Preparation method of recombinant engineering bacterium for expressing XVII type collagen COL17A1

Abstract

The invention discloses a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, and relates to the technical field of gene engineering, in particular to a preparation method of a recombinant engineering bacterium for expressing XVII type collagen COL17A1, which comprises the following steps: gene synthesis and vector construction: synthesizing an optimized sequence SEQ ID No.1 of human source XVII type collagen COL17A1; adding proper restriction enzyme cutting sites at two ends of the sequence; the synthesized gene sequence is cloned to a pGAPZ A-alpha A vector, and the expression of the gene under the control of a GAP promoter is ensured. According to the preparation method of the recombinant engineering bacterium for expressing the XVII type collagen COL17A1, by using a GAP promoter, the constructed recombinant engineering bacterium can keep a stable expression level under different environmental conditions, the GAP promoter serving as a constitutive promoter can continuously and stably express recombinant protein in pichia pastoris, an inducer does not need to be added, the production process is simplified, and the production cost is reduced. The production cost is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for preparing a recombinant engineering bacterium expressing type XVII collagen COL17A1. Background Art

[0002] Type XVII collagen alpha 1 chain protein (COL17A1), also known as type XVII collagen, is present as a component of hemidesmosomes in the basement membrane region of the skin connecting the epidermis and dermis. It is a transmembrane protein widely expressed in the human body and has multiple biological functions such as cell adhesion, morphogenesis, neuromuscular signaling, and host defense. In addition, mutations in the gene encoding this protein may lead to diseases such as generalized atrophic benign and junctional epidermolytic bullous. The 56 exons of the COL17A1 gene encode a 1497 amino acid protein that consists of three α (α1) chains forming a homotrimer, each with a molecular weight of 180 kDa. These chains consist of a globular amino-terminal intracellular domain, a short transmembrane domain, and a flexible rod-shaped carboxyl-terminal extracellular domain. Among them, the extracellular domain consists of 15 collagen (COL1-COL15) sequences, containing repeated Gly-XY tripeptides, forming a characteristic collagen triple helix in the homotrimer. It is flanked by 16 non-collagen sequences (NC1-NC16) with multiple protease binding sites. Protease catalysis causes this region to fall off the cell surface, so it exists in the body in a transmembrane or shed form, acting as both a cell membrane surface receptor and an extracellular matrix component.

[0003] The extraction of proteins from animals usually involves adjusting the acidity and alkalinity or hydrolyzing animal tissues with proteases. However, the content of COL17A1 in humans and animals is extremely low, and it is difficult to extract. Traditional methods can only extract a small amount to meet the needs of laboratory research, and cannot be mass-produced, so the application prospects are limited. There are many factors that affect the expression of exogenous genes in host bacteria, including the characteristics of the exogenous genes themselves, the copy number of the expression cassette, the stability of the product, and post-translational modifications. Pichia pastoris is one of the commonly used cell factories for the production of recombinant products. It is widely used in industrial protein expression and metabolite production, such as feed enzymes, antibodies, vaccines, cosmetic ingredients, biofuels, and other chemicals. The engineering of protein secretion pathways and methanol metabolic pathways has been shown to be beneficial for improving the expression level of natural proteins. However, although the methanol-nutritional 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 feeding 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.

[0004] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be stably expressed in a variety of biological cells such as Pichia pastoris. Recently, the GAPDH protein encoding gene promoter (GAP) has been studied in depth and can continuously express recombinant proteins at high levels in Pichia pastoris. pGAPZA vector (2.9KB) uses the GAP promoter to stably express recombinant proteins in Pichia pastoris. It is a constitutive gene expression vector that can maintain a stable expression rate regardless of changes in the environment within the cell. Summary of the invention

[0005] The present invention provides a method for preparing a recombinant engineering bacterium expressing type XVII collagen COL17A1, which solves the problems raised by the above background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing a recombinant engineering bacterium expressing type XVII collagen COL17A1, the method comprising the following steps:

[0007] S1: Gene synthesis and vector construction

[0008] 1.1. Synthesize the optimized sequence of human type XVII collagen COL17A1 SEQ ID No.1, and add appropriate restriction enzyme sites at both ends of the sequence;

[0009] 1.2. Clone the synthesized gene sequence into the pGAPZ A-αA vector to ensure that the gene is expressed under the control of the GAP promoter;

[0010] 1.3. Transform the vector into E. coli DH5α for amplification and extract the recombinant plasmid.

[0011] S2: Construction of recombinant Pichia pastoris

[0012] 2.1. The recombinant plasmid was transformed into Pichia pastoris strain GS115 by electroporation;

[0013] 2.2. Screen on a culture medium containing screening antibiotics, pick out resistant clones and perform PCR verification and sequencing confirmation.

[0014] S3: Fermentation culture and expression optimization

[0015] 3.1. Inoculate the verified recombinant Pichia pastoris into YPD medium and culture at 28°C and 220 rpm for 24 hours;

[0016] 3.2. Transfer the seed culture medium to a fermentation medium containing bleomycin for expansion culture, and gradually increase the cell density;

[0017] 3.3. Efficient expression of recombinant protein by controlling fermentation temperature, pH and dissolved oxygen level.

[0018] S4: Protein extraction and purification

[0019] 4.1. Collect the supernatant. After the fermentation is completed, remove the cells and cell debris by centrifugation or filtration, and collect the supernatant, which contains the secretory expression protein;

[0020] 4.2. Use ultrafiltration to concentrate the supernatant to increase the protein concentration;

[0021] 4.3. According to the molecular weight of the protein, select gel filtration chromatography to separate proteins of different molecular weights;

[0022] 4.4. 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;

[0023] 4.5. 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.

[0024] S5: Functional activity verification

[0025] 5.1. Determine the activity of recombinant type XVII collagen in in vitro cell adhesion assay to verify its physiological function;

[0026] 5.2. Conduct skin model experiments to verify the functional effect of recombinant type XVII collagen in the basement membrane area of ​​the skin.

[0027] The present invention has the following beneficial effects:

[0028] 1. The preparation method of the recombinant engineered bacteria expressing type XVII collagen COL17A1 is based on ensuring the homology of the amino acid sequence: selecting an amino acid sequence with high homology to natural COL17A1 to ensure that the recombinant collagen can retain the biological function of the natural protein. Ensure protein structure and stability: Consider the influence of the amino acid sequence on the protein structure and stability, and select a sequence that can form a stable tertiary structure. Ensure high expression efficiency: Select a sequence suitable for expression in host cells to improve the expression efficiency of the recombinant protein. Ensure high functional activity: The selected sequence should be able to give the recombinant protein similar physiological functional activity to natural COL17A1, such as cell adhesion and skin repair. In order to solve the technical problems of optimizing the selection of the human type XVII collagen COL17A1 protein sequence, its sustained, stable and high expression in Pichia pastoris, and large-scale production of collagen.

[0029] 2. The preparation method of the recombinant engineering bacteria expressing type XVII collagen COL17A1 is to optimize and select the sequence of human type XVII collagen COL17A1, and use the repeated Gly-XY tripeptide in the basic skeleton of the natural COL17A1 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, select the recombinant protein sequence, use the yeast expression system to optimize the codon of the target sequence, and increase the expression of the recombinant protein. Finally, when cloning the target nucleic acid sequence, insert as few enzyme cutting sites as possible to ensure the stability of the recombinant protein, select the pGAPZ A-αA vector, and 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 XVII collagen in the fields of medicine and beauty.

[0030] 3. The preparation method of the recombinant engineered bacteria expressing type XVII collagen COL17A1, through the use of GAP promoter, the recombinant engineered bacteria constructed by the present invention can maintain a stable expression level under different environmental conditions. The GAP promoter, as a constitutive promoter, can continuously and stably express the recombinant protein in Pichia pastoris without the need for an external inducer, thereby simplifying the production process, reducing production costs, and improving production efficiency.

[0031] 4. The preparation method of the recombinant engineered bacteria expressing type XVII collagen COL17A1, the recombinant collagen undergoes a multi-step purification process, and maintains the natural tertiary structure and physiological function activity. The results of in vitro cell experiments and skin model experiments show that the recombinant collagen has significant physiological function activity in cell adhesion and skin repair, and has important application value in the fields of medical cosmetology, tissue engineering and wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The recombinant type XVII collagen protein map is COL17A1-585aa;

[0033] Figure 2 The recombinant type XVII collagen protein map is COL17A1-486aa;

[0034] Figure 3 The recombinant type XVII collagen plasmid of COL17A1-36aa is shown;

[0035] Figure 4 This is the result of nucleic acid sequencing of COL17A1-585aa plasmid after codon optimization;

[0036] Figure 5 This is the result of nucleic acid sequencing of COL17A1-486aa plasmid after codon optimization;

[0037] Figure 6 This is the result of nucleic acid sequencing of COL17A1-36aa plasmid after codon optimization;

[0038] Figure 7 This is the result of single enzyme digestion of recombinant type XVII collagen protein;

[0039] Figure 8 This is the result of SDS-PAGE gel electrophoresis of recombinant type XVII collagen;

[0040] Fig. 9 The results show that recombinant type XVII collagen promotes cell adhesion;

[0041] Fig.10 This is the thermal stability test result of recombinant type XVII collagen;

[0042] Fig.11 It is the physical property index of recombinant type XVII collagen. DETAILED DESCRIPTION

[0043] 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.

[0044] See also Figures 1 to 10 The present invention provides a technical solution: a method for preparing a recombinant engineering bacterium expressing type XVII collagen COL17A1, the method comprising the following steps:

[0045] S1: Gene synthesis and vector construction

[0046] Through the use of the GAP promoter, combined with the constructed recombinant engineered bacteria, a stable expression level can be maintained under different environmental conditions. As a constitutive promoter, the GAP promoter can continuously and stably express recombinant proteins in Pichia pastoris without the need for external inducers, thereby simplifying the production process, reducing production costs, and improving production efficiency.

[0047] 1.1. Synthesize the optimized sequence of human type XVII collagen COL17A1 (SEQ ID No. 1) and add appropriate restriction enzyme sites at both ends of the sequence;

[0048] 1.2. Clone the synthesized gene sequence into the pGAPZ A-αA vector to ensure that the gene is expressed under the control of the GAP promoter;

[0049] 1.3. Transform the vector into E. coli DH5α for amplification and extract the recombinant plasmid.

[0050] S2: Construction of recombinant Pichia pastoris

[0051] 2.1. The recombinant plasmid was transformed into Pichia pastoris strain GS115 by electroporation;

[0052] 2.2. Screen on a culture medium containing screening antibiotics, pick out resistant clones and perform PCR verification and sequencing confirmation.

[0053] S3: Fermentation culture and expression optimization

[0054] 3.1. Inoculate the verified recombinant Pichia pastoris into YPD medium and culture at 28°C and 220 rpm for 24 hours;

[0055] 3.2. Transfer the seed culture medium to a fermentation medium containing bleomycin for expansion culture, and gradually increase the cell density;

[0056] 3.3. Efficient expression of recombinant protein by controlling fermentation temperature, pH and dissolved oxygen level.

[0057] S4: Protein extraction and purification

[0058] Recombinant collagen has undergone a multi-step purification process and has maintained its natural tertiary structure and physiological functional activity. The results of in vitro cell experiments and skin model experiments showed that recombinant collagen has significant physiological functional activity in cell adhesion and skin repair, and has important application value in medical cosmetology, tissue engineering, and wound repair.

[0059] 4.1. Collect the supernatant. After the fermentation is completed, remove the cells and cell debris by centrifugation or filtration, and collect the supernatant, which contains the secretory expression protein;

[0060] 4.2. Use ultrafiltration to concentrate the supernatant to increase the protein concentration;

[0061] 4.3. According to the molecular weight of the protein, select gel filtration chromatography to separate proteins of different molecular weights;

[0062] 4.4. 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;

[0063] 4.5. 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.

[0064] S5: Functional activity verification

[0065] 5.1. Determine the activity of recombinant type XVII collagen in in vitro cell adhesion assay to verify its physiological function;

[0066] 5.2. Conduct skin model experiments to verify the functional effect of recombinant type XVII collagen in the basement membrane area of ​​the skin.

[0067] Example 1 Design of recombinant type XVII collagen amino acid sequence and DNA sequence optimization Human type XVII collagen COL17A1 amino acid sequence reference:

[0068]

[0069] The basic skeleton of the natural COL17A1 protein sequence contains repeated Gly-XY tripeptides as basic units, and these tripeptides form a characteristic collagen triple helix structure in the homotrimer. This triple helix structure has high stability. The present invention selects part of the natural collagen sequence rich in the basic unit of Gly-XY tripeptide, and at the same time ensures that the recombinant collagen contains more hydrophobic structures as the basic principle, as the target recombinant collagen sequence, and the selected protein sequence is shown below.

[0070] Scheme 1: The sequence of 585 homologous amino acids is named 585aa, and the amino acid sequence is as follows:

[0071] GEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKP GLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGLPGHQEVLNLQGPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPG PRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPSGPSEGGSSSTMYVSGPPGPPGPPGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGVQGPPGPPGPPGPVTTITGETFDYSELASHVVSYLRT SGYGVSLFSSSISSEDILAVLQRDDVRQYLRQYLMGPRGPPGPPGASGDGSLLSLDYAELSSRILSYMSSSGISIGLPGPPGPPGLPGTSYEELLSLLRGSEFRGIVGPPGPPGPPGIPGNVWSSISVEDLSSYLHTAGLSFIPGPP

[0072] The 585aa amino acid sequence corresponds to the nucleic acid sequence:

[0073]

[0074] Selected codon-optimized nucleic acid sequence:

[0075]

[0076] Solution 2: Select 486 homologous amino acids in the recombinant collagen sequence and name it 486aa. The amino acid sequence is as follows:

[0077] GPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGLPGHQEVLNLQGPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSFLSNSET FLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPSGPSEGGSSSTMYVSGPPGPPGPPGPPGSISSSGQEIQQYISEYMQS DSIRSYLSGVQGPPGPPGPPGPVTTITGETFDYSELASHVVSYLRTSGYGVSLFSSSISSEDILAVLQRDDVRQYLRQYLMMGPRGPPGPPGASGDGSLLSLDYAELSSRILSYMSSSGISI GLPGPPGPPGLPGTSYEELLSLLRGSEFRGIVGPPGPPGPPGIPGNVWSSISVEDLSSYLHTAGLSFIPGPPGPPGPPGPRGPPGVSGALATYAAENSDSFRSELISYLTSPDVRSFIVGPP

[0078] The 486aa amino acid sequence corresponds to the nucleic acid sequence:

[0079]

[0080] Selected codon-optimized nucleic acid sequence:

[0081]

[0082] Solution 3: Select 36 homologous amino acids in the recombinant collagen sequence and connect them 15 times in series, named 36aa. The amino acid sequence is as follows:

[0083] GMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQ GLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKP GLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPP GPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRP

[0084] The corresponding nucleic acid sequence of 36aa amino acid tandem 15 times is:

[0085]

[0086] Selected codon-optimized nucleic acid sequence:

[0087]

[0088] Example 2 Expression of recombinant type XVII collagen amino acid sequence

[0089] Gene synthesis and vector construction:

[0090] 1. Chemically synthesize recombinant human type XVII collagen gene. Add KpnI and NotI recognition sites at the 5' and 3' ends respectively, and add a signal peptide recognition site at the 5' end;

[0091] 2. The synthesized gene was cloned into the expression vector pGAPZ A-αA and introduced into Pichia pastoris GS115 by electroporation;

[0092] 3. Select high-copy positive clones through resistance screening and PCR verification;

[0093] 4. Inoculate the positive clones into YPD medium and culture at 28°C and 180 rpm until OD600 = 25 ± 3;

[0094] 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;

[0095] 6. Collect the fermentation supernatant by centrifugation and detect the protein concentration using a BCA kit;

[0096] 7. Purify recombinant type XVII collagen using steps such as NaCl precipitation, ultrafiltration membrane concentration and desalting, cation exchange chromatography and freeze drying;

[0097] 8. Verify protein purity and molecular weight by SDS-PAGE electrophoresis.

[0098] Example 3 Physical property determination test of recombinant type XVII collagen:

[0099] 1. Stability analysis

[0100] The stability of recombinant type XVII 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 analyzer, slowly heated to a predetermined temperature of 105°C, and the heat flow and weight change data are recorded.

[0101] 2. Molecular weight determination

[0102] The molecular weight of recombinant collagen was determined by mass spectrometry.

[0103] Example 4 In vitro cell adhesion experiment of recombinant type XVII collagen

[0104] 1. Dissolve recombinant type XVII collagen in PBS and adjust the concentration to 1 mg / mL. Add 100 μL of collagen solution to each well of a 96-well culture plate. Coat the plate overnight at 4°C to allow collagen to adsorb to the well wall;

[0105] 2. On the second 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;

[0106] 3. Meanwhile, prepare the target cell suspension and adjust the cell concentration to about 5×104 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% CO for 2-4 hours to allow cells to attach.

[0107] 4. Gently wash the plate with PBS to remove unattached cells. Fix the cells with 4% formaldehyde for 15 minutes;

[0108] 5. Stain with 0.1% crystal violet for 20 minutes to mark attached cells. Wash again with PBS to remove excess dye. Add appropriate amount of deionized water or 10% acetic acid to dissolve crystal violet and release cell stain into the solution. Use an absorbance spectrophotometer to measure the absorbance of each well at OD 492nm to quantify cell adhesion.

[0109] 6. 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 the adhesion effect.

[0110] In summary, the preparation method of the recombinant engineered bacteria expressing type XVII collagen COL17A1 provided by the present invention successfully solves the technical problems of optimizing the selection of the human type XVII collagen COL17A1 protein sequence, as well as its sustained, stable and high expression in Pichia pastoris and large-scale production of collagen. The recombinant collagen prepared by the method has high physiological function activity and has broad application prospects in the fields of medical cosmetology, tissue engineering and wound repair.

[0111] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific position, be constructed and operated in a specific position, and therefore cannot be understood as limiting the present invention; the terms "first", "second", and "third" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance. In addition, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or also includes elements inherent to such process, method, article, or apparatus.

[0112] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a recombinant engineered bacterium expressing type XVII collagen COL17A1, characterized in that the method comprises the following steps: S1: Gene synthesis and vector construction 1.

1. Synthesize the optimized sequence of human type XVII collagen COL17A1 SEQ ID No.1, and add appropriate restriction enzyme sites at both ends of the sequence; 1.

2. Clone the synthesized gene sequence into the pGAPZ A-αA vector to ensure that the gene is expressed under the control of the GAP promoter; 1.

3. Transform the vector into E. coli DH5α for amplification and extract the recombinant plasmid. S2: Construction of recombinant Pichia pastoris 2.

1. The recombinant plasmid was transformed into Pichia pastoris strain GS115 by electroporation; 2.

2. Screen on a culture medium containing screening antibiotics, pick out resistant clones and perform PCR verification and sequencing confirmation. S3: Fermentation culture and expression optimization 3.

1. Inoculate the verified recombinant Pichia pastoris into YPD medium and culture at 28°C and 220 rpm for 24 hours; 3.

2. Transfer the seed culture medium to a fermentation medium containing bleomycin for expansion culture, and gradually increase the cell density; 3.

3. Efficient expression of recombinant protein by controlling fermentation temperature, pH and dissolved oxygen level. S4: Protein extraction and purification 4.

1. Collect the supernatant. After the fermentation is completed, remove the cells and cell debris by centrifugation or filtration, and collect the supernatant, which contains the secretory expression protein; 4.

2. Use ultrafiltration to concentrate the supernatant to increase the protein concentration; 4.

3. According to the molecular weight of the protein, select gel filtration chromatography to separate proteins of different molecular weights; 4.

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; 4.

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. S5: Functional activity verification 5.

1. Determine the activity of recombinant type XVII collagen in in vitro cell adhesion assay to verify its physiological function; 5.

2. Conduct skin model experiments to verify the functional effect of recombinant type XVII collagen in the basement membrane area of ​​the skin.

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