Preparation method of recombinant engineering bacterium for expressing III-type collagen COL17A1

Abstract

The invention relates to the technical field of collagen, and discloses a preparation method of recombinant engineering bacteria for expressing III-type collagen COL17A1, which comprises the following steps: synthesizing an optimized sequence of human III-type collagen, and adding proper restriction enzyme cutting sites at two ends of the sequence. According to the preparation method of the recombinant engineering bacterium for expressing the III-type collagen COL17A1, codon optimization is carried out on a target sequence by adopting a yeast expression system, OptimGeneTM codon optimization is provided according to codon preference of yeast strains such as pichia pastoris, and the yield of recombinant protein is further improved. And a proper vector is selected to realize high-efficiency expression in pichia pastoris. For example, a pGAPZ A-alpha A vector stably expresses recombinant protein in pichia pastoris by using a GAP promoter, is a constitutive gene expression vector, and can maintain a stable expression rate in cells no matter how the environment changes. An inducer does not need to be added, the production process is simplified, the production cost is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for preparing an engineering bacterium expressing type III recombinant collagen. Background Art

[0002] Type III collagen, as an important component of human skin, plays an important role in maintaining skin elasticity, protecting blood vessels and nerves, improving cell microenvironment, promoting wound healing, and reducing skin inflammation. At present, there are two main methods for obtaining collagen: animal tissue extraction and genetic engineering method preparation. Collagen extracted from animal tissue is usually a mixture of collagens with different molecular weights, which is insoluble in water and has poor biocompatibility, and there is also a risk of immunogenicity. Collagen prepared by genetic engineering methods can avoid these problems. Through high-density fermentation of Escherichia coli or Pichia pastoris, high expression levels can be achieved, which is more conducive to industrialization. Pichia pastoris is one of the commonly used cell factories, but the methanol nutrition yeast expression system has limitations. The present invention aims to achieve the goal of sustained, stable, high expression and large-scale production of human type III collagen in Pichia pastoris.

[0003] This patent uses Pichia pastoris as an expression host to construct a recombinant engineered bacterium to express type III collagen. Pichia pastoris has many advantages, such as being simple and easy to culture, low cost and high expression, and the eukaryotic expression system can modify exogenous proteins and does not contain bacterial endotoxins.

[0004] Pichia pastoris is a methylotrophic yeast that has the ability to efficiently express exogenous proteins, with the expression level of exogenous proteins reaching 30% of the total cell protein. It has the strongest promoter pAOX1 in eukaryotes, which can be used for high-level expression of rate-limiting enzymes such as CYPs, and has unique advantages in the synthesis of plant natural products. However, although the methanol-trophic yeast expression system is considered to be a highly promising eukaryotic expression system, it still has certain limitations. For example, large-scale protein production depends on the optimization of process parameters such as methanol feed rate, induction temperature and specific growth rate, and the use of flammable methanol as a raw material requires consideration of the hygienic safety of the expression product.

[0005] In order to solve the problem of sustained, stable, high expression and large-scale production of human type III collagen in Pichia pastoris, the present invention optimizes and selects the human type III collagen sequence, and takes the repeated Gly-XY tripeptide in the basic skeleton of the natural type III collagen protein sequence as the basic unit. These tripeptides form a characteristic collagen triple helix structure in the homotrimer. This collagen triple helix structure has high stability. At the same time, the recombinant protein contains more hydrophobic structures as the basic criterion, selects the recombinant protein sequence, uses the yeast expression system to optimize the codon of the target sequence, improves the expression of the recombinant protein, and finally inserts as few enzyme cutting sites as possible when cloning the target nucleic acid sequence to ensure the stability of the recombinant protein. The pGAPZ A-αA vector is selected to achieve efficient recombinant protein expression in Pichia pastoris. Compared with the traditional method, the optimization strategy of the present invention significantly improves the expression level, biological activity and storage stability of the protein, makes large-scale production possible, and broadens the application prospects of type III collagen in the fields of medicine and beauty. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a recombinant engineered bacterium expressing type III human collagen, which is necessary to ensure the homology of the amino acid sequence, protein structure and stability, high expression efficiency and high functional activity, so as to solve the technical problems of optimizing the selection of the protein sequence of type III human collagen, and its difficulty in sustained stable high expression and large-scale production of collagen in Pichia pastoris. The present invention is designed to select a fragment of 378 amino acids at positions 177-554 of type III collagen, which is repeated twice in series, with a total of 756 amino acids, and construct a secretory expression vector with Pichia pastoris as the host. The protein is verified to be expressed by shaking flask culture, with an apparent molecular weight of about 68KDa, and a peptide coverage rate of more than 95% detected by mass spectrometry, confirming that the expressed protein is the target protein. The protein expression level of three batches fermented in a 5L fermenter was stable at around 8.1g / L. After hollow fiber clarification, ultrafiltration and chromatography separation, the purity of the target protein was over 95%. Cytological experiments showed that it had a significant effect on promoting the adhesion, proliferation and migration of skin fibroblasts. Injection into the dermis of rat skin can promote the secretion and expression of collagen in rat skin. In vitro cell experiments showed that the addition of 0.5mg / mL can significantly promote the proliferation of outer root sheath cells. Compared with the blank group, the proliferation rate increased by more than 29%.

[0007] The preparation method of the present invention is as follows:

[0008] (1) Synthesize an optimized sequence of human type III collagen and add appropriate restriction enzyme sites at both ends of the sequence; select the recombinant protein sequence by optimizing the sequence of human type III collagen, using the repeated Gly-XY tripeptide in the basic skeleton of the natural type III collagen protein sequence as the basic unit to ensure that the recombinant protein contains more hydrophobic structures; at the same time, according to the codon preference of yeast strains such as Pichia pastoris, codon optimization is performed on the target sequence to increase the expression of the recombinant protein; clone the synthesized gene sequence into a specific vector to ensure that the gene is expressed under the control of a suitable promoter;

[0009] (2) The optimized gene sequence was cloned into the pGAPZ A-αA vector, which uses the GAP promoter to stably express the recombinant protein in Pichia pastoris. This vector is a constitutive gene expression vector that can maintain a stable expression rate regardless of changes in the intracellular environment. No external inducer is required, which simplifies the production process, reduces production costs, and improves production efficiency.

[0010] (3) The transformation vector is introduced into Escherichia coli for amplification and the recombinant plasmid is extracted; The transformation vector is introduced into Escherichia coli DH5α for amplification and the recombinant plasmid is extracted to prepare for the subsequent construction of recombinant Pichia pastoris;

[0011] (4) Construction of recombinant Pichia pastoris

[0012] The recombinant plasmid was transformed into the Pichia pastoris strain GS115 by electroporation, so that the recombinant plasmid was integrated into the Pichia pastoris chromosome, replicated and inherited along with the chromosome, and the loss of exogenous genes would not occur.

[0013] Screening was performed on a medium containing screening antibiotics, and resistant clones were picked and verified by PCR and sequencing. Screening was performed on a medium containing screening antibiotics, and resistant clones were picked and verified by PCR and sequencing to ensure that the recombinant Pichia pastoris contained the correct human type III collagen gene sequence.

[0014] (5) Fermentation culture and expression optimization

[0015] The verified recombinant Pichia pastoris was inoculated into the YPD medium for cultivation; the verified recombinant Pichia pastoris was inoculated into the YPD medium and cultured at 28°C and 220rpm for 24 hours to allow the recombinant Pichia pastoris to adapt to the culture environment and grow and reproduce rapidly; the seed culture solution was transferred to a specific fermentation medium for amplification culture, and the fermentation conditions were controlled to achieve efficient expression of the recombinant protein;

[0016] The seed culture medium is transferred to a fermentation medium containing bleomycin for amplification culture, and the cell density is gradually increased; the fermentation temperature, pH and dissolved oxygen level are controlled to achieve efficient expression of the recombinant protein; for example, the fermentation temperature can be controlled at 27°C-29°C, the pH of the basic fermentation culture medium can be controlled at 4.8-5.2, and the dissolved oxygen level can be controlled at 30-32.5 to increase the protein expression of the recombinant human type III collagen engineering bacteria;

[0017] (6) Protein extraction and purification

[0018] Collect the supernatant and remove cells and cell debris;

[0019] After the fermentation is finished, the cells and cell debris are removed by centrifugation or filtration, and the supernatant is collected; the supernatant contains the secretory expressed protein;

[0020] The supernatant was concentrated using ultrafiltration;

[0021] The supernatant is concentrated by ultrafiltration to increase the protein concentration for subsequent purification operations;

[0022] Choose gel filtration chromatography to separate proteins of different molecular weights;

[0023] According to the molecular weight of the protein, gel filtration chromatography is selected to separate proteins of different molecular weights, remove impurities, and improve the purity of recombinant type III collagen;

[0024] (7) Ensure protein quality through testing methods;

[0025] The purity, molecular weight and activity of the protein can be tested by SDS-PAGE gel electrophoresis and other methods to ensure the quality of the protein; if the purity, molecular weight and activity of the protein do not meet the requirements, the purification method can be adjusted or the fermentation culture can be repeated;

[0026] The purified secretory expression protein can be appropriately stored as needed, such as frozen or with the addition of stabilizers, to maintain the stability and activity of the protein;

[0027] (8) Functional activity verification

[0028] Determine the activity of recombinant collagen in in vitro cell adhesion assay;

[0029] Dissolve recombinant human type III collagen in PBS and adjust to an appropriate concentration (e.g., 10 μg / mL); add 100 μL of collagen solution to each well of a 96-well culture plate; coat the culture plate overnight at 4°C to allow collagen to adsorb to the well wall; on the next day, replace the collagen solution in each well with PBS buffer containing 2% BSA to block the surface not covered by collagen; incubate at room temperature for 1 hour; prepare the target cell suspension at the same time and adjust the cell concentration to about 5×10 4 cells / mL; wash the culture plate, remove the BSA solution, and add 100 μL of cell suspension to each well; incubate at 37°C, 5% CO2 for 2-4 hours to allow cells to attach; gently wash the plate with PBS to remove unattached cells; fix the cells with 4% formaldehyde for 15 minutes; stain with 0.1% crystal violet for 20 minutes to mark attached cells; wash again with PBS to remove excess dye; add an appropriate amount of deionized water or 10% acetic acid to dissolve crystal violet and release cell staining into the solution; use an absorbance spectrophotometer to measure the absorbance OD 492nm of each well to quantify cell adhesion; compare the absorbance difference between the collagen-treated group and the control group (wells not coated or coated with other proteins); use statistical analysis (such as t-test) to evaluate the significance of adhesion effect;

[0030] Conduct stability testing;

[0031] The stability of collagen is determined using a differential scanning calorimeter (DSC) or a thermogravimetric analyzer (TGA); a small amount of sample (e.g., 5-10 mg) is placed in a DSC or TGA analyzer, the temperature is slowly increased (e.g., 10°C per minute) to a predetermined temperature (e.g., 200°C), and the heat flow and weight change data are recorded;

[0032] Conduct skin model experiments to verify the functional effects of recombinant collagen;

[0033] Conduct skin model experiments to verify the functional effects of recombinant collagen in the basement membrane zone of the skin; for example, recombinant collagen can be applied to a skin model to observe its effects on the growth, proliferation and differentiation of skin cells, as well as its repair effects on the skin barrier function.

[0034] Compared with the existing preparation method of recombinant engineering bacteria expressing type III collagen COL17A1, the present invention has the following beneficial effects:

[0035] 1. The preparation method of the recombinant engineered bacteria expressing type III collagen COL17A1 uses a yeast expression system to optimize the codons of the target sequence, and provides OptimumGeneTM codon optimization according to the codon preference of yeast strains such as Pichia pastoris, so as to further improve the yield of the recombinant protein. Select a suitable vector to achieve efficient expression in Pichia pastoris. For example, the pGAPZ A-αA vector uses the GAP promoter to stably express the recombinant protein in Pichia pastoris. It is a constitutive gene expression vector that can maintain a stable expression rate in the cell regardless of changes in the environment. No external inducer is required, which simplifies the production process, reduces production costs, and improves production efficiency.

[0036] 2. The preparation method of the recombinant engineered bacteria expressing type III collagen COL17A1 significantly improves the expression level, biological activity and storage stability of the protein through optimization strategies, making large-scale production possible and broadening the application prospects of type III collagen in the fields of medicine and cosmetology.

[0037] 3. The preparation method of the recombinant engineered bacteria expressing type III collagen COL17A1 optimizes and selects the human type III collagen sequence, uses a yeast expression system to perform codon optimization on the target sequence, improves the expression amount of the recombinant protein, and selects a suitable vector to achieve efficient expression in Pichia pastoris. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the map of recombinant type III collagen protein III-378aa;

[0039] Figure 2 The nucleic acid sequencing result of III-378aa recombinant type III collagen plasmid after codon optimization is 1;

[0040] Figure 3 The second result of nucleic acid sequencing of III-378aa recombinant type III collagen protein after codon optimization;

[0041] Figure 4 This is the result of single enzyme digestion of recombinant type III collagen protein;

[0042] Figure 5 This is the result of SDS-PAGE gel electrophoresis of recombinant type III collagen;

[0043] Figure 6 These are the results of an experiment in which recombinant type III collagen promoted the adhesion of human skin fibroblasts. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1 Design of recombinant type III collagen amino acid sequence and DNA sequence optimization

[0047] Human type III collagen amino acid sequence reference:

[0048]

[0049] Implementation plan: 378 homologous amino acids in the type III collagen sequence were selected and connected in series twice and named 378aa. The amino acid sequence is as follows:

[0050] GPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAP GQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPP GPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAP GQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPP

[0051] The corresponding nucleic acid sequence of the 378aa amino acid tandem sequence is:

[0052]

[0053] Selected codon-optimized nucleic acid sequence:

[0054]

[0055] Example 2 Expression of recombinant type III collagen amino acid sequence

[0056] Gene synthesis and vector construction:

[0057] 1. Chemically synthesize the recombinant human type III collagen gene. Add appropriate restriction enzyme sites at the 5' and 3' ends respectively;

[0058] 2. The synthesized gene was cloned into the expression vector pGAPZ A-αA and introduced into Pichia pastoris GS115;

[0059] 3. Select high-copy positive clones through resistance screening and PCR verification;

[0060] 4. Inoculate the positive clones into YPD medium and culture at 28°C and 180 rpm until OD600 = 25 ± 3;

[0061] 5. Inoculate the seed liquid into a 5L fermentation tank at a 10% inoculation rate, with a culture temperature of 28°C, pH = 5.5, dissolved oxygen controlled at 30-32.5, and ferment for 72 hours;

[0062] 6. Collect the fermentation supernatant by centrifugation and detect the protein concentration using a BCA kit;

[0063] 7. Purify recombinant collagen using steps such as NaCl precipitation, ultrafiltration membrane concentration and desalting, cation exchange chromatography and freeze drying;

[0064] 8. Verify protein purity and molecular weight by SDS-PAGE electrophoresis;

[0065] Example 3 Fermentation and expression optimization of recombinant type III collagen

[0066] 1. Inoculate the verified recombinant Pichia pastoris into YPD medium and culture at 28°C and 220rpm for 24 hours to allow the recombinant Pichia pastoris to adapt to the culture environment and grow and reproduce rapidly;

[0067] 2. Transfer the seed culture medium to a fermentation medium containing bleomycin for amplification culture, and gradually increase the cell density. By controlling the fermentation temperature, pH and dissolved oxygen level, the recombinant protein can be efficiently expressed. For example, the fermentation temperature can be controlled at 27°C-29°C, the pH of the basic fermentation medium can be controlled at 4.8-5.2, and the dissolved oxygen level can be controlled at 30-32.5 to increase the protein expression of the recombinant human type III collagen engineered bacteria.

[0068] Example 4 Protein extraction and purification of recombinant type III collagen

[0069] 1. Collect the supernatant. After the fermentation is completed, remove the cells and cell debris by centrifugation or filtration, and collect the supernatant. The supernatant contains secretory expression proteins;

[0070] 2. Use ultrafiltration to concentrate the supernatant to increase the protein concentration for subsequent purification operations;

[0071] 3. According to the molecular weight of the protein, select gel filtration chromatography to separate proteins of different molecular weights, remove impurities, and improve the purity of recombinant type III collagen;

[0072] 4. Detect the purity, molecular weight and activity of the protein by SDS-PAGE gel electrophoresis and other methods to ensure the quality of the protein. If the purity, molecular weight and activity of the protein do not meet the requirements, the purification method can be adjusted or the fermentation culture can be repeated;

[0073] 5. The purified secretory expression protein can be appropriately stored as needed, such as frozen or added with stabilizers, to maintain the stability and activity of the protein.

[0074] Example 5 Verification of the functional activity of recombinant type III collagen

[0075] 1. Determine the activity of recombinant type III collagen in an in vitro cell adhesion assay. Dissolve the purified recombinant type III collagen in PBS at concentrations of 1 mg / mL, 2 mg / mL, and 5 mg / mL. Add 100 μL of recombinant collagen solution to each well of a 96-well culture plate. Coat the plate overnight at 4°C to allow the recombinant collagen to adsorb to the well wall. On the second day, replace the recombinant collagen solution in each well with PBS buffer containing 2% BSA to block the surface not covered by recombinant collagen. Incubate at room temperature for 1 hour. At the same time, prepare the target cell suspension and adjust the cell concentration to approximately 5×10 4 cells / mL. Wash the culture plate, remove the BSA solution, and add 100 μL of the cell suspension to each well. Incubate at 37°C, 5% CO2 for 2-4 hours to allow cells to attach. Gently wash the well plate with PBS to remove unattached cells. Fix the cells with 4% formaldehyde for 15 minutes. Stain with 0.1% crystal violet for 20 minutes to mark the attached cells. Wash again with PBS to remove excess dye. Add an appropriate amount of deionized water or 10% acetic acid to dissolve the crystal violet and release the cell stain into the solution. Use an absorbance spectrophotometer to measure the absorbance OD 492nm of each well to quantify cell adhesion. Compare the absorbance differences between each recombinant collagen treatment group and the control group (wells not coated or coated with other proteins). The blank group is taken as 100%, and the adhesion effect is calculated and evaluated. The results are as follows Figure 6 .

[0076] In summary, the method for preparing a recombinant engineered bacterium expressing type III collagen provided by the present invention successfully solves the technical problem of sustained, stable and high expression and large-scale production of a specific sequence of human type III collagen in Pichia pastoris. The recombinant collagen prepared by this method has high physiological function activity and has broad application prospects in the fields of medical cosmetology, tissue engineering and wound repair.

Claims

1. A method for preparing a recombinant engineered bacterium expressing type III collagen COL17A1, comprising Pichia pastoris, characterized in that: The preparation method is as follows: S1; synthesize the optimized sequence of human type III collagen and add appropriate restriction enzyme sites at both ends of the sequence; through the optimization and selection of the human type III collagen sequence, the repetitive Gly-XY tripeptide in the basic skeleton of the natural type III collagen protein sequence is used as the basic unit to ensure that the recombinant protein contains more hydrophobic structures and select the recombinant protein sequence; at the same time, according to the codon preference of yeast strains such as Pichia pastoris, the codon of the target sequence is optimized to increase the expression of the recombinant protein; clone the synthesized gene sequence into a specific vector to ensure that the gene is expressed under the control of a suitable promoter; S2; The optimized gene sequence is cloned into the pGAPZ A-αA vector, which uses the GAP promoter to stably express the recombinant protein in Pichia pastoris. It is a constitutive gene expression vector that can maintain a stable expression rate in the cell regardless of changes in the environment; S3; transform the vector into E. coli for amplification and extract the recombinant plasmid; The transformation vector was introduced into E. coli DH5α for amplification, and the recombinant plasmid was extracted to prepare for the subsequent construction of recombinant Pichia pastoris; S4; Construction of recombinant Pichia pastoris The recombinant plasmid was transformed into the Pichia pastoris strain GS115 by electroporation, so that the recombinant plasmid was integrated into the Pichia pastoris chromosome and replicated and inherited along with the chromosome; Screening was performed on a medium containing screening antibiotics, and resistant clones were picked and verified by PCR and sequencing. Screening was performed on a medium containing screening antibiotics, and resistant clones were picked and verified by PCR and sequencing to ensure that the recombinant Pichia pastoris contained the correct human type III collagen gene sequence. S5; Fermentation culture and expression optimization The verified recombinant Pichia pastoris was inoculated into the YPD medium for cultivation; the verified recombinant Pichia pastoris was inoculated into the YPD medium and cultured at 28°C and 220rpm for 24 hours to allow the recombinant Pichia pastoris to adapt to the culture environment and grow and reproduce rapidly; the seed culture solution was transferred to a specific fermentation medium for amplification culture; The seed culture medium is transferred to a fermentation medium containing bleomycin for amplification culture, and the cell density is gradually increased; the fermentation temperature, pH and dissolved oxygen level are controlled to achieve efficient expression of the recombinant protein; for example, the fermentation temperature can be controlled at 27°C-29°C, the pH of the basic fermentation culture medium can be controlled at 4.8-5.2, and the dissolved oxygen level can be controlled at 30-32.5 to increase the protein expression of the recombinant human type III collagen engineering bacteria; S6; Protein extraction and purification includes the following steps: Step 1: Collect the supernatant and remove cells and cell debris; Step 2: After the fermentation is completed, the cells and cell debris are removed by centrifugation or filtration, and the supernatant is collected. The supernatant contains the secretory expressed protein; Step 3: Concentrate the supernatant using ultrafiltration to increase the protein concentration; Step 4: Select gel filtration chromatography to separate proteins of different molecular weights; Step 5: According to the molecular weight of the protein, gel filtration chromatography is selected to separate proteins of different molecular weights, remove impurities, and improve the purity of recombinant type III collagen; S7; Detect the purity, molecular weight and activity of the protein by SDS-PAGE gel electrophoresis and other methods to ensure the quality of the protein; if the purity, molecular weight and activity of the protein do not meet the requirements, the purification method can be adjusted or the fermentation culture can be repeated; The purified secretory expression protein can be appropriately stored as needed, such as frozen or with the addition of stabilizers, to maintain the stability and activity of the protein; S8; A method for preparing a recombinant engineered bacterium expressing type III collagen COL17A1 according to claim 1, characterized in that: the activity of the recombinant collagen in an in vitro cell adhesion experiment is determined, and the specific steps are: Dissolve recombinant human type III collagen in PBS and adjust to an appropriate concentration (e.g., 10 μg / mL); add 100 μL of collagen solution to each well of a 96-well culture plate; coat the culture plate overnight at 4°C to allow collagen to adsorb to the well wall; on the next day, replace the collagen solution in each well with PBS buffer containing 2% BSA to block the surface not covered by collagen; incubate at room temperature for 1 hour; prepare the target cell suspension at the same time and adjust the cell concentration to about 5×10 4 cells / mL; wash the culture plate, remove the BSA solution, and add 100 μL of cell suspension to each well; incubate at 37°C, 5% CO2 for 2-4 hours to allow cells to attach; gently wash the plate with PBS to remove unattached cells; fix the cells with 4% formaldehyde for 15 minutes; stain with 0.1% crystal violet for 20 minutes to mark attached cells; wash again with PBS to remove excess dye; add an appropriate amount of deionized water or 10% acetic acid to dissolve crystal violet to release cell staining into the solution; use an absorbance spectrophotometer to measure the absorbance OD 492nm of each well to quantify cell adhesion; compare the absorbance difference between the collagen-treated group and the control group (wells not coated or coated with other proteins); use statistical analysis (such as t-test) to evaluate the adhesion effect; S9: Perform stability test, the operation steps are as follows; Step 1: Use a differential scanning calorimeter (DSC) or a thermogravimetric analyzer (TGA) to determine the stability of collagen; place a small amount of sample (e.g., 5-10 mg) in the DSC or TGA analyzer, slowly increase the temperature (e.g., 10°C per minute) to a predetermined temperature (e.g., 200°C), and record the heat flow and weight change data; Step 2: Conduct skin model experiments to verify the functional effects of recombinant collagen; Step 3: Conduct a skin model experiment to verify the functional effect of recombinant collagen in the basement membrane zone of the skin; for example, recombinant collagen can be applied to a skin model to observe its effect on the growth, proliferation and differentiation of skin cells.

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