WO2025140277A1 - Recombinant human type iv collagen and preparation method therefor - Google Patents

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

A recombinant human type IV collagen and its preparation method Technical Field

The invention relates to recombinant human type IV collagen and a preparation method thereof, belonging to the technical field of bioengineering.

Background Art

Collagen is the main extracellular matrix of the human body, accounting for about 85% of the extracellular matrix. It is also the main structural protein of human tissues and organs, accounting for about 30%-40% of the total protein in the human body. It is commonly found in the skin, blood vessels, tendons, fascia and other parts, and plays a variety of important biological functions. So far, 28 different types of collagen have been discovered. The members of the collagen family have a unique trimeric helical structure. According to whether collagen fibrils with periodic horizontal stripes can be formed, they are divided into fibrillar collagen and nonfibrillar collagen. Common fibrillar collagens include type I, II, III, V and XI collagens, and common nonfibrillar collagens include type IV and X collagens. Among them, type IV collagen is the main structural component of the basement membrane, which is composed of six types of α-peptide chains: α1, α2, α3, α4, α5, and α6. The six types of α-peptide chains form three different heterotrimers, α1α1α2, α3α4α5, and α5α5α6, of which 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, thereby activating intracellular signal transduction pathways and participating in important physiological processes such as cell adhesion, migration, growth, proliferation and differentiation.

At present, the main source of collagen is animal collagen, most of which are terrestrial and marine animals. Terrestrial animal collagen has been cross-linked and embedded in native tissues, which requires relatively strict extraction and purification technology; in addition, pathogen contamination and allergic risks have also become unavoidable problems for terrestrial animal collagen; marine collagen can effectively avoid the problems of pathogen contamination and allergic risks of terrestrial animal collagen, but it has disadvantages such as difficult extraction and high purification costs. The content of type IV collagen in animals is low, making it difficult to extract a single component. With the development of genetic engineering technology, recombinant collagen has become the best substitute for animal collagen in biomedicine and tissue engineering. Recombinant collagen is a protein obtained by cloning the human collagen gene into a selected expression vector and transforming it into expression cells, and finally by purification technology. Compared with collagen obtained by traditional extraction, recombinant collagen mainly has the advantages of processability, no virus risks, good water solubility, batch stability and low rejection reaction.

At present, 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 few studies and patent applications on the expression of human type IV collagen by recombinant expression systems. At present, the expression system is mainly based on Escherichia coli and yeast. Escherichia coli cannot 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 carried endotoxins and peptidoglycans produced when the bacteria are lysed require a complex purification process to be removed; and 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 have a sequence that is a natural human type IV collagen sequence, and may cause rejection reactions when applied to the human body.

Summary of the invention

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.

In order to achieve the above technical purpose, the present invention adopts the following technical solutions:

The present invention first provides a recombinant human type IV collagen monomer, 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. on the basis of 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 in the α2 chain of human type IV collagen.

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 a basic unit.

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 Ste 13 enzyme is KKREA non-natural collagen sequence amino acid.

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

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.

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 shown in SEQ ID No: 2 and the activity of natural human type IV collagen is retained.

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

The present invention also provides a polynucleotide encoding the recombinant human type IV collagen, and the polynucleotide sequence is as shown in SEQ ID No: 5 or its degenerate sequence.

The present invention also provides a recombinant expression vector, which comprises the polynucleotide.

The present invention also provides a recombinant engineered bacterium or cell, wherein the recombinant engineered bacterium or cell comprises the recombinant expression vector, and preferably the host cell of the recombinant engineered bacterium or cell is preferably Pichia pastoris.

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 National Microbiological Culture Collection Administration with the deposit number CGMCC No. 25815.

Furthermore, the recombinant engineered bacteria or cells are deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No.29210.

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

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 are cultured to induce the expression of the target protein; the target protein is collected and purified and identified.

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

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

Design of type IV collagen sequence: The present invention designs a recombinant human type IV collagen sequence 2R2 based on a partial active amino acid sequence in the α2 chain of human type IV collagen, and adopts a design of repeated tandem expression of the protein sequence, adding amino acids of non-natural collagen sequences as linkers between each repeated tandem sequence. 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.

Construction of a recombinant expression vector: Synthesize a 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).

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

Protein expression identification: The expressed protein was preliminarily identified by SDS-PAGE electrophoresis. The results 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 compared, proving that they were completely consistent with the designed sequence.

The present invention also verifies the cell adhesion activity and cell migration promoting activity of the recombinant collagen by 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 more excellent biological adhesion activity and cell migration promoting activity.

The present invention also provides a composition comprising the collagen.

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.

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

Beneficial effects of the present invention:

(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 G-X-Y 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.

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, and 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 in vitro protease cleavage, and can also shorten the time and cost of subsequent purification process.

(2) The collagen protein of the present invention greatly increases the expression of collagen protein through the design of tandem repeats, and the present invention adopts the strain HCPB-PPKEX2, does not introduce exogenous proteins, and does not use any protease for cutting in vitro, thereby avoiding the risk of exogenous protein residues, and can also shorten the time and cost of the subsequent purification process. The linker used in the sequence design of the present invention will be cut and removed during the process of collagen secretion in cells, and a recombinant collagen protein with 100% homology to the corresponding region of natural collagen protein is obtained. In other words, the method of the present invention uses the HCPB-PPKEX2 strain, and the protein obtained does not need to be additionally added with the corresponding enzyme for enzymatic cutting, and the target protein can be directly obtained.

(3) The host bacteria of the present invention is Pichia pastoris, which can secrete proteins for extracellular expression, and can effectively avoid problems such as impurities caused by lysing bacteria when extracting products. As a eukaryotic organism, it can perform post-translational modifications on the secreted recombinant proteins, such as glycosylation and phosphorylation, and the produced recombinant proteins do not contain pathogens, viral inclusion bodies or heat sources, have high safety, and low fermentation costs.

(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 application value in the field of biomaterials.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 2 is a diagram showing the SDS-PAGE results of the recombinant human IV collagen 2R2 collagen expression supernatant (induced for 48 hours).

FIG3 is the result of NC terminal sequence alignment of recombinant human IV collagen 2R2.

FIG. 4 shows the results of cell adhesion assay-centrifugation method, where * indicates significant difference in adhesion ratio with human collagen (P<0.05), Dunnett method.

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

FIG6 is a comparison of the cell migration results of recombinant human type IV collagen, natural collagen and blank control.

DETAILED DESCRIPTION

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

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

All culture media or other reagent materials involved in the present invention are conventionally prepared or purchased unless otherwise specified, such as BMGY culture medium, BSM culture medium, serum-free DMEM culture medium (brand Gibco/item number 11995065), complete culture medium and other culture media involved, which are conventional commercial products or prepared by known public formulas. Some of the formulas are as follows:

BMGY medium formula: by volume percentage, peptone 2%, yeast extract 1%, glycerol 1%, K ₂ HPO ₄ 0.3%, KH ₂ PO ₄ 1.18%, stored at room temperature after sterilization. Add 10×YNB and 500×biotin in the clean bench before use.

BSM inorganic salt medium: by volume percentage, NH ₄ H ₂ PO ₄ 2.38%, KH ₂ PO ₄ 0.25%, CaSO ₄ ·2H ₂ O0.03%, K ₂ SO ₄ 0.46%, MgSO ₄ ·7H ₂ O 0.37%, stored at room temperature after sterilization. Before use, add 0.4% (v/v) PTM1 and 0.5% (v/v) anhydrous methanol in the clean bench.

YPD solid medium: peptone 2% (w/v), yeast extract 1% (w/v), glucose (D-glucose) 2% (w/v), 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.

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

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

In the present invention, any DNA sequence that can encode 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 the expression vector, as long as the selected expression vector has a signal peptide sequence and can introduce the translated protein into the endoplasmic reticulum, commonly used expression vectors such as pPICZαB, pFLDα, pPIC9K, and other expression vectors based on similar effects are also available; 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 obtain technical effects similar to those of the present invention, and the biological activity of the obtained protein should also be basically the same.

The amino acid sequence of the recombinant human type IV collagen in 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, but n can be any other integer, that is, the number of tandem repeats is not limited, and in theory, similar effects as those of the present invention can be achieved;

Artificially designed non-natural collagen sequences can also be applied to the repeated tandem expression system of the present invention in theory.

In the present invention, the selected engineering 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 China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.25815.

Embodiment 1:

(1) Design of amino acid sequence

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

GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGL

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

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 KKR and EA in each KKREA, and then the two amino acids of EA at the amino terminal will be cut and removed by Ste 13 protease. The expressed sequence is shown in SEQ ID No:3:

GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKKR

Then the three amino acids of KKR will be cleaved and removed by 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):

GLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGL

After optimization, the gene DNA sequence encoding the amino acid sequence of recombinant human type IV collagen 2R2-12 was added with double termination codon TGATAA at the 3' end of the sequence. The sequence is shown in SEQ ID No: 5:

(2) Construction of recombinant expression vector and engineered strain

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.

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 Biotechnology (Shanghai) Co., Ltd.) was used to recover the linearized plasmid, and the DNA concentration in the resulting solution was controlled as much as possible above 100 ng/μL.

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

Add 2 mL of sterile double distilled water to the surface of the MD plate, then use a sterile triangular applicator to gently scrape off the His+ transformants on the surface of the plate and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double distilled water, take 10 ⁶ cells and spread them on a YPD plate containing 0.5 mg/mL G418, and invert and culture at 30°C for 3 to 4 days until a single colony appears.

The engineered bacteria samples containing pPIC9K-2R2-12 were sent to the China National Center for Microbiological Culture Collection for preservation.

The engineered strain expressing 2R2 and containing pPIC9K-2R2-12 constructed by the present invention is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, and the deposit number is: NO.29210. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Deposit Date: December 1, 2023. Classification Name: Pichia pastoris Komagataella phaffii.

In this embodiment, the selected engineering 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), which expresses human CPB enzyme, and recombinant human carboxypeptidase HCPB can specifically cut the basic amino acids (especially K and R) at the carboxyl terminus of the protein until the basic amino acids at the C terminus are cut off, and other non-basic amino acids are exposed at the C terminus of the protein. That is, the non-human KKR sequence introduced by Linker can be removed.

(3) Induced expression and identification of recombinant collagen

Take the Pichia yeast engineering strain expressing 2R2, place it in a 100mL Erlenmeyer flask with 15mL BMGY medium, and culture it at 28-30℃/220rpm until _OD600 is about 10 (24h). Centrifuge at 1500-3000×g for 5min at room temperature, collect the bacteria, resuspend the bacteria with BSM medium to make _OD600 about 10, place it on a shaker at 28-30℃/220rpm and continue to grow for 2 days. Add 100% methanol to the culture medium every 24h to a final concentration of 1.0%. After inducing with methanol for more than 16h, the bacterial liquid sample can be collected, and induction for 48h is better; take an appropriate amount of sample and place it in a centrifuge tube, centrifuge it at 12000×g for 5min at 4℃, collect the supernatant, and test it immediately or store it at -80℃ for later use.

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.

The SDS-PAGE of the expression supernatant is shown in FIG2 . As can be seen from the figure, the recombinant human IV collagen 2R2 is effectively secreted and expressed in the supernatant, and the electrophoresis 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).

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

(4) Detection of biological activity of recombinant collagen

① Recombinant collagen cell adhesion activity experiment

The cell adhesion activity experiment of recombinant collagen was carried out in accordance with "YY/T 1849-2022 Recombinant Collagen Appendix B Cell Adhesion Assay-Centrifugation Method".

Preparation of test samples and positive control samples:

Under sterile conditions, take collagen 2R2-12 sample, weigh it in proportion, and dissolve it in ultrapure water to 5 mg/mL; take control sample natural human collagen (Sigma, item number C7774), weigh it in proportion, dissolve it in ultrapure water, and add acetic acid to pH 3.0 (concentration 5 mg/mL). When using, dilute the sample to a concentration of 0.5 mg/mL with serum-free DMEM medium, and filter through a 0.22 μm sterile filter for sterilization.

Coating preparation:

Add 100 μL of the sample to be tested and the D-PBS blank control to the 96-well plate. Prepare 4 wells for each sample coating and incubate in a 37°C, 5% CO ₂ incubator for 1 hour. Remove the excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate in a 37°C, 5% CO ₂ incubator for 1 hour. After removing the liquid in the wells, wash three times with D-PBS, discard the washing solution, seal with sealing film, and place at 4°C for use.

Cell preparation:

NIH/3T3 cells (ATCC CRL-1658 mouse embryonic fibroblasts) were cultured in a 37°C, 5% _CO2 cell culture incubator, and the cell density and status were observed under an inverted microscope every day. When the cells grew to 80% to 90% of the culture flask, cell passage or cell inoculation was performed. Cells were diluted to 5× ¹⁰⁴ /mL using complete medium that had been premixed with Hoechst-33342 fluorescent stain (10%). 100 μL of cells were added to the wells, covered with aluminum foil, and incubated at 37°C, 5% _CO2 for 1 hour. Three replicate samples were measured, and the fourth well was used to adjust the microscope parameters, and its measured values were not used.

Detection:

Use an inverted microscope to capture at least 5×5 digital tile images (fluorescence) of each of the 3 wells. Each well is filled with D-PBS to form a "reverse meniscus", purged of bubbles and covered with sealing film. Centrifuge the plate (inverted) at 22°C for 5 min at a relative centrifugal force (RCF) of 300g. 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, take a total of 25 fluorescent tile digital images (at least 5×5 matrix is recommended, with 10% tile overlap). Count approximately 2400 to 3600 cells for each sample (800-1200 cells/well × 3 wells).

Result calculation and data processing:

The cell numbers before and after centrifugation were determined. The percentage of adhesion was calculated according to the formula V = Nt/Nc (Nt: cell number after centrifugation, Nc: cell number before centrifugation), and the data differences of each experimental group were analyzed using one-way ANOVA (Dunnett method).

Test results:

Table 1. Cell adhesion assay - centrifugation assay results

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.

②Cell migration assay

Cell culture:

Use a marker pen to draw straight and even lines on the back, with the interval between each two lines being 0.5 cm to 1 cm. The lines are drawn horizontally through the holes, ensuring that there are 3 lines in each hole. Add about 5×10 ⁵ cells to the wells for culture.

Scratch test:

On the second day of cell culture, use a ruler to scratch the horizontal line on the back of the 6-well plate with the tip of a pipette as vertically as possible. The tip should be upright and not tilted. Rinse the cells 3 times with PBS to fully remove the scratched cells, and add serum-free DMEM medium containing the test sample as the experimental group, with a concentration of 0.05%. Place in a 37°C, 5% _CO2 incubator for culture, and take samples and take photos at 0h, 6h, and 24h.

Data processing:

Image J image processing software was used to calculate the scratch area of each picture, and the migration rate of each group of cells was calculated by dividing the total area of migrating cells in the fixed scratch area by the initial area of the fixed scratch area. The time was used as the horizontal axis and the migration area ratio was used as the vertical axis to draw a graph, and the photos of the experimental group and the control group at the initial time 0 and the end of the experiment were compared. The chi-square test was used to analyze the differences in the data of each experimental group.

Test results:

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.

In the present invention, there are recognition and cutting sites for Kex2 enzyme, CPB enzyme and STE13 enzyme between each two adjacent basic units of recombinant type IV collagen, and these enzymes can cut and remove the non-natural collagen sequence (i.e., Linker) between the two tandem sequences; in addition, the design of repeated tandem expression of amino acid sequences can achieve the purpose of indirectly increasing the copy number, so as to achieve the purpose of increasing the expression amount of exogenous protein. After transcription and translation, the sequence in the present invention retains the protein sequence 100% consistent with the human type IV collagen sequence. The reason why such an effect can be achieved is that the sequence of Linker is determined as LKKREA in the present invention, wherein L comes from the type IV collagen sequence itself, and KKREA is an additional non-natural collagen sequence. Linker can be completely cut and removed by Kex2 enzyme, CPB enzyme and STE13 enzyme in the secretion pathway of the protein.

Kex2 enzyme is a calcium ion-dependent serine protease expressed by yeast microorganisms (naturally including Pichia pastoris) itself, which 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 a cutting efficiency that meets expectations, and K can be cut by CPB enzyme, so in the present invention, when the amino acid KKREA is added at the end of 2R2 to form the sequence LKKREA as Linker for repeated tandem expression, KKREA can be completely cut and removed, and non-natural collagen sequences will not be introduced in the final recombinant protein product.

Yeast microorganisms have the STE 13 gene and express the Ste 13 protease (strictly speaking, it belongs to the dipeptidyl aminopeptidase) in the cell, which can cut the amino acid sequence of EA and EAEA at the amino terminus of the protein. Recombinant carboxypeptidase B (CPB enzyme) can specifically cut the basic amino acids (especially K and R) at the carboxyl terminus of the protein until the basic amino acids at the C terminus are cut off and other non-basic amino acids are exposed at the C terminus of the protein. The three proteases work together to remove the non-collagenous amino acids such as KR and EA at the N terminus, respectively, to maintain the sequence homology between the recombinant collagen and the human collagen.

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 the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. 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 (14)

A recombinant human type IV collagen monomer, characterized in that the amino acid sequence of the recombinant human type IV collagen monomer is as shown in SEQ ID No: 1. A recombinant human type IV collagen, characterized in that the amino acid sequence of the recombinant human type IV collagen contains n monomers according to claim 1, n is an integer greater than or equal to 3 and less than or equal to 20; wherein each monomer sequence is identical and repeated in series based on the monomer as a basic unit; the amino acid sequence of the recombinant human type IV collagen contains an amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste13 enzyme; The amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste 13 enzyme is KKREA; The amino acid sequence that can be cut and removed by Kex2 enzyme, CPB enzyme and Ste 13 enzyme is directly connected to the basic unit; 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. The recombinant human type IV collagen according to claim 2 is characterized in that the amino acid sequence of the recombinant human type IV collagen is as shown in SEQ ID No: 2. A polynucleotide encoding the recombinant human type IV collagen according to any one of claims 2 to 3. The polynucleotide according to claim 4 is characterized in that the polynucleotide sequence is as shown in SEQ ID No: 5 or its degenerate sequence. A recombinant expression vector, characterized in that the recombinant expression vector comprises the polynucleotide according to claim 4 or 5. A recombinant engineered bacterium or cell, characterized in that the recombinant engineered bacterium or cell comprises the recombinant expression vector according to claim 6; and the host cell of the recombinant engineered bacterium or cell is Pichia pastoris. The recombinant engineered bacteria or cells according to claim 7 are characterized in that the host cell of the recombinant engineered bacteria or cells is the engineered strain HCPB-PPKEX2, and the engineered strain HCPB-PPKEX2 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number CGMCC No. 25815. The recombinant engineered bacteria or cells according to claim 8 are characterized in that the recombinant engineered bacteria or cells are deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No. 29210. A method for preparing the recombinant human type IV collagen, characterized in that the method comprises: Transform the recombinant expression vector of claim 6 into competent Escherichia coli DH5α cells, screen positive clones and extract the recombinant expression vector plasmid; transform the plasmid into the engineering strain HCPB-PPKEX2 after linearization to obtain the recombinant engineering bacteria; culture the obtained recombinant engineering bacteria to induce the expression of the target protein; collect the target protein, purify it and identify it; The engineered strain HCPB-PPKEX2 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.25815. The method according to claim 10 is characterized in that the recombinant engineered bacteria is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.29210. A composition, characterized in that the composition comprises 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. A product, characterized in that the product comprises 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 product includes a drug, a pharmaceutical composition, a medical device, a biomaterial, a tissue engineering product, a cosmetic or a health product. Use of 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, or the polynucleotide according to any one of claims 4-5, the recombinant expression vector according to any one of claims 6-7, the engineered bacteria or cells according to any one of claims 8-9, or the composition according to claim 12 in the preparation of finished products, wherein the finished products include drugs, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.