Construction of recombinant humanized A-type XVII collagen strain as well as preparation and application of recombinant humanized A-type XVII collagen strain

Abstract

The invention relates to the technical field of recombinant humanized type A (X < VII >) collagen, and particularly discloses construction of a recombinant humanized type A XVII collagen strain as well as preparation and application of a product of the recombinant humanized type A XVII collagen strain. The recombinant bacterium for efficiently synthesizing the humanized A-type and XVII-type collagen is obtained, and the humanized collagen produced by using the recombinant bacterium does not contain any gene sequence of a non-human collagen source. In addition, the collagen prepared by the method has excellent water solubility and stability as well as good hydrophilicity and moisture retention, and has a good application prospect in the fields of delayed treatment of alopecia and the like. Therefore, the preparation method of the recombinant humanized type A (X VII) collagen provided by the invention has a relatively good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of recombinant humanized type A XVII collagen technology, specifically to a recombinant humanized type A XVII collagen, its preparation method and application, as well as a recombinant bacterium capable of expressing the recombinant humanized type A XVII collagen and a method for constructing the recombinant bacterium. Background Technology

[0002] Collagen is a family of proteins widely distributed in animal connective tissues, playing a vital role in maintaining the normal physiological functions of cells, tissues, and organs, as well as in damage repair. Due to its excellent physical and biological properties, collagen has wide applications in medicine, tissue engineering, food, and cosmetics.

[0003] To address the issue of non-standard terminology in the industry, the National Medical Products Administration (NMPA) issued the "Guidelines for Naming Recombinant Collagen Biomaterials" on March 15, 2021. This guideline classifies recombinant collagen into three categories: recombinant human collagen, recombinant humanized collagen, and recombinant collagen-like proteins. Recombinant humanized collagen is further divided into type A and type B. Type A refers to materials that do not contain non-human collagen amino acid sequences; type B refers to materials that combine functional fragments of human collagen with the addition of linking amino acids, tag amino acids, and other non-human collagen amino acid sequences. Because type A contains entirely human collagen sequences, it significantly reduces the risk of allergic reactions after use.

[0004] Type XVII collagen (COL17) is a transmembrane protein in the basal layer of the epidermis. It maintains the tight junction between the epidermis and dermis, promoting epidermal turnover and stabilizing the basement membrane. It can achieve anti-aging repair through the basement membrane zone. Evidence suggests that COL17 hydrolysis can cause hair follicle miniaturization, ultimately leading to hair loss. Due to its unique physiological functions, COL17 shows great promise in delaying and treating hair loss.

[0005] Currently, collagen is mainly prepared through animal extraction. However, collagen obtained through animal extraction may exhibit batch-to-batch variations due to individual animal differences. Furthermore, COL17 is scarce in animals, leading to difficulties in extraction, low production capacity, and high prices, making it unsuitable for large-scale industrial production and hindering sustainable production. Some zoonotic viruses may also exist in animals, posing significant risks to the application of animal-derived collagen.

[0006] Therefore, there is an urgent need to provide a method for efficiently synthesizing humanized type A XVII collagen, and the type XVII collagen obtained by this method does not contain any gene sequences of non-human collagen origin, which has high industrial application prospects. Summary of the Invention

[0007] To address the problems existing in the prior art, the first objective of this invention is to provide a recombinant humanized type A XVII collagen, its preparation method, and its applications. This preparation method integrates peptides with 100% identical sequences from human type XVII collagen into a host bacterium to obtain a recombinant bacterium that efficiently synthesizes humanized type A XVII collagen. Using this recombinant bacterium to produce humanized collagen, the prepared humanized collagen contains no gene sequences of non-human collagen origin and exhibits excellent water solubility, stability, and good hydrophilicity and moisturizing properties, showing great application potential in areas such as delaying and treating hair loss. The second objective of this invention is to provide a recombinant bacterium expressing recombinant humanized type A XVII collagen and its construction method. This recombinant bacterium can efficiently synthesize humanized type A XVII collagen.

[0008] To solve the above problems, the present invention adopts the following technical solution.

[0009] A method for preparing recombinant humanized type A XVII collagen includes the following steps: S1: The target gene with the nucleotide sequence shown in SEQ ID NO. 2 is ligated into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is electroporated into the expression host, and then positive cloning is performed to obtain recombinant bacteria. S2: After methanol induction, the recombinant bacteria obtained in step S1 are inoculated into BSM medium for fermentation to obtain fermentation broth. The bacterial cells are collected by centrifugation, the cells are disrupted, and the supernatant is obtained by centrifugation. The supernatant is the crude protein solution. S3: The crude protein solution obtained in step S2 is purified and dialyzed to obtain recombinant humanized type A and type XVII collagen.

[0010] Furthermore, in step S1, the expression vector is pPICZa A, and the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0011] Furthermore, the specific operation of the positive clone in step S1 is as follows: the expression host transformed with recombinant plasmid is plated on yeast extract peptone glucose medium (YPD plate) supplemented with bleomycin and cultured for 2-3 days; in step S2, the fermentation pH is 5 and the fermentation time is 120 h.

[0012] Furthermore, the purification operation in step S2 involves sequentially passing the crude enzyme solution through a desalting column and a cation exchange chromatography column; the desalting column is a HiPre. TM 26 / 10 Desalting; the cation exchange column chromatography column is SP-FF; the dialysate composition is: 10 mM Tris and 5 mM NaCl; the dialysate pH is 8.0.

[0013] The present invention also provides a strain expressing recombinant humanized type A XVII collagen, wherein the strain is a recombinant bacterium and includes a recombinant plasmid and an expression host; the recombinant plasmid contains a target gene and an expression vector; the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, and the molecular weight of the protein product is 12 kDa.

[0014] Furthermore, the recombinant plasmid is pPICZa A-COL17, the expression host is Pichia pastoris, and the expression vector is pPICZa A.

[0015] The present invention also provides a method for constructing a strain expressing recombinant humanized type A XVII collagen. The method involves ligating the target gene into an expression vector to obtain the recombinant plasmid pPICZa A-COL17, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacteria, i.e., the target strain.

[0016] Furthermore, the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the expression vector is pPICZa A, and the expression host is Pichia pastoris, preferably Pichia pastoris X33.

[0017] Furthermore, the specific method for constructing the recombinant bacteria is as follows: S1: Simultaneously digest with EcoRI and NotI enzymes to ligate the target gene into pPICZa A to obtain the recombinant plasmid pPICZa A-COL17; S2: After transforming the pPICZa A-COL17 obtained in step S1 into E. coli for amplification, the plasmid was screened and extracted; then linearized. S3: The linearized pPICZa A-COL17 from step S2 is transferred into the expression host for positive cloning. The resulting bacterial strain is the recombinant bacteria.

[0018] Furthermore, in step S2, the culture medium used for amplification is LB plates containing zeocin (the host in step S2 is Escherichia coli, and LB plates are used for screening), and the culture is carried out overnight at 37°C; the linearization enzyme is Sac I enzyme; in step S3, the culture dish for positive clones is YPD plates with added zeocin (bleomycin), the culture temperature for positive clones is 28°C, and the cloning time is 2-3 days.

[0019] Furthermore, the Escherichia coli in step S2 is Escherichia coli DH5α.

[0020] The present invention also provides a recombinant humanized type A XVII collagen, which is prepared by the above-mentioned method for preparing recombinant humanized type A XVII collagen or expressed by the above-mentioned strain expressing recombinant humanized type A XVII collagen.

[0021] Furthermore, the amino acid sequence of the recombinant humanized type A XVII collagen is shown in SEQ ID NO. 1.

[0022] Furthermore, the nucleotide sequence of the recombinant humanized type A XVII collagen is shown in SEQ ID NO. 2.

[0023] This invention also provides the application of recombinant humanized type A XVII collagen in drugs for delaying or treating hair loss.

[0024] The present invention also provides the application of recombinant humanized type A XVII collagen in topical coatings for delaying or treating hair loss.

[0025] This invention also provides the application of recombinant humanized type A and type XVII collagen in medical excipients for delaying or treating hair loss.

[0026] The present invention also provides a polypeptide having the amino acid sequence shown in SEQ ID NO. 1.

[0027] The present invention also provides a DNA molecule encoding the aforementioned polypeptide or the aforementioned recombinant humanized type A XVII collagen, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0028] The specific contents of the present invention are as follows: In a first aspect, the present invention provides a recombinant bacterium that efficiently synthesizes humanized type A XVII collagen, including a recombinant plasmid and an expression host; the recombinant plasmid contains a target gene and an expression vector; the target gene is a gene with a 100% identical sequence to human type XVII collagen.

[0029] In order to obtain higher yields of recombinant humanized type A and type XVII collagen, the inventors analyzed the structure of COL17 through a protein database. Based on gene fragments known in the field that are related to anti-hair loss, they screened several candidate fragments related to hair follicle cell adhesion or signal transduction. These fragments were then sent to Shanghai Sangon Biotech to synthesize the encoding DNA of the candidate fragments. Subsequently, the DNA was loaded into host cells, and functional fragments that could be highly expressed in recombinant bacteria were screened.

[0030] Specifically, the amino acid sequence of the screened functional fragment is shown in SEQ ID NO. 1: GTYDATILDALPSHHVWSSTLPAGSSMGTYHNNMTTQSSSLLNTNAYSAGSVFGVPNNMASCSPTLHPGLSTSSVFGMQNNLAPSLTTLSHGTTTTSTAYGVKKNMPQSPAAVNTGVST.

[0031] Because different species and cells exhibit codon bias, in order to enable host cells to utilize their codon bias to obtain stable and highly expressed recombinant humanized type A XVII collagen, this invention utilizes the online design tool Jcat to reverse-engineer gene sequences and optimize the codons of the gene sequences of the functional fragments obtained from the above screening. In some embodiments, the nucleotide sequence of humanized type A XVII collagen is as shown in SEQ ID NO. 2: GGAACCTATGACGCTACTATCCTTGACGCCAACCTGCCATCCCACGTTTGGTCCTCTACTCTACCCGCTGGTAGTTCAATGGGTACGTATCACAACATGACCACGCAATCCTCCTCACTATTGAACACTAACGCCTATAGTGCCGGTAGTGTTTTTGGTGTACCTAATAACATGGCATCATGTTCCCCTACTCTACATCCAGGTCTGTCAACTTCTTCCTCCGTCTTTGGTATGCAG AACAATCTGGCCCCATCTCTGACCACTTTGAGTCATGGAACTACAACCACTTCTACCGCCTACGGAGTTAAGAAAAACATGCCACAATCTCCAGCTGCTG TTAATACTGGAGTTTCCACC.

[0032] In some embodiments, the expression host is Pichia pastoris.

[0033] In some embodiments, the expression host is Pichia pastoris X33.

[0034] In some embodiments, the expression vector is pPICZa A.

[0035] Secondly, the present invention provides a method for constructing the above-mentioned recombinant bacteria, which includes: ligating the target gene into an expression vector to obtain a recombinant plasmid, and then transforming the recombinant plasmid into an expression host to obtain recombinant bacteria.

[0036] In some embodiments, the recombinant plasmid is pPICZa A-COL17, and the expression host is Pichia pastoris X33.

[0037] Thirdly, the present invention provides the application of the above-mentioned recombinant bacteria in the production of humanized type A, X, and VII collagen.

[0038] Fourthly, the present invention also provides a method for producing recombinant humanized type A XVII collagen, comprising constructing the above-mentioned recombinant bacteria, then inoculating the recombinant bacteria into BSM medium for fermentation to obtain fermentation broth, collecting the bacterial cells by centrifugation, disrupting the bacterial cells, centrifuging to obtain supernatant, and the supernatant is crude protein solution.

[0039] In some embodiments, the fermentation conditions of the recombinant bacteria in BSM medium are: pH=5.0, fermentation time is 120h.

[0040] In some embodiments, the method further includes purifying and dialyzing the crude protein solution.

[0041] In some embodiments, the purification process includes passing the crude protein solution sequentially through a desalting column and a cation exchange chromatography column.

[0042] In some embodiments, the desalination column is a HiPre. TM 26 / 10Desalting.

[0043] In some embodiments, the cation exchange chromatography column is SP-FF.

[0044] In some embodiments, the dialysate used for dialysis specifically comprises 10 mM Tris and 5 mM NaCl, with a pH of 8.0.

[0045] Compared with the prior art, the advantages of this invention are: This invention discloses a method for producing and purifying recombinant humanized type A and type VII collagen. The method involves using collagen derived from human (…) Homo Sapiens A peptide segment with a 100% identical sequence to type XVII collagen was integrated into a host bacterium to obtain a highly efficient recombinant bacterium for synthesizing recombinant humanized type A XVII collagen. The recombinant humanized type A XVII collagen produced using this bacterium does not contain any gene sequences derived from non-human collagen, and this collagen exhibits excellent water solubility and stability, as well as good hydrophilicity and moisturizing properties, showing great promise for applications in delaying and treating hair loss. Therefore, the production and purification method of recombinant humanized type A XVII collagen provided by this invention has good prospects for industrial application. Attached Figure Description

[0046] Figure 1 This is a schematic diagram illustrating the construction of the recombinant plasmid pPICZa A-COL17 in Example 1; Figure 2This is a DNA identification diagram of the recombinant plasmid pPICZa A-COL17 molecule constructed in Example 1; Figure 3 SDS-PAGE image of recombinant Pichia pastoris X33 / pPICZa A-COL17 expressed protein in Example 2; Figure 4 The image shows the SDS-PAGE spectrum of recombinant humanized type A and type XVII collagen purified by ion exchange column in Example 2. Figure 5 This is an SDS-PAGE image of the recombinant humanized type A XVII collagen used in Experiment Example 1 to verify its thermal stability. Figure 6 This is a graph showing the test results for verifying the water solubility of recombinant humanized type A and type XVII collagen in Experiment Example 2; Figure 7 The figure shows the results of verifying the biocompatibility of recombinant humanized type A and type XVII collagen in Experiment Example 3. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] The *Escherichia coli* DH5α used in the following examples was purchased from Sangon Biotech (Shanghai) Co., Ltd.; *Pichia pastoris* X33 was purchased from Miaoling Plasmid Platform; and the plasmid mini-preparation kit was purchased from Aisjin Biotechnology (Hangzhou) Co., Ltd.

[0049] The culture media used in the following examples are: Yeast Extract Peptone Glucose Medium (YPD), purchased from Sangon Biotech (Shanghai) Co., Ltd.; BMGY medium, purchased from Sangon Biotech (Shanghai) Co., Ltd.; BMMY medium, purchased from Sangon Biotech (Shanghai) Co., Ltd.; and BSM medium, purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0050] The restriction endonucleases and ligases involved in the following examples were purchased from New England Bioland (Beijing) Co., Ltd.

[0051] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0052] Example 1:

[0053] This embodiment describes a method for constructing recombinant humanized type A XVII collagen recombinant bacteria, the steps of which are as follows: (1) The target gene and vector pPICZa A as shown in SEQ ID NO .2 were double-digested with EcoRI enzyme and NotI enzyme, respectively. The DNA fragments obtained by the digestion reaction were cleaned and recovered using the DNA gel recovery kit of Aisjin Biotechnology (Hangzhou) Co., Ltd., according to the instructions. Then, the double-digested vector and target gene were ligated with T4 DNA ligase overnight at 16 ºC to obtain the recombinant plasmid pPICZa A-COL17. (2) The recombinant plasmid pPICZa A-COL17 was transformed into Escherichia coli DH5α competent cells (competent cells were commercially available and purchased directly) by chemical transformation. The cells were then plated on LB agar plates containing bleomycin and incubated overnight at 37°C to obtain single colonies. Colony PCR was performed on the single colonies to verify the correctness of the recombinant plasmid and screen for positive clones, i.e., E. coli carrying the vector pPICZa A-COL17. These were then inoculated into LB liquid medium plated with bleomycin and cultured at 37°C with shaking for 12-16 hours to amplify the bacterial cells and plasmid. Plasmids were extracted using a plasmid mini-preparation kit according to the product instructions, and the extracted genome and plasmid pPICZa A-COL17 were stored at -20°C. The obtained plasmids were verified by electrophoresis (electrophoresis results are shown in Figure 1). Figure 2 As shown in the figure, the electrophoresis operation is a routine procedure and will not be described in detail; then the obtained plasmid was sent to Suzhou Genewise Biotechnology Co., Ltd. for gene sequencing verification, and the sequence alignment proved that the construction was successful.

[0054] (3) Linearize the obtained plasmid pPICZa A-COL17 using Sac I enzyme to obtain the linearized plasmid; take 500 μL of the obtained linearized plasmid into a clean 1.5 mL centrifuge tube; add 500 μL of nucleic acid extraction reagent and mix thoroughly; centrifuge at 13000 rpm for 10 min at 4℃ and take 400 μL of the supernatant into a new centrifuge tube, then add 1 mL of 4℃ pre-cooled anhydrous ethanol and 40 μL of 3M sodium acetate solution to adjust the pH to 5.2, mix thoroughly, and let stand at -20℃ for more than half an hour; centrifuge at 14000 rpm for 15 min at 4℃; then carefully remove the centrifuge tube, and fine DNA precipitation can be observed at the bottom of the tube with the naked eye; carefully remove the supernatant, and slowly add 1 mL of 75% ethanol along the tube wall to rinse; centrifuge at 13000 rpm for 5 min at 4℃; carefully remove the supernatant, open the cap and let it air dry for 3 min to allow the ethanol to evaporate completely; add 10 μL The plasmid was resuspended and dissolved in sterile ddH2O to obtain linearized pPICZa A-COL17; (4) Linearized pPICZa A-COL17 was transferred into Pichia pastoris X33 by electroporation and spread on YPD plates with 300 μg / mL zeocin. The plates were cultured at 28°C for 2-3 days, and positive clones were selected from the YPD plates with 300 μg / mL zeocin. The strains obtained from the positive clones were the recombinant bacteria.

[0055] in Figure 1 This is a schematic diagram illustrating the construction of the recombinant plasmid pPICZa A-COL17; Figure 2 This is a verification diagram showing the construction results of the recombinant plasmid pPICZa A-COL17. In the diagram, M represents the DNA marker; 1, 2, and 3 all represent the results after double enzyme digestion of the constructed target vector. Taking the electrophoresis result of the recombinant plasmid pPICZa A-COL17 in lane 2 of the diagram as an example, it can be seen that the electrophoretic band is the same size as the target gene, indicating that the COL17 gene has been correctly inserted into the multiple cloning site of the vector pPICZa A, proving the successful construction.

[0056] Example 2:

[0057] This embodiment describes a method for producing and purifying recombinant humanized type A XVII collagen, with the following steps: (2) The recombinant plasmid pPICZa A-COL17 was transformed into Escherichia coli DH5α competent cells (competent cells were commercially available and purchased directly) by chemical transformation. The cells were then plated on LB agar plates containing bleomycin and incubated overnight at 37°C to obtain single colonies. Colony PCR was performed on the single colonies to verify the correctness of the recombinant plasmid and screen for positive clones, i.e., E. coli carrying the vector pPICZa A-COL17. These were then inoculated into LB liquid medium plated with bleomycin and cultured at 37°C with shaking for 12-16 hours to amplify the bacterial cells and plasmid. Plasmids were extracted using a plasmid mini-preparation kit according to the product instructions, and the extracted genome and plasmid pPICZa A-COL17 were stored at -20°C. The obtained plasmids were verified by electrophoresis (electrophoresis results are shown in Figure 1). Figure 2 As shown in the figure, the electrophoresis operation is a routine procedure and will not be described in detail; then the obtained plasmid was sent to Suzhou Genewise Biotechnology Co., Ltd. for gene sequencing verification, and the sequence alignment proved that the construction was successful.

[0058] (3) Linearize the obtained plasmid pPICZa A-COL17 using Sac I enzyme to obtain the linearized plasmid; take 500 μL of the obtained linearized plasmid into a clean 1.5 mL centrifuge tube; add 500 μL of nucleic acid extraction reagent and mix thoroughly; centrifuge at 13000 rpm for 10 min at 4℃ and take 400 μL of the supernatant into a new centrifuge tube, then add 1 mL of 4℃ pre-cooled anhydrous ethanol and 40 μL of 3M sodium acetate solution to adjust the pH to 5.2, mix thoroughly, and let stand at -20℃ for more than half an hour; centrifuge at 14000 rpm for 15 min at 4℃; then carefully remove the centrifuge tube, and fine DNA precipitation can be observed at the bottom of the tube with the naked eye; carefully remove the supernatant, and slowly add 1 mL of 75% ethanol along the tube wall to rinse; centrifuge at 13000 rpm for 5 min at 4℃; carefully remove the supernatant, open the cap and let it air dry for 3 min to allow the ethanol to evaporate completely; add 10 μL The plasmid was resuspended and dissolved in sterile ddH2O to obtain linearized pPICZa A-COL17; (4) Linearized pPICZa A-COL17 was transferred into Pichia pastoris X33 by electroporation and spread on YPD plates with 300 μg / mL zeocin. The plates were cultured at 28°C for 2-3 days, and positive clones were selected from the YPD plates with 300 μg / mL zeocin. The strains obtained from the positive clones were the recombinant bacteria. (5) Recombinant bacteria were screened from YPD plates containing 300 μg / mL zeocin and fermented. The fermentation medium was BMGY medium. After culturing for 24 h, the medium was changed to BMMY medium and expression was induced with 1% methanol. (6) After methanol induction for 5 days, the supernatant was collected and the protein expression level was detected by SDS-PAGE. The bacteria corresponding to the thickest protein band were screened out, that is, the bacterial species with the highest expression level (numbered: A1). (7) The strain with the highest expression level selected was subjected to pilot-scale fermentation. The fermentation medium was BSM, the fermentation pH was controlled at 5.0, and the fermentation time was 120 h. (8) After fermentation, centrifuge the fermentation broth at 10,000 r·min. -1 Centrifuge for 30 min and collect the supernatant, which is the crude protein solution; (9) The purification method was ion exchange column purification. The crude protein solution was sequentially purified using HiPre. TM The purified collagen solution was obtained after passing through a 26 / 10 desalting column and an SP-FF cation exchange chromatography column. (10) The purified collagen solution was dialyzed with a dialysate containing 10 mM Tris, 5 mM NaCl and pH=8.0 to obtain recombinant humanized type A XVII collagen solution.

[0059] The obtained recombinant humanized type A XVII collagen solution was sent to Suzhou Genewise Biotech Co., Ltd. for LC-MS / MS analysis. Sequence alignment confirmed that the obtained recombinant humanized type A XVII collagen had the same amino acid sequence as SEQ ID NO. 1, and no non-human collagen gene sequence was detected.

[0060] Figure 3 The image shows an SDS-PAGE image of the recombinant Pichia pastoris X33 / pPICZa A-COL17 expressed protein. The image shows the expressed protein at 12 kDa, consistent with the expected theoretical molecular weight of the target protein COL17, indicating successful expression of the SEQ ID NO. 2 gene in Pichia pastoris X33. Furthermore, only a single band was observed in the SDS-PAGE, without any other contaminating protein bands, indicating high purity of the expressed protein. Figure 3 The blue arrow indicates the target protein expressed by strain A1.

[0061] Figure 4 This is an SDS-PAGE spectrum of recombinant humanized type A and type VII collagen purified by ion exchange column. In the spectrum, M: Marker; 1 is lane 1: fermentation supernatant; 2 is lane 2; E0: buffer A elution buffer; 3 is lane 3: 50 mM NaCl elution buffer; 4 is lane 4: 100 mM NaCl elution buffer; 5 is lane 5: 150 mM NaCl elution buffer; 6 is lane 6: 200 mM NaCl elution buffer; 7 is lane 7: 250 mM NaCl elution buffer. In the graph, the brightest band at 65 kDa is the protein marker, clearly indicating the location of the target protein. The graph shows that the target protein is mainly located in lane 5, while other proteins are found in lanes 3 and 4.

[0062] Experimental Example 1: The purified recombinant humanized type A XVII collagen solution from Example 2 was subjected to a thermal stability test, and the test method is as follows: The purified protein solution was placed in a 4°C refrigerator, and samples were taken at different times for SDS-PAGE analysis to detect any degradation of the collagen solution. The test results are as follows: Figure 5 As shown.

[0063] Figure 5 In the diagram, M represents the DNA marker, and 1 / 2 / 3 / 4 represent storage times of 0 days, 3 days, 7 days, and 14 days at 4 degrees Celsius, respectively. Figure 5 The results showed that the recombinant humanized type A XVII collagen bands were consistent in position and intensity, and no small molecular weight impurities or faded bands were observed. This indicates that the recombinant humanized type A XVII collagen was stable within 14 days at 4℃ and did not undergo degradation; that is, the recombinant humanized type A XVII collagen has good thermal stability.

[0064] Experimental Example 2: Water solubility test: The recombinant humanized type A XVII collagen solution obtained in Example 2 was frozen, and 65 mg was weighed and freeze-dried. It was then dissolved in 2 mL of pure water for a water solubility test. The test results are as follows: Figure 6 As shown.

[0065] Depend on Figure 6 It can be seen that recombinant humanized type A XVII collagen can be completely dissolved in pure water to form a transparent solution, that is, the recombinant humanized type A XVII collagen solution prepared in Example 2 has good water solubility.

[0066] Experimental Example 3: The recombinant humanized type A XVII collagen obtained in Example 2 was subjected to biocompatibility testing. The experimental steps are as follows: 1. HEK293 cells (hereinafter referred to as cells) were cultured at a rate of 8 × 10⁻⁶. 5 Inoculated at a cell / mL density in 90 mm culturedish, the cells settled to the bottom after 6 hours; 2. Tilt the culture dish and vacuum-suck away the supernatant, avoiding contact with the cell layer at the bottom of the dish; 3. Add fresh whole culture medium and wash once (gently add the medium along the wall to wash the cells and then remove it) to remove residual culture medium and metabolic waste; 4. Add trypsin solution (to cover the cell layer at the bottom of the plate), and digest at 37°C for 2 minutes to break down the cell-cell contact and allow the cells to detach from the bottom of the plate. Then add fresh whole culture medium to stop the digestion. 5. After removing the supernatant, mix the cells with fresh whole culture medium, load them into EP tubes of a centrifuge, centrifuge at 5000×g for 2 min, remove the supernatant, mix again with fresh whole culture medium, dilute initially, and count using a small flow cytometer (it needs to be shaken constantly during the process). Repeat 2-3 times, set the final cell concentration and volume on the flow cytometer, and then dilute. 6. The density is 3x10 5 Cells were injected at a rate of 100 μL per well (3 × 10⁶ cells per well). 4 (cell / mL) was seeded into a 96-well plate, incubated at 37°C for 6 hours, the supernatant was removed, and fresh complete culture medium was added; 7. The cells were divided into experimental and control groups and cultured in a 37°C incubator for 24 hours. The experimental groups were supplemented with 0.1 mg / mL and 5 mg / mL of recombinant humanized type A XVII collagen obtained in Example 2, respectively. The control group was cultured in whole culture medium only. 8. Use CCK-8 kit to check the cell viability of the experimental group and the control group respectively.

[0067] Conclusion: From Figure 7 As can be seen, the recombinant humanized type A XVII collagen solution in this study, based on HEK293 cells, did not exhibit significant cytotoxicity at concentrations of 0.1 mg / mL and 5 mg / mL, indicating good biocompatibility.

[0068] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing recombinant humanized type A XVII collagen, characterized in that: Includes the following steps: S1: The target gene with the nucleotide sequence shown in SEQ ID NO. 2 is ligated into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is electroporated into the expression host, and then positive cloning is performed to obtain recombinant bacteria. S2: After methanol induction, the recombinant bacteria obtained in step S1 are inoculated into BSM medium for fermentation to obtain fermentation broth. The bacterial cells are collected by centrifugation, the cells are disrupted, and the supernatant is obtained by centrifugation. The supernatant is the crude protein solution. S3: The crude protein solution obtained in step S2 is purified and dialyzed to obtain recombinant humanized type A and type XVII collagen.

2. The method for preparing recombinant humanized type A XVII collagen according to claim 1, characterized in that: In step S1, the expression vector is pPICZa A, and the expression host is Pichia pastoris.

3. The method for preparing recombinant humanized type A XVII collagen according to claim 1, characterized in that: The specific operation of the positive clone in step S1 is as follows: the expression host transformed with recombinant plasmid is plated on yeast extract peptone glucose medium supplemented with bleomycin and cultured for 2-3 days; in step S2, the fermentation pH is 5.0 and the fermentation time is 120 h.

4. The method for preparing recombinant humanized type A XVII collagen according to claim 1, characterized in that: The purification operation in step S3 involves passing the crude protein solution sequentially through a desalting column and a cation exchange chromatography column; the desalting column is a HiPre column. TM 26 / 10 Desalting; the cation exchange column chromatography column is SP-FF; the dialysate composition is: 10 mM Tris and 5 mM NaCl; the dialysate pH is 8.

0.

5. A bacterial strain expressing recombinant humanized type A and type XVII collagen, characterized in that: The strain is a recombinant bacterium, comprising a recombinant plasmid and an expression host; the recombinant plasmid contains a target gene and an expression vector; the nucleotide sequence of the target gene is shown in SEQ ID NO. 2, and the molecular weight of the protein product is 12 kDa.

6. The bacterial strain expressing recombinant humanized type A XVII collagen according to claim 5, characterized in that: The recombinant plasmid is pPICZa A-COL17, the expression host is Pichia pastoris, and the expression vector is pPICZaA.

7. A method for constructing a strain expressing recombinant humanized type A XVII collagen according to any one of claims 5-6, characterized in that: The construction method involves ligating the target gene into an expression vector to obtain the recombinant plasmid pPICZaA-COL17, and then transforming the recombinant plasmid into an expression host to obtain the recombinant bacteria, i.e., the target strain.

8. The method for constructing a strain expressing recombinant humanized type A XVII collagen according to claim 7, characterized in that: The nucleotide sequence of the target gene is shown in SEQ ID NO. 2, the expression vector is pPICZa A, and the expression host is Pichia pastoris.

9. A recombinant humanized type A XVII collagen, characterized in that: It is prepared by the method for preparing recombinant humanized type A XVII collagen according to any one of claims 1-4, or expressed by a strain expressing recombinant humanized type A XVII collagen according to any one of claims 5-6.

10. A recombinant humanized type A XVII collagen according to claim 9, characterized in that: The amino acid sequence of the recombinant humanized type A XVII collagen is shown in SEQ ID NO.

1.

11. The application of the recombinant humanized type A XVII collagen according to claim 10 in a drug for delaying or treating hair loss.

12. The application of the recombinant humanized type A XVII collagen as described in claim 10 in a topical coating for delaying or treating hair loss.

13. The application of the recombinant humanized type A XVII collagen according to claim 10 in a medical excipient for delaying or treating hair loss.

14. A polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

15. A DNA molecule encoding a recombinant humanized type A XVII collagen as described in claim 10 or encoding a polypeptide as described in claim 14, characterized in that: The nucleotide sequence is shown in SEQ ID NO. 2.

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