Recombinant humanized XVII type collagen CO1703 and preparation method thereof

Abstract

The invention provides a recombinant humanized XVII type collagen CO1703 and a preparation method thereof, belongs to the technical field of genetic engineering, and mainly provides the recombinant humanized XVII type collagen CO1703, the amino acid sequence of the recombinant humanized XVII type collagen is as shown in SEQ ID NO.1, and the recombinant humanized XVII type collagen CO1703 is as shown in SEQ ID NO.2. The invention also provides a preparation method of the recombinant humanized XVII type collagen CO1703. The sequence of the coding gene is as shown in SEQ ID NO. 2; the recombinant humanized XVII type collagen CO1703 provided by the invention can be effectively expressed in saccharomycetes, and has relatively strong cell adhesion activity.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a recombinant humanized type XVII collagen CO1703 and its preparation method. Background Technology

[0002] Collagen is a fibrous, large protein molecule found in all mammals. It is secreted by connective tissue cells and other cell types (such as liver, lung, spleen, and brain cells) and is widely distributed in the extracellular matrix, accounting for about one-third of total protein. Studies have identified 28 different types of collagen in the human body. All collagens contain repeating proline tripeptide Gly-XY domains, which are fundamental to the formation of the triple helix structure. Collagen's unique molecular structure gives it a strong binding affinity to certain tissues and excellent biocompatibility. Its extremely low antigenicity, biodegradability, bioabsorption, hemostatic properties, and cell growth-promoting functions have attracted increasing attention from researchers.

[0003] Type XVII collagen (COL17) is a rare type II transmembrane protein containing 1497 amino acids. Its N-terminus is located in the cytoplasm, and its C-terminus is located in the extracellular matrix. COL17 has 15 collagen domains in the extracellular matrix, and its epitopes are tightly packed within the non-collagenous 16A (NC16A) domain. Its N-terminus (N-terminus) is located intracellularly and consists of 501 amino acids, containing four tandem repeat sequences of 24–26 amino acids each. The C-terminus (C-terminus) is a relatively long extracellular polypeptide chain that attaches to the anchor fibers of the lamina lucidum of the skin's basement membrane. Heterologous expression of transmembrane proteins has many drawbacks, including low expression levels, complex structures, instability, and low solubility, significantly increasing the difficulty of transmembrane protein extraction and purification. Chinese patent document CN113185604A expressed the full-length sequence of type XVII collagen in Pichia pastoris and found no target band in the fermentation supernatant after induced expression, but a large amount of degraded collagen was found in the cell lysate with an apparent molecular weight of 120 kDa. This indicates that for transmembrane proteins, recombinant expression will fix them on the cell membrane and they can only be released into the cell after degradation. They cannot be secreted into the extracellular space, which greatly increases the difficulty of protein extraction and purification, making it difficult to achieve large-scale preparation and application.

[0004] Because collagen has a wide range of uses, it is of great significance to develop and research collagen that can be effectively expressed and has certain functions. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a recombinant humanized type XVII collagen CO1703 and its preparation method.

[0006] This invention provides a recombinant humanized type XVII collagen CO1703 that can be efficiently and stably expressed in yeast and has corresponding functional effects.

[0007] The technical solution of the present invention is as follows:

[0008] A recombinant humanized type XVII collagen CO1703, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] The gene sequence encoding the above-mentioned recombinant humanized type XVII collagen CO1703 is shown in SEQ ID NO.2.

[0010] According to a preferred embodiment of the present invention, the gene sequence encoding the above-mentioned amino acid sequence SEQ ID NO.1 is modified at both ends, wherein a DNA sequence encoding a start codon, a DNA sequence encoding a His tag, and a DNA sequence encoding a TEV protease are added to the amino terminus, and a DNA sequence encoding a stop codon is added to the carboxyl terminus, and the encoded amino acid sequence is as shown in SEQ ID NO.3.

[0011] According to a preferred embodiment of the present invention, the gene sequence encoding the above-mentioned amino acid sequence SEQ ID NO.3 includes the gene sequence shown in SEQ ID NO.4 and the gene sequence shown in SEQ ID NO.5.

[0012] A recombinant vector comprising the gene sequence SEQ ID NO.2, gene sequence SEQ ID NO.4, or gene sequence SEQ ID NO.5.

[0013] According to a preferred embodiment of the present invention, the carrier used includes any one of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pYAM75P and their modified carriers.

[0014] Further preferred, the carrier used is pPIC9K.

[0015] A recombinant bacterium comprising the gene sequence SEQ ID NO.2, gene sequence SEQ ID NO.4, or gene sequence SEQ ID NO.5.

[0016] According to a preferred embodiment of the present invention, the host used is bacteria or yeast.

[0017] Further preferred, the host used is Pichia pastoris GS115.

[0018] A species of Pichia pastoris GS115 CO1703 was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242196.

[0019] The application of the above gene sequences SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, recombinant vector, recombinant bacteria or Pichia pastoris GS115 CO1703 in the preparation of recombinant humanized type XVII collagen CO1703 with the amino acid sequence shown in SEQ ID NO.1.

[0020] The preparation method of the above-mentioned recombinant humanized type XVII collagen CO1703 includes the following steps:

[0021] (1) The gene sequence SEQ ID NO.5 was cloned into the EcoRI and NotI restriction sites of the pPIC9K vector and transformed into Escherichia coli DH5α. After DNA sequencing verification, the recombinant expression vector pPIC9K-CO1703 was successfully constructed.

[0022] (2) The recombinant expression vector pPIC9K-CO1703 obtained in step (1) was digested with sal I enzyme and the linearized product was recovered.

[0023] (3) The linearized product obtained in step (2) was transformed into Pichia pastoris GS115 competent cells, recombinant engineered bacteria were screened and verified to obtain recombinant engineered bacteria Pichia pastoris GS115 CO1703.

[0024] (4) The recombinant engineered bacteria Pichia pastoris GS115 CO1703 obtained in step (3) was fermented to prepare recombinant humanized type XVII collagen CO1703.

[0025] Alternatively, the above-mentioned Pichia pastoris GS115 CO1703 can be fermented to prepare recombinant humanized type XVII collagen CO1703.

[0026] According to a preferred embodiment of the present invention, in step (4), the recombinant engineered bacteria Pichia pastoris GS115 CO1703 is inoculated into BMGY liquid medium and cultured at 28-30℃ and 200-250rpm until the OD value is 5.0-6.0. The bacterial cells are collected by centrifugation and transferred to BMGY liquid medium. After induction with methanol at 22-28℃ and 200-250rpm for 48-120h, the supernatant is collected and purified to obtain recombinant humanized type XVII collagen CO1703.

[0027] The beneficial effects of this invention are:

[0028] 1. The recombinant humanized type XVII collagen provided by this invention can be effectively expressed in yeast and has strong cell adhesion activity.

[0029] 2. The recombinant humanized type XVII collagen provided by this invention has 100% homology with the corresponding part of the amino acid sequence of natural collagen, is non-immunogenic, non-endotoxin, and has a wide range of applications. Attached Figure Description

[0030] Figure 1 A graph showing the codon fitness index (CAI) after codon optimization for CO1703.

[0031] Figure 2 The plasmid map constructed for the pPIC9K-CO1703 vector.

[0032] Figure 3 Electrophoresis diagram for verification of recombinant Pichia pastoris genome;

[0033] In the figure: M is the marker, and 1-6 are the PCR products of recombinant engineered bacteria genome.

[0034] Figure 4 The image shows the SDS-PAGE electrophoresis results of the CO1703 fermentation supernatant.

[0035] In the diagram: M stands for Marker, and 1 stands for CO17O3 fermentation supernatant.

[0036] Figure 5 Figure 1. Western blot results of CO1703 fermentation supernatant using anti-His tag antibody;

[0037] In the diagram: M stands for Marker, and 1 stands for CO17O3 fermentation supernatant.

[0038] Figure 6 The image shows the HPLC detection results of purified CO17O3.

[0039] Figure 7The image shows the results of cell adhesion activity assay for recombinant type XVII collagen CO1703. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0041] Unless otherwise described in the embodiments, all contents are based on existing conventional techniques in the art; experimental materials and reagents not described in detail are all common commercially available products.

[0042] Example 1: Sequence selection and codon optimization of recombinant humanized type XVII collagen CO1703

[0043] (1) Based on the full-length sequence of human type XVII collagen in GenBank (GenBank accession number: Q9UMD9.3), all collagen regions of the extracellular domain were extracted and named 1703, totaling 563 amino acids, linked end to end. The specific amino acid sequence is shown in SEQ ID NO.1:

[0044] SEQ ID NO.1:

[0045] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIGPPGPPGAMGPPGPPGAPGPAGPAGLPGHQGPPGPPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGSFGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGPPGPPGPPGSIGVQGPPGPPGPPGPVGPRGPPGPPGASGDGSLGLPGPPGPPGLPGTSGIVGPPGPPGPPGIPGNVGPPGPPGPPGPRGPPGVSGALGPPGPPGPQGPPGDSGGAGSLGAGGAFGEAGPPGQPGPQGPPGISGPPGQKGEMGTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ

[0046] The DNA sequence encoding 1703 is shown in SEQ ID NO.2:

[0047]

[0048] (2) Modifications were made at both ends of 1703, with a start codon sequence, a His-tagged DNA sequence, and a TEV protease sequence added to the amino terminus, and a stop codon sequence added to the carboxyl terminus, resulting in a total of 576 amino acids. The optimized amino acid sequence of 1703 is shown in SEQ ID NO.3. Modifications were made at both ends of 1703, with an EcoR I restriction site, a start codon sequence (ATG), a His-tagged DNA sequence, and a TEV protease sequence added to the amino terminus, and a stop codon (TAA) and Not I restriction site added to the carboxyl terminus, as shown in SEQ ID NO.4. The underlined parts are restriction sites. The total length of the amino acid sequence is 1728 bp. The restriction sites and stop codons at both ends of the sequence shown in SEQ ID NO.4 were removed.

[0049] TEV protease is a commonly used enzyme for cleaving affinity tags on fusion proteins. It exhibits strong site specificity and strictly recognizes the heptapeptide sequence EXXYXQ. ↓ (G / S), the cleavage site is between glutamine and glycine / serine. After specific recognition and cleavage by the TEV protease, the His tag can be digested, thereby minimizing the tag's impact on the structure and function of the target protein.

[0050] SEQ ID NO.3

[0051] *

[0052] SEQ ID NO.4

[0053] GAATTC GCGGCCGC

[0054] Beijing Liuhe BGI Genomics Co., Ltd. was commissioned to optimize the codons of the above gene sequence for the Pichia pastoris expression system. The optimized sequence is shown in SEQ ID NO.5, with the underlined portions representing restriction enzyme sites. The codon fitness index (CAI) analysis of the optimized sequence is shown in the figure below. Figure 1 As shown.

[0055] SEQ ID NO.5

[0056] GAATTC GCG GCCGC Example 2: Synthesis of recombinant humanized type XVII collagen CO1703 gene and construction and screening of recombinant expression strains

[0057] (1) The optimized gene sequence SEQ ID NO.5 was synthesized by Beijing Liuhe Huada Genomics Co., Ltd. and cloned into the EcoRI and NotI restriction sites of the pPIC9K vector, and then transformed into E. coli DH5α. DNA sequencing confirmed the successful construction of the recombinant expression vector pPIC9K-CO1703. The plasmid map of the constructed vector is shown below. Figure 2 As shown.

[0058] (2) Extract the verified recombinant plasmid pPIC9K-CO1703, take 10ug of plasmid, digest it with sal I fast digestion enzyme (reagent purchased from TaKaRa, specific operation according to the kit instructions) at 37℃ for 10-30 minutes, and then use the PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to recover the linearized product, with an elution volume of 10ul.

[0059] (3) Prepare Pichia pastoris GS115 competent cells. Pick single colonies of Pichia pastoris GS115 (purchased from Beyotime Biotechnology Co., Ltd.) into YPD liquid medium and culture overnight at 30℃ and 220rpm. Transfer 1% by volume to YPD liquid medium (formulation: yeast extract 10g / L, peptone 20g / L, glucose 20g / L) and culture until OD600 is 1.3-1.5. Take 50ml of bacterial culture, incubate on ice for 10 minutes, centrifuge at low speed to collect the cells, add pre-cooled 1M D-sorbitol and wash twice, add 5ml of 1M D-sorbitol, mix well and dispense into 100ul / vial.

[0060] (4) Electroporation: Take one vial of Pichia pastoris GS115 competent cells, add 10 μL of the recovered product, select the PIC program on the electroporator (purchased from Bio-Rad) for one electroporation, quickly remove and add 1 ml of pre-cooled 1M D-sorbitol, mix well, and culture statically for 2-3 hours. After centrifugation at low speed, remove part of the supernatant, mix the remaining liquid by pipetting, and spread it on MD (formulation: amino-free yeast nitrogen source (YNB) 13.4 g / L, glucose 10 g / L, agar 2 g / L, biotin 4 × 10⁻⁶). -4 Incubate g / L plates at 30°C for 2-4 days until single colonies appear.

[0061] (5) G418 screening: Prepare G418 YPD solid medium with a concentration of 4 g / L, draw 1 cm*1 cm grids and number them, transfer the above single colony points to the screening medium, and incubate at 30℃ for 2-4 days until the colony size is significantly different.

[0062] (6) Larger colonies were selected for shake-flask culture. Genomes were extracted and verified by PCR. The correctly verified strains were preserved to obtain the recombinant engineered strain *Pichia pastoris* GS115 CO1703. Electrophoresis verification results are shown below. Figure 3 As shown, the gel image shows a band at around 1700 bp that corresponds to the size of the target fragment, indicating that the 1703 gene has been successfully introduced into the host Pichia pastoris GS115.

[0063] The recombinant engineered bacterium *Pichia pastoris* GS115 CO1703 has been deposited at the China Center for Type Culture Collection (CCTCC). The deposit information is as follows:

[0064] A species of Pichia pastoris GS115 CO1703 was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242196.

[0065] Example 3: Induced expression of recombinant engineered bacteria Pichia pastoris GS115 CO1703

[0066] The recombinant engineered strain Pichia pastoris GS115 CO1703 obtained in Example 2 was inoculated into YPD medium and cultured overnight at 30°C and 220 rpm. Then, it was inoculated into 50 ml of BMGY medium (10 g / L yeast extract, 20 g / L peptone, 100 mM potassium phosphate buffer pH 6.0, YNB 13.4 g / L, biotin 4 × 10⁻⁶) at a volume ratio of 2%. -4 Incubate in a solution of yeast extract (10 g / L, glycerol 10 g / L), at 30°C and 220 rpm for 24 h (OD value 5.0-6.0). After incubation, transfer the culture to a centrifuge tube, centrifuge at 5000 rpm for 5 min, wash once with sterile water, and add 5 ml of BMMY medium (10 g / L yeast extract, 20 g / L peptone, 100 mM potassium phosphate buffer pH 6.0, YNB 13.4 g / L, biotin 4 × 10⁻⁶ g / L). -4 After resuspending the culture medium (5 g / L methanol) in 100 ml BMMY medium, the culture was incubated at 25 °C and 220 rpm. Every 24 h, 1% methanol was added and samples were taken and stored at -20 °C until the induction was completed after 96 h.

[0067] Example 4: SDS-PAGE and WB analysis of recombinant humanized type XVII collagen CO1703

[0068] After centrifugation at 12000 rpm for 1 min, supernatant protein samples from different induction times in Example 3 were collected. 5× loading buffer was added at a 4:1 ratio, mixed thoroughly, and then boiled in a water bath for 5-10 min before cooling. Samples were loaded onto a 12% separating gel and electrophoresed at 120V until the bromophenol blue band reached the bottom of the gel. After staining with Coomassie Brilliant Blue G250, the gel was destained with water overnight. Images were taken to analyze the expression. Specific results are as follows: Figure 4 As shown, the SDS-PAGE electrophoresis results indicate that the supernatant protein sample has a specific band at 70 kDa, indicating that the protein has been correctly expressed.

[0069] Because the target protein 1703 carries a 6×His tag, Western blotting can be performed using the His Tag Mouse Monoclonal Antibody (purchased from Beyotime). The specific method should be followed according to the manufacturer's instructions. Specific results are as follows: Figure 5 As shown, the WB results showed a specific band at 70 kDa, indicating that the protein can bind to His antibody, carries a His tag, and is expressed normally.

[0070] Example 5: Fermentation preparation of recombinant humanized type XVII collagen CO1703

[0071] The recombinant engineered bacteria *Pichia pastoris* GS115 CO1703 obtained in Example 2 was inoculated into YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain primary seed culture. This seed culture was then transferred to YPD medium at a volume ratio of 2% to form secondary seed culture in shake flasks and cultured at 30°C and 220 rpm until the OD600 reached approximately 6.0. Fermentation was then carried out using BSM fermentation medium. The successfully activated secondary seed culture was inoculated into a fermenter at a volume ratio of 10%. The initial culture temperature was 30°C, the stirring speed was 300 rpm, the aeration was 2 vvm, and the pH was adjusted to 5.0 using 50% ammonia. Dissolved oxygen levels in the fermenter were maintained above 20% by adjusting the dissolved oxygen-dependent stirring speed. After 18-20 hours of cultivation, the glycerol in the BSM fermentation medium was depleted, and the dissolved oxygen (DO) value rose to above 80%. Glycerol feeding was then initiated to promote secondary cell growth, with dissolved oxygen feedback-coordinated stirring. When the DO value exceeded 30%, feeding was increased to 1.0 mL / min. Feeding was stopped when the wet cell weight reached 300 g / L, and the stirring speed was increased to 900 rpm to begin starvation culture. Once the DO value reached 100% and stabilized, methanol was added for induction. The induction temperature was lowered to 25°C. For the first 4 hours of induction, the methanol feed rate was 0.06 mL / min to promote cell adaptation to methanol as a carbon source. After 4 hours of induction, the feed rate was adjusted to 0.12 mL / min, using dissolved oxygen feedback feeding. Feeding was initiated when the DO value exceeded 30% and induction continued for 96 hours to obtain the fermentation broth.

[0072] The BSM fermentation medium formula is as follows: 26.7 ml / L phosphate (85%), 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate, 4.13 g / L potassium hydroxide, and 40 g / L glycerol. After sterilization at 121℃ for 20 min, and after the temperature drops to room temperature, add trace element stock solution PTM1 at 4.35 ml / L. Adjust the pH value to 5.0 with 50% (v / v) ammonia water.

[0073] The formula for the trace element mother liquor PTM1 is as follows: copper sulfate 6 g / L, sodium iodide 0.08 g / L, manganese sulfate 3 g / L, sodium molybdate 14.9 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate heptahydrate 65 g / L, biotin 0.2 g / L, and sulfuric acid 5 ml / L; it is sterilized by filtration through a 0.22 μm filter membrane and stored at 4℃ for later use.

[0074] Glycerin replenishment solution: 50% (w / w) glycerin, sterilized at 121℃ for 20 min, and after the temperature drops to room temperature, add trace element stock solution PTM1 at 12 ml / L and mix well;

[0075] Methanol feed solution: 100% methanol, add trace element mother liquor PTM1 at 12ml / L and mix well.

[0076] Example 6: Purification of recombinant humanized type XVII collagen CO1703

[0077] The recombinant protein CO1703 was purified using a nickel column.

[0078] (1) Sample preparation: The fermentation broth obtained in Example 5 was centrifuged at 8000 rpm for 10 min at 4℃, the precipitate was discarded, the supernatant was collected, and the supernatant was filtered using a 0.45 μm filter and placed on ice.

[0079] (2) Nickel column pretreatment: Place the nickel column vertically and add 5 column volumes of binding buffer (20 mmol / L NaH2PO4, 20 mmol / L Na2HPO4, 500 mmol / L NaCl, 10 mmol / L imidazole) to equilibrate the column (the binding buffer should be placed on ice).

[0080] (3) Binding of the target protein to the nickel column: Slowly add the supernatant of the bacterial culture to the column, control the flow rate at 5s / drop, collect the flow-through liquid, and pass it through the nickel column again;

[0081] (4) Nickel column washing: Add 15 column volumes of washing buffer (20 mmol / L NaH2PO4, 20 mmol / L Na2HPO4, 500 mmol / L NaCl, 20 mmol / L imidazole) to wash the column (the washing buffer should be placed on ice, this step is to elute impurities).

[0082] (5) Elution of target protein: Add 10 column volumes of elution buffer (20 mmol / L NaH2PO4, 20 mmol / L Na2HPO4, 500 mmol / L NaCl, 200 mmol / L imidazole) to elute the protein slowly, and then collect the protein.

[0083] (6) Nickel column preservation: Wash the column with 3 column volumes of binding buffer, wash the column with 5 column volumes of deionized water, and preserve the column with 3 column volumes of 20% ethanol at 4°C.

[0084] (7) Desalting and Lyophilization: The purified protein solution was replaced with pure water using a dialysis bag with a molecular weight cutoff of 20 kDa. The dialysis solution was changed every 4 hours, and dialysis was continued for 24 hours to remove salt from the purified protein solution. The resulting solution was lyophilized to obtain lyophilized powder for later use. The purity of the product was determined by HPLC. The chromatographic column was BioCore SEC-150, the mobile phase was 50 mM phosphate buffer (pH 6.8) containing 300 mM sodium chloride, isocratic elution, flow rate 0.5 ml / min, injection volume 5 μl, column temperature 30 °C, and the lyophilized powder was diluted to 1 mg / ml before injection. Detection was performed at 220 nm. The chromatogram is shown below. Figure 6 As shown, the purity is 93.256%.

[0085] Example 7: Cell adhesion activity assay of recombinant humanized type XVII collagen CO1703

[0086] The purified recombinant protein CO1703 prepared in Example 6 and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) prepared in Example 6 were dissolved and diluted to 0.1 mg / mL using PBS buffer at pH 7.0. 100 μL of the above protein solution and blank PBS solution control were added to each well of a 96-well cell culture plate and incubated at room temperature for 60 min; then 1 × 10⁻⁶ ppm was added to each well. 5 One well-cultured NIH / 3T3 cell line (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GMN6; culture and passage methods were performed according to the cell instructions) was incubated at 37°C and 5% CO2 for 60 min. Cells were washed four times with PBS, and the absorbance at OD492 nm was measured using a cell adhesion assay kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). Cell adhesion rate was calculated based on the blank control.

[0087] Cell adhesion rate = [(Experimental group cell OD - blank OD) / (Control group cell OD - blank OD)] × 100%

[0088] The results are as follows Figure 7 As shown, recombinant humanized type XVII collagen COL1703 has strong cell adhesion activity.

Claims

1. A recombinant humanized type XVII collagen CO1703, characterized in that, The amino acid sequence of the recombinant humanized type XVII collagen is shown in SEQ ID NO.

1.

2. The gene sequence encoding the recombinant humanized type XVII collagen CO1703 as described in claim 1 is shown in SEQ ID NO.

2.

3. The recombinant humanized type XVII collagen CO1703 as described in claim 1, characterized in that, The gene sequence encoding the amino acid sequence SEQ ID NO.1 was modified at both ends, with a start codon DNA sequence, a His tag DNA sequence, and a TEV protease DNA sequence added to the amino terminus, and a stop codon DNA sequence added to the carboxyl terminus, resulting in the amino acid sequence shown in SEQ ID NO.

3.

4. The gene sequence of the amino acid sequence SEQ ID NO.3 in claim 3 includes the gene sequence shown in SEQ ID NO.4 and the gene sequence shown in SEQ ID NO.

5.

5. A recombinant vector, characterized in that, Includes the gene sequence SEQ ID NO.2 of claim 2, the gene sequence SEQ ID NO.4 of claim 4, or the gene sequence SEQ ID NO.5; Preferably, the vector used includes any one of pPIC9, pPIC9K, pHIL-S1, pPICZαA, pYAM75P and their modified vectors; Preferably, the carrier used is pPIC9K.

6. A recombinant bacterium, characterized in that, Includes the gene sequence SEQ ID NO.2 of claim 2, the gene sequence SEQ ID NO.4 of claim 4, or the gene sequence SEQ ID NO.5; Preferably, the host used is bacteria or yeast; Preferably, the host used is Pichia pastoris GS115.

7. A species of Pichia pastoris GS115 CO1703 was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242196.

8. The use of the gene sequence SEQ ID NO.2 of claim 2, the gene sequence SEQ ID NO.4 of claim 4, the gene sequence SEQ ID NO.5, the recombinant vector of claim 5, the recombinant bacteria of claim 6, or the Pichia pastoris GS115 CO1703 of claim 7 in the preparation of recombinant humanized type XVII collagen CO1703 with the amino acid sequence shown in SEQ ID NO.

1.

9. The method for preparing recombinant humanized type XVII collagen CO1703 according to claim 1, comprising the following steps: (1) The gene sequence SEQ ID NO.5 was cloned into the EcoRI and NotI restriction sites of the pPIC9K vector and transformed into Escherichia coli DH5α. After DNA sequencing verification, the recombinant expression vector pPIC9K-CO1703 was successfully constructed. (2) The recombinant expression vector pPIC9K-CO1703 obtained in step (1) was digested with sal I enzyme and the linearized product was recovered. (3) The linearized product obtained in step (2) was transformed into Pichia pastoris GS115 competent cells, recombinant engineered bacteria were screened and verified to obtain recombinant engineered bacteria Pichia pastoris GS115 CO1703. (4) The recombinant engineered bacteria Pichia.pastoris GS115 CO1703 obtained in step (3) was fermented to prepare recombinant humanized type XVII collagen CO1703. Alternatively, the Pichia pastoris GS115 CO1703 described in claim 7 can be fermented to prepare recombinant humanized type XVII collagen CO1703.

10. The preparation method according to claim 9, characterized in that, In step (4), the recombinant engineered bacteria Pichia pastoris GS115 CO1703 was inoculated into BMGY liquid medium and cultured at 28-30℃ and 200-250rpm until the OD value reached 5.0-6.

0. The bacterial cells were collected by centrifugation and transferred to BMGY liquid medium. After induction with methanol at 22-28℃ and 200-250rpm for 48-120h, the supernatant was collected and purified to obtain recombinant humanized type XVII collagen CO1703.

Images