

[0009] Furthermore, the amino acid sequence of the recombinant human type IV collagen also contains an amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste13 enzyme, and the amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste13 enzyme is KKREA non-natural collagen sequence amino acid.

[0010] Preferably, the amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste 13 enzyme is directly linked to the basic unit.

[0011] Further preferably, at least one amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste 13 enzyme is added between every two adjacent basic units in the recombinant human type IV collagen, that is, KKREA non-natural collagen sequence amino acids are added between every two adjacent basic units in the recombinant human type IV collagen.

[0012] Further preferably, the amino acid sequence of the recombinant human type IV collagen is as shown in SEQ ID No: 2, or an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the amino acid sequence as shown in SEQ ID No: 2 and the activity of natural human type IV collagen is retained.

[0013] When the recombinant human type IV collagen of the present invention enters the protein secretion pathway composed of the endoplasmic reticulum and the Golgi apparatus through transcription and translation, the KKREA in the sequence will be cut and removed by the Kex2 enzyme, the CPB enzyme and the Ste 13 enzyme, and the 2R2 portion which is 100% identical to the human type IV collagen sequence will be retained.

[0014] The present invention also provides a polynucleotide encoding the recombinant human type IV collagen, wherein the polynucleotide sequence is shown as SEQ ID No: 5 or a degenerate sequence thereof.

[0015] The present invention also provides a recombinant expression vector comprising the polynucleotide.

[0016] The present invention also provides a recombinant engineered bacterium or cell, wherein the recombinant engineered bacterium or cell comprises the recombinant expression vector. Preferably, the host cell of the recombinant engineered bacterium or cell is Pichia pastoris.

[0017] Further preferably, the host cell of the recombinant engineered bacteria or cells is preferably the engineered strain HCPB-PPKEX2, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 25815.

[0018] Furthermore, the recombinant engineered bacteria or cells are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 29210.

[0019] The present invention also provides a method for preparing the recombinant human type IV collagen, the method comprising:

[0020] The recombinant expression vector is linearized with Sal 1 and then transformed into the engineering strain HCPB-PPKEX2 to obtain the recombinant engineering bacteria; the obtained recombinant engineering bacteria is cultured to induce the expression of the target protein; the target protein is collected, purified and identified.

[0021] The recombinant engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number of CGMCC No.29210.

[0022] Specifically, the method for preparing the recombinant human type IV collagen of the present invention comprises:

[0023] Design of type IV collagen sequence: The present invention designs a recombinant human type IV collagen sequence 2R2 based on the partial active amino acid sequence in the α2 chain of human type IV collagen, and adopts the design of repeated tandem expression of protein sequences. Amino acids of non-natural collagen sequences are added between each repeated tandem as a linker. The specific amino acid sequence added is KKREA, which can be completely removed by Kex2, Ste13 and CPB enzymes, maintaining 100% homology between the recombinant collagen and the human collagen sequence.

[0024] Construction of a recombinant expression vector: Synthesize the DNA sequence encoding 2R2(n), connect the exogenous DNA into the expression vector pPIC9K, and construct a recombinant expression vector pPIC9K-2R2(n) that expresses 2R2(n).

[0025] Construction of recombinant engineering strains, induced expression and strain screening: The recombinant expression vector was linearized with Sal I and electroporated into Pichia pastoris competent cells. After induction of expression, engineering strains with high expression of the target protein were selected for subsequent experiments.

[0026] Protein expression identification: Preliminary identification of the expressed protein by SDS-PAGE electrophoresis showed that 2R2(n) could be completely cleaved by Kex2, Ste13, and CPB enzymes and efficiently secreted and expressed extracellularly. The N-terminal and C-terminal amino acid sequences of the 2R2 induced expression supernatant were aligned, demonstrating that they were completely consistent with the designed sequence.

[0027] The present invention also verifies the cell adhesion activity and cell migration promoting activity of the recombinant collagen through in vitro cell experiments: compared with the blank group and the commercial natural human collagen control group, the recombinant human type IV collagen of the present invention has better biological adhesion activity and cell migration promoting activity.

[0028] The present invention also provides a composition comprising the collagen.

[0029] The present invention also provides a product, wherein the product comprises the collagen or the composition, and the product includes a medicine, a pharmaceutical composition, a medical device, a biomaterial, a tissue engineering product, a cosmetic or a health product.

[0030] The present invention also provides uses of the collagen, the polynucleotide, the recombinant expression vector, the recombinant engineered bacteria or cells, or the composition in preparing finished products, including drugs, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0031] Beneficial effects of the present invention:

[0032] (1) The present invention constructs a recombinant human type IV collagen and can express it in Pichia pastoris. The amino acid sequence of the recombinant human type IV collagen obtained by the present invention has a typical GXY triplet structure, which is 100% consistent with the sequence of human type IV collagen. The theoretical molecular weight is 3.41 kDa, which is a relatively small value. In addition, the present invention has experimentally verified that 2R2 has the activity of promoting cell adhesion and migration, and has value in the field of biomaterials.

[0033] The expression system or method constructed by the present invention obtains recombinant collagen that is 100% consistent with the sequence of human type IV collagen, which will not produce an immune response when applied to the human body. The solution of the present invention avoids the cost and risk of exogenous protein residue caused by the use of protease cleavage in vitro, and can also shorten the time and cost of the subsequent purification process.

[0034] (2) The collagen protein of the present invention greatly increases the expression level of collagen protein through the design of tandem repeats. In addition, the present invention uses the strain HCPB-PPKEX2, does not introduce exogenous proteins, and does not use any proteases for in vitro cleavage, thereby avoiding the risk of exogenous protein residues and shortening the time and cost of subsequent purification processes. The linker used in the sequence design of the present invention will be cut and removed during the process of collagen secretion in the cell, obtaining a recombinant collagen protein with 100% homology to the corresponding region of natural collagen protein. In other words, the method of the present invention uses the HCPB-PPKEX2 strain, and the protein obtained does not need to be further added with the corresponding enzyme for enzymatic cleavage, and the target protein can be directly obtained.

[0035] (3) The host bacteria of the present invention is Pichia pastoris, which can secrete proteins for extracellular expression, effectively avoiding problems such as impurities introduced by bacterial cell lysis during product extraction. As a eukaryotic organism, it can perform post-translational modifications on the secreted recombinant proteins, such as glycosylation and phosphorylation. The resulting recombinant proteins are free of pathogens, viral inclusion bodies, or pyrogens, have high safety, and low fermentation costs.

[0036] (4) The sequence of the recombinant human type IV collagen of the present invention is selected from the α2 chain of natural type IV collagen, with a theoretical molecular weight of 3.41 kDa, and will not produce an immune response when applied to the human body; in addition, experimental results show that 2R2 has the activity of promoting cell adhesion and migration, and has value in the field of biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a map of the pPIC9K-2R2 vector.

[0038] FIG2 is a graph showing the SDS-PAGE results of the recombinant human IV collagen 2R2 collagen expression supernatant (induced for 48 hours).

[0039] FIG3 shows the NC terminal sequence alignment results of recombinant human IV collagen 2R2.

[0040] FIG4 shows the results of cell adhesion assay-centrifugation method, where * indicates significant difference in adhesion ratio compared with human collagen (P<0.05), Dunnett method.

[0041] FIG5 shows the statistical results of the cell migration area ratio, where * indicates a significant difference in migration rate compared with native collagen (P<0.05), chi-square test.

[0042] FIG6 is a comparison of cell migration results of recombinant human type IV collagen, natural collagen and blank control. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solutions of the present invention, preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0044] In the embodiments of the present invention, those that are not described in detail are all completed using conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0045] Unless otherwise specified, all culture media or other reagent materials involved in the present invention are conventionally prepared or purchased. For example, the culture media involved, such as BMGY medium, BSM medium, serum-free DMEM medium (Gibco brand / product number 11995065), complete medium, etc., are all conventional commercially available products or prepared according to publicly known formulas. Some of the formulas are as follows:

[0046] BMGY medium formula: by volume: 2% peptone, 1% yeast extract, 1% glycerol, 0.3% K₂HPO₄, and 1.18% KH₂PO₄. Sterilize and store at room temperature. Add 10× YNB and 500× biotin in a clean room before use.

[0047] BSM Inorganic Salts Medium: By volume, the following components are: NH₄H₂PO₄ 2.38%, KH₂PO₄ 0.25%, CaSO₄·2H₂O 0.03%, K₂SO₄ 0.46%, and MgSO₄·7H₂O 0.37%. Sterilize and store at room temperature. Add 0.4% (v / v) PTM1 and 0.5% (v / v) anhydrous methanol in a clean room before use.

[0048] YPD solid medium: 2% (w / v) peptone, 1% (w / v) yeast extract, 2% (w / v) glucose (D-glucose), 2% (w / v) agar powder; after high-pressure sterilization, the resistance screening medium was added with G418 (Geneticin) at a final concentration of 0.5 mg / mL.

[0049] MD solid medium: 2% agar powder, after sterilization, add glucose (D-glucose) 2% (w / v), 10×YNB, 500×biotin in a clean bench.

[0050] Complete culture medium: 88% DMEM + 10% FBS + 1% glutamine + 1% sodium pyruvate.

[0051] In the present invention, any DNA sequence encoding the amino acid sequence of recombinant human type IV collagen 2R2 can be optimized as long as the final encoded amino acid sequence is the same; the present invention is not limited to expression vectors, as long as the selected expression vector has a signal peptide sequence and can import the translated expressed protein into the endoplasmic reticulum, commonly used expression vectors such as pPICZαB, pFLDα, and pPIC9K can be used, and other expression vectors based on similar functions can also be used; the host cell in the present invention can be a eukaryotic cell (such as fungi and yeast) or a prokaryotic cell (such as Enterobacteriaceae). In theory, the above schemes can all achieve technical effects similar to those of the present invention, and the biological activity of the obtained protein should also be basically the same.

[0052] The amino acid sequence of the recombinant human type IV collagen of the present invention comprises n repeating sequences, where n is an integer greater than or equal to 3 and less than or equal to 20. However, n can be any other integer, i.e., the number of tandem repeats is not limited, and in theory, similar effects to those of the present invention can be achieved.

[0053] Artificially designed non-natural collagen sequences can also theoretically be applied to the repeated tandem expression system of the present invention.

[0054] In the present invention, the selected engineered strain HCPB-PPKEX2 is disclosed in the patent applied for by the inventor team of the present invention (the invention name is: Recombinant small molecule collagen and its expression system and preparation method, application number is 202310481706.3), and is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number: CGMCC No. 25815.

[0055] Example 1:

[0056] (1) Design of amino acid sequence

[0057] The amino acid sequence of human type IV collagen 2R2 is derived from the 497-531AA portion of the sequence P08572 in the Uniprot database (https: / / www.uniprot.org / Uniprotkb / P08572 / entry#sequences), which contains 35 amino acids and has a theoretical molecular weight of 3.41 kDa. It can be used for a tandem monomer (repeating sequence), and its sequence is shown in SEQ ID No: 1:

[0058] GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGL

[0059] Based on the above SEQ ID No: 1, a repeating tandem sequence was designed and repeated 12 times. The amino acid sequence KKREA, which can be removed by Kex2, Ste13, and CPB enzymes, was added between each tandem monomer. The resulting sequence totaled 475 amino acids and was named 2R2-12. Its sequence is shown in SEQ ID No: 2:

[0060]

[0061] When 2R2-12 enters the protein secretion pathway composed of the endoplasmic reticulum and Golgi apparatus through transcription and translation, the Kex2 enzyme will split it between the KKR and EA in each KKREA. Then, the two amino acids of EA at the amino terminal will be cleaved and removed by Ste 13 protease. The expressed sequence is shown in SEQ ID No: 3:

[0062] GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKR

[0063] The three amino acids of KKR are then cleaved and removed by the CPB enzyme, and the final secreted protein is a 35-amino acid recombinant collagen protein, the sequence of which is shown in SEQ ID No: 4 (same as SEQ ID No: 1):

[0064] GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGL

[0065] The gene DNA sequence encoding the amino acid sequence of recombinant human type IV collagen 2R2-12 was optimized and a double stop codon TGATAA was added to the 3' end of the sequence. The sequence is shown in SEQ ID No: 5:

[0066]

[0067] (2) Construction of recombinant expression vector and engineered strain

[0068] Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the DNA fragment SEQ ID No: 5 of the SEQ ID No: 2 gene. The synthesized fragment SEQ ID No: 5 was recombined into the pPIC9K empty vector (purchased from Thermo Fisher Scientific). After cloning, the 39 bp sequence at sites 1210-1248 in the vector was replaced, so that the target fragment was accurately inserted into the secretory vector reading frame containing the secretion signal α-factor, and the pPIC9K-2R2-12 recombinant expression vector plasmid expressing 2R2 was obtained. The map is shown in Figure 1.

[0069] 10 μg of the above-mentioned recombinant expression vector plasmid was digested with SalⅠ (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C for 2 h to linearize it, and then a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used to recover the linearized plasmid. The DNA concentration in the resulting solution was controlled as much as possible to be above 100 ng / μL.

[0070] The linearized plasmid was electroporated into the competent cells of the engineered strain HCPB-PPKEX2 (General Microbiology Center of China National Microorganism Culture Collection Administration, deposit number: CGMCC No. 25815). The electroporated bacterial solution was spread on MD plates, with 100 μL to 200 μL spread on one plate. The plate was allowed to stand at room temperature for 10 minutes and then inverted and cultured at 30°C for 2-5 days until a single colony (positive transformant) appeared.

[0071] Add 2 mL of sterile double-distilled water to the surface of the MD plate, then gently scrape the His+ transformants on the surface of the plate with a sterile triangular applicator and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water and take 10 6 The cells were spread on YPD plates containing 0.5 mg / mL G418 and cultured upside down at 30°C for 3-4 days until single colonies appeared.

[0072] The engineered bacteria samples containing pPIC9K-2R2-12 were sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms for preservation.

[0073] The engineered strain expressing 2R2, containing pPIC9K-2R2-12, constructed in the present invention, is deposited with the General Microbiology Center of the China Culture Collection Administration under the deposit number 29210. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is December 1, 2023. The yeast is designated as Pichia pastoris Komagataella phaffii.

[0074] In this example, the engineered strain HCPB-PPKEX2 was selected, as disclosed in a patent application filed by the inventors (titled "Recombinant Small Molecule Collagen and Its Expression System and Preparation Method, Application No. 202310481706.3"). This strain expresses the human CPB enzyme. The recombinant human carboxypeptidase HCPB can specifically cleave basic amino acids (especially K and R) at the carboxyl terminus of proteins until all the basic amino acids at the C-terminus are cleaved, leaving the remaining non-basic amino acids exposed at the C-terminus of the protein. This means that the non-human KKR sequence introduced by the linker can be removed.

[0075] (3) Induced expression and identification of recombinant collagen

[0076] Take the Pichia pastoris engineered strain expressing 2R2 and place it in a 100 mL Erlenmeyer flask containing 15 mL of BMGY medium. Culture at 28-30°C / 220 rpm until the OD 600 The cells were centrifuged at 1500-3000×g for 5 min at room temperature, and the cells were resuspended in BSM medium to an OD of 0. 600 Once the cell count is approximately 10, continue growing on a shaker at 28-30°C / 220 rpm for 2 days. Add 100% methanol to the culture medium every 24 hours to a final concentration of 1.0%. Harvest the culture medium after induction with methanol for at least 16 hours, preferably 48 hours. Place an appropriate amount of the sample in a centrifuge tube and centrifuge at 12,000 × g for 5 minutes at 4°C. Collect the supernatant and analyze immediately or store at -80°C for later use.

[0077] The collected expression supernatant was added with 2× loading buffer, heated in a 100°C metal bath for 10 min, and then subjected to SDS-PAGE detection.

[0078] The SDS-PAGE of the expression supernatant is shown in Figure 2. As can be seen in the figure, recombinant human IV collagen 2R2 is effectively secreted and expressed in the supernatant, and the electrophoretic band is single (collagen has a certain electrophoretic migration delay during electrophoresis, so its apparent molecular weight during electrophoresis will be higher than the theoretical value).

[0079] The supernatant of recombinant human type IV collagen 2R2, obtained after 48 hours of induced expression, was subjected to N-terminal and C-terminal sequence alignment (commissioned by Suzhou Putai Biotechnology Co., Ltd.). The data comparison results are shown in Figure 3, demonstrating that the sequence alignment of recombinant human type IV collagen 2R2 obtained by the present invention is consistent with the theoretical sequence. The first row in the figure shows the results of protein sequence detection, the second row shows the N-terminal alignment results, and the third and fourth rows show the C-terminal alignment results.

[0080] (4) Detection of biological activity of recombinant collagen

[0081] ① Recombinant collagen cell adhesion activity experiment

[0082] The cell adhesion activity test of recombinant collagen was performed in accordance with "YY / T 1849-2022 Recombinant Collagen Appendix B Cell Adhesion Assay-Centrifugation Method".

[0083] Preparation of test samples and positive control samples:

[0084] Under sterile conditions, weigh a sample of collagen 2R2-12 and dissolve it in ultrapure water to 5 mg / mL. Weigh a control sample of native human collagen (Sigma, Cat. No. C7774) and dissolve it in ultrapure water to a pH of 3.0 (5 mg / mL) with acetic acid. Dilute the sample to 0.5 mg / mL in serum-free DMEM medium and sterilize by filtering through a 0.22 μm sterile filter.

[0085] Coating preparation:

[0086] Add 100 μL of the test sample and a D-PBS blank control to a 96-well plate. Coat four wells for each sample and incubate in a 37°C, 5% CO2 incubator for 1 hour. Remove excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate in a 37°C, 5% CO2 incubator for 1 hour. After removing the liquid from the wells, wash three times with D-PBS, discard the wash solution, seal with parafilm, and store at 4°C until ready for use.

[0087] Cell preparation:

[0088] NIH / 3T3 cells (ATCC CRL-1658 mouse embryonic fibroblasts) were cultured in a 37°C, 5% CO2 cell culture incubator. The cell density and status were observed daily under an inverted microscope. When the cells grew to 80% to 90% of the culture flask, the cells were passaged or seeded. The cells were diluted to 5×10 using complete culture medium pre-mixed with Hoechst-33342 fluorescent dye (10%). 4Cells were added to the wells (100 μL), covered with aluminum foil, and incubated at 37°C, 5% CO₂ for 1 h. Three replicates were measured; the fourth well was used to adjust microscope parameters and its value was not used.

[0089] Detection:

[0090] Use an inverted microscope to capture at least 5×5 digital tile images (fluorescence) of each of the 3 wells. Each well was filled with D-PBS to form a "reverse meniscus", purged of bubbles and covered with sealing film. Centrifuge the plate (inverted) at a relative centrifugal force (RCF) of 300g at 22°C for 5 minutes. After centrifugation, remove the sealing film and remove the supernatant from the wells. After washing once with D-PBS, add 100μL D-PBS. For each of the 3 wells, a total of 25 fluorescent tile digital images were taken (at least a 5×5 matrix with a 10% tile overlap is recommended). Approximately 2400 to 3600 cells were calculated for each sample (800-1200 cells / well × 3 wells).

[0091] Result calculation and data processing:

[0092] The cell counts before and after centrifugation were determined. The percentage of adherence was calculated using the formula V = Nt / Nc (Nt: cell count after centrifugation, Nc: cell count before centrifugation). The data differences among the experimental groups were analyzed using one-way ANOVA (Dunnett's method).

[0093] Test results:

[0094] Table 1. Cell adhesion assay - centrifugation assay results

[0095]

[0096]

[0097] The results are shown in FIG4 . Compared with the blank group and the commercial natural human collagen control group, the recombinant human type IV collagen of the present invention has better bioadhesion activity.

[0098] ②Cell migration assay

[0099] Cell culture:

[0100] Use a marker to draw straight and even lines on the back, with a distance of 0.5cm to 1cm between each line. Make sure that there are 3 straight lines across each hole. Add about 5×10 5 cells were cultured.

[0101] Scratch test:

[0102] On the second day of cell culture, use a ruler to scratch the horizontal line on the back of the 6-well plate with a pipette tip as perpendicular as possible to the horizontal line. Keep the pipette tip upright and not tilted. Rinse the cells three times with PBS to thoroughly remove the scratched cells. Add serum-free DMEM containing the test sample at a concentration of 0.05% as the experimental group. Incubate in a 37°C, 5% CO2 incubator. Samples are taken at 0, 6, and 24 hours and photographed.

[0103] Data processing:

[0104] The scratch area of ​​each image was calculated using Image J software. The migration rate of each group was calculated by dividing the total area of ​​migrating cells in the fixed scratch zone by the initial area of ​​the fixed scratch zone. Graphs were plotted with time on the horizontal axis and the migration area ratio on the vertical axis. Images of the experimental and control groups at time zero and at the end of the experiment were compared. Data differences between experimental groups were analyzed using the chi-square test.

[0105] Test results:

[0106] The results are shown in FIG5 and FIG6 . Compared with the blank group and the commercial natural human collagen control group, the recombinant human type IV collagen of the present invention has a more excellent cell migration promoting activity.

[0107] In the present invention, between each two adjacent basic units of the recombinant type IV collagen, there are recognition and cleavage sites for the enzymes Kex2, CPB, and STE13. These enzymes can cleave and remove the non-native collagen sequence (i.e., linker) between the two tandem sequences. In addition, the design of repeated tandem expression of the amino acid sequence can indirectly increase the copy number, thereby achieving the goal of increasing the expression level of the exogenous protein. After transcription and translation, the sequence of the present invention retains a protein sequence that is 100% identical to the human type IV collagen sequence. This effect is achieved because the linker sequence is determined to be LKKREA in the present invention, where L comes from the type IV collagen sequence itself and KKREA is an additional non-native collagen sequence. The linker can be completely cleaved and removed by the enzymes Kex2, CPB, and STE13 in the protein's secretory pathway.

[0108] The Kex2 enzyme is a calcium-dependent serine protease expressed by yeast microorganisms (naturally including Pichia pastoris) that can specifically recognize and cut the carboxyl-terminal peptide bonds of dibasic amino acids such as RR and KR in the amino acid sequence. Taking KR as an example, the position of R is set to p1, the position of K is set to p2, and so on. In order to improve the cutting efficiency, amino acid sequences such as EA and EAEA are added after p1. At the same time, the amino acids of p3 and p4 also have different acidity and alkalinity and charge, etc., so that the Kex2 enzyme exhibits different cutting efficiencies. It has been verified that in Pichia pastoris, when p3 is K, Kex2 has the expected cutting efficiency, and K can be cut by the CPB enzyme. Therefore, in the present invention, when the amino acid KKREA is added at the end of 2R2 to form the sequence LKKREA as a linker for repeated tandem expression, KKREA can be completely cut and removed, and non-native collagen sequences will not be introduced into the final recombinant protein product.

[0109] Yeast microorganisms harbor the STE13 gene, which expresses the Ste13 protease (strictly speaking, a dipeptidyl aminopeptidase) intracellularly. This protease cleaves the amino acid sequences EA and EAEA at the amino termini of proteins. Recombinant carboxypeptidase B (CPB) specifically cleaves basic amino acids (especially K and R) at the carboxyl terminus of proteins, until all the basic amino acids at the C-terminus are cleaved, leaving the remaining non-basic amino acids exposed at the C-terminus. These three proteases synergistically remove non-collagenous amino acids such as K, R, and EA at the N-terminus, maintaining the sequence homology of the recombinant collagen to human collagen.

[0110] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

1.

2. A recombinant human type IV collagen, characterized in that, The amino acid sequence of the recombinant human type IV collagen contains n monomers as described in claim 1, where n is an integer greater than or equal to 3 and less than or equal to 20; each monomer sequence is the same and is tandemly repeated based on the monomer; the amino acid sequence of the recombinant human type IV collagen contains an amino acid sequence that can be cleaved and removed by Kex2 enzyme, CPB enzyme, and Ste13 enzyme; The amino acid sequence that can be cleaved and removed by Kex2 enzyme, CPB enzyme, and Ste13 enzyme is KKREA; The amino acid sequence that can be cleaved and removed by Kex2 enzyme, CPB enzyme, and Ste13 enzyme is directly linked to the said basic unit; at least one amino acid sequence that can be cleaved and removed by Kex2 enzyme, CPB enzyme, and Ste13 enzyme is added between every two adjacent basic units in the recombinant human type IV collagen.

3. The recombinant human type IV collagen according to claim 2, wherein The amino acid sequence of the recombinant human type IV collagen is shown in SEQ ID No:

2.

4. A polynucleotide encoding the recombinant human type IV collagen according to any one of claims 2 - 3.

5. The polynucleotide according to claim 4, wherein The polynucleotide sequence is shown in SEQ ID No: 5 or its degenerate sequence.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide according to claim 4 or 5.

7. A recombinant engineering bacterium or cell, characterized in that, The recombinant engineered bacterium or cell contains the recombinant expression vector according to claim 6; the host cell of the recombinant engineered bacterium or cell is Pichia pastoris.

8. The recombinant engineered bacterium or cell according to claim 7, wherein The host cell of the recombinant engineered bacterium or cell is the engineered strain HCPB - PPKEX2, and the engineered strain HCPB - PPKEX2 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 25815.

9. The recombinant engineered bacterium or cell according to claim 8, wherein The recombinant engineered bacterium or cell is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 29210.

10. A method for preparing the recombinant human type IV collagen, characterized in that, The method includes: Transforming the recombinant expression vector according to claim 6 into competent Escherichia coli DH5α cells, screening for positive clones and extracting the recombinant expression vector plasmid; after linearizing the plasmid, transforming it into the engineered strain HCPB - PPKEX2 to obtain a recombinant engineered bacterium; culturing the obtained recombinant engineered bacterium to induce the expression of the target protein; collecting the target protein and purifying and identifying it; The engineered strain HCPB - PPKEX2 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 25815.

11. The method according to claim 10, wherein The recombinant engineered bacterium is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 29210.

12. A composition, characterized in that, The composition contains the monomer according to claim 1, or the recombinant human type IV collagen according to any one of claims 2 - 3, or the recombinant human type IV collagen prepared by the method according to any one of claims 10 - 11.

13. An article, characterized in that, The article contains the monomer described in 1, or the recombinant human type IV collagen described in any one of claims 2-3, or the recombinant human type IV collagen prepared by the method described in any one of claims 10-11, or the composition described in claim 12, and the article includes drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

14. Use of the monomer described in claim 1, or the recombinant human type IV collagen described in any one of claims 2-3, or the recombinant human type IV collagen prepared by the method described in any one of claims 10-11, or the polynucleotide described in any one of claims 4-5, the recombinant expression vector described in any one of claims 6-7, the engineered bacterium or cell described in any one of claims 8-9, or the composition described in claim 12 in the preparation of finished products, the finished products including drugs, medical devices, biomaterials, tissue engineering products, cosmetics, health products.

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