A strain expressing recombinant human type III collagen with full chain length and triple helical structure, and its preparation method and application

Abstract

The present invention relates to a recombinant type III human collagen expression strain with a full chain length and a triple helix structure, a preparation method thereof, and an application thereof, and belongs to the technical field of genetically engineered bacteria. The recombinant Pichia pastoris genetically engineered bacteria of the present invention expresses a recombinant type III human collagen with a full chain length and a triple helix structure, and the ku70 gene of the recombinant Pichia pastoris genetically engineered bacteria is knocked out and a recombinant 4-proline hydroxylase encoding gene and a FKBP22 encoding gene are knocked in. The recombinant Pichia pastoris genetically engineered bacteria provided by the present invention can obtain a high expression level of recombinant full-chain length triple helix recombinant type III human collagen through high-density fermentation, does not contain heterologous sequences, and has a high hydroxyproline content and thermal stability, which is close to natural human collagen, and has a stable triple helix structure and high thermal stability, which can meet the application in medical health and medical beauty fields such as medical beauty filling and tissue repair.

Description

Technical Field

[0001] The present invention relates to the technical field of genetically engineered bacteria, and in particular to a recombinant human type III collagen expression strain with a full-chain length and a triple-helix structure, and a preparation method and application thereof. Background Art

[0002] In recent years, market demand for collagen products has surged and seen robust growth, with recombinant collagen materials accounting for a growing share of the market annually. Human type III collagen, with its exceptional performance in skin rejuvenation, supporting skin elasticity, and repairing its functions, has become a mainstream choice for industrial research and end-use applications, particularly in the beauty, skincare, and healthcare sectors.

[0003] The core competitiveness of natural collagen lies in its triple helix structure (formed by three α chains), which imparts mechanical stability and biological activity. Collagen with a triple helix structure not only offers significant bioactivity advantages but also exhibits significantly improved resistance to degradation. Furthermore, based on this basic triple helix structure, it can be assembled into higher-dimensional structures (collagen fibrils), further enhancing the product's support and thus more effectively resisting degradation. It can be said that the triple helix structure is the foundation of collagen's biological activity, and only a triple helix structure can be considered authentic collagen. Furthermore, the ability of triple helix collagen molecules to assemble into supramolecular structures is fundamental to the commercial application of collagen in medical applications (such as cosmetic fillers and tissue repair). Therefore, how to achieve triple helix formation in recombinant collagen has become a focus of industry attention.

[0004] However, the natural sources of human type III collagen are extremely rare and cannot be obtained commercially on a large scale. Currently, the vast majority of human type III collagen on the market is produced through genetic recombination technology. Structurally, this type of recombinant collagen is mainly a truncated polypeptide fragment consistent with the amino acid sequence of natural human collagen, or a combination of multiple functional fragments, ranging in size from a few kDa to tens of kDa. This type of recombinant collagen product has deficiencies in the stability and molecular weight of the three-dimensional helical structure, which may affect its support effect and efficacy in clinical applications. For example, most recombinant collagen has a small molecular weight, lacks the triple helical structure of natural human collagen, and does not have complete biological activity; secondly, existing recombinant collagen products generally have challenges such as poor stability, rapid degradation in the body, short maintenance time, and lack of mechanical support, which cannot meet the needs of long-term applications such as medical aesthetic injection filling and tissue repair.

[0005] Existing recombinant technologies remain limited in achieving stable expression of full-chain triple-helical structures, facing technical bottlenecks such as low expression levels and high production costs. While Escherichia coli, yeast, insect cells, and mammalian cells, commonly used as platforms for recombinant protein expression and production, hold some promise for synthesizing full-chain triple-helical collagen, they also have significant limitations. For example, while the E. coli expression system offers a short production cycle and relatively high productivity, it lacks endogenous hydroxylation, preventing it from effectively hydroxylating proline or lysine residues on the collagen chain, thereby preventing the formation of a stable triple-helical structure. While existing eukaryotic systems (such as insect and mammalian cells) are the closest to human-derived modification and expression systems, theoretically offering the easiest way to obtain active recombinant human collagen with a complete triple-helical structure, recombinant human collagen expression often suffers from long production cycles, low yields, high costs, and difficulties in scale-up. Pichia pastoris is an efficient eukaryotic expression system with certain post-translational modification and processing capabilities compared to the prokaryotic system of Escherichia coli. Although its natural hydroxylase activity is insufficient, it can be modified through genetic engineering technology to co-express exogenous hydroxylase genes (such as human prolyl-4-hydroxylase, P4H), significantly improving the hydroxylation levels of proline and lysine and enhancing triple helix stability. Secondly, Pichia pastoris mainly uses inexpensive carbon sources such as glycerol and methanol, and its growth and culture conditions are relatively simple, with costs far lower than mammalian cell or insect cell culture systems. In addition, Pichia pastoris has a relatively mature fermentation process and has been widely used in biopharmaceuticals, facilitating rapid scale-up of production.

[0006] Patents CN118440975A, CN118834284A, CN119331079A, and CN119431554A also disclose different methods for preparing recombinant humanized type III collagen with triple-helical structures. However, the molecular weight of these recombinant collagens is much smaller than that of natural full-length collagen and their hydroxylation levels are limited, posing significant challenges to their biological functional integrity and mechanical support. Patent CN118652924A achieves the preparation of recombinant human type III collagen with a triple-helical structure by co-expressing the human type III collagen α1 chain and P4H in yeast cells, but the stability and expression level of this recombinant collagen are unknown. Summary of the Invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a strain expressing recombinant human type III collagen with full chain length and triple helical structure, as well as a preparation method and application thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a recombinant genetically engineered Pichia pastoris, wherein the recombinant genetically engineered Pichia pastoris expresses recombinant type III human collagen with a full-chain length and a triple-helical structure, and the ku70 gene of the recombinant genetically engineered Pichia pastoris is knocked out and recombinant 4-proline hydroxylase encoding genes and FKBP22 encoding genes are knocked in;

[0010] The amino acid sequence of the recombinant human type III collagen with full chain length and triple helical structure is shown in SEQ ID NO: 3;

[0011] The ku70 gene is numbered in NCBI as Gene ID: 8199462;

[0012] The recombinant 4-proline hydroxylase comprises a recombinant 4-proline hydroxylase α1 subunit and a recombinant 4-proline hydroxylase β subunit. The nucleotide sequence of the gene encoding the recombinant 4-proline hydroxylase α1 subunit is shown in SEQ ID NO: 5, and the nucleotide sequence of the gene encoding the recombinant 4-proline hydroxylase β subunit is shown in SEQ ID NO: 6.

[0013] The nucleotide sequence of the FKBP22 encoding gene is shown in SEQ ID NO: 10.

[0014] The present invention fuses a special signal peptide sequence sPC1 and a folding domain sequence Foldon to the N-terminus and C-terminus of the full-chain type III human collagen α1 chain sequence, respectively, to promote the formation of a triple helical structure.

[0015] The ku70 gene encodes a key protein in the non-homologous end joining (NHEJ) repair pathway, involved in the direct joining of broken DNA ends. This invention, through knockout of the ku70 gene, inhibits the NHEJ repair pathway, forcing DNA damage repair to rely on homologous recombination, significantly improving the accuracy and efficiency of gene editing, thereby enabling precise knockout or integration of targeted genes.

[0016] By knocking in the recombinant 4-proline hydroxylase encoding gene, hydroxylated recombinant collagen can be obtained, so that the proline of the recombinant full-chain humanized collagen is 4-hydroxyproline, and the recombinant Pichia pastoris genetically engineered bacteria has hydroxylation modification ability, which is beneficial to improving the stability of collagen during the fermentation process.

[0017] FKBP22 is a peptidylproline cis-trans isomerase that catalyzes the folding of type III collagen and acts as a molecular chaperone for type III collagen.

[0018] The present invention transforms the recombinant type III human collagen encoding gene with full chain length and triple helix structure into the modified chassis strain to construct an engineering strain that co-expresses molecular chaperone, hydroxylase and recombinant full chain length collagen.

[0019] Furthermore, the FKBP22 encoding gene is knocked into the lnt39 gene site of the Pichia pastoris chassis strain, the nucleotide sequence of the lnt39 gene is shown in SEQ ID NO: 40, and the Pichia pastoris chassis strain includes at least one of Pichia pastoris X33, Pichia pastoris GS115, Pichia pastoris KM71H and Pichia pastoris SMD1168.

[0020] In a specific embodiment of the present invention, experiments were conducted using Pichia pastoris X33 as a Pichia pastoris chassis strain.

[0021] Most preferably, the recombinant Pichia pastoris genetically engineered bacterium is Pichia pastoris komagataellaphaffiiXF / 3A1ncF-68#, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 21, 2025, with the deposit number: GDMCC No: 66181.

[0022] In a second aspect, the present invention provides the use of the recombinant Pichia pastoris genetically engineered bacteria in the preparation of collagen.

[0023] In a third aspect, the present invention provides a method for preparing the recombinant Pichia pastoris genetically engineered bacteria, comprising the following steps:

[0024] S1: Knockout the ku70 gene of Pichia pastoris X33 to obtain the recombinant Pichia pastoris genetically engineered strain X33△ku70;

[0025] S2: knocking in the FKBP22 encoding gene into the recombinant Pichia genetically engineered bacteria X33Δku70 described in step S1 to obtain the recombinant Pichia genetically engineered bacteria X33Δku70:FKBP22;

[0026] S3: knocking in the recombinant 4-proline hydroxylase encoding gene into the recombinant Pichia genetically engineered bacteria X33Δku70:FKBP22 in step S2 to obtain the recombinant Pichia genetically engineered bacteria X33Δku70:FKBP22 / α1β;

[0027] S4: Knock-in the recombinant type III human collagen encoding gene with full chain length and triple helical structure into the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β described in step S2 to obtain the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β-3A1ncF. The nucleotide sequence of the recombinant type III human collagen encoding gene with full chain length and triple helical structure is shown in SEQ ID NO: 4.

[0028] Furthermore, in step S1, the ku70 gene in Pichia pastoris X33 is knocked out using CRISPR / Cas9 gene editing technology. Pichia pastoris X33, a CRISPR / Cas9 gene editing vector containing the sgRNA1 target sequence, and a △ku70-Donor fragment fused with the upstream and downstream homology arms of the ku70 gene are mixed and transformed by electroporation to obtain a recombinant Pichia pastoris genetically engineered strain X33△ku70;

[0029] The nucleotide sequence of the sgRNA2 target sequence is shown in SEQ ID NO: 29;

[0030] The nucleotide sequence of the Δku70-Donor fragment is shown in SEQ ID NO: 39.

[0031] Furthermore, during the electroporation transformation process, the ratio of Pichia pastoris X33, CRISPR / Cas9 gene editing vector containing sgRNA1 target sequence, and △ku70-Donor fragment was 50~100μL: 2~4μg: 2~4μg.

[0032] In a specific embodiment of the present invention, the ratio of Pichia pastoris X33 to the CRISPR / Cas9 gene editing vector containing the sgRNA1 target sequence and the Δku70-Donor fragment is 50 μL: 2 μg: 2 μg, and the OD of Pichia pastoris X33 is 0. 600 It is 40~100.

[0033] Furthermore, during the electroporation transformation process, the voltage is 1.2-1.8 kV, and the electroporation time is 4-6 mSec; the preferred voltage is 1.5 kV.

[0034] Furthermore, in step S2, the recombinant Pichia pastoris genetic engineering bacteria X33△ku70 described in step S1, the CRISPR / Cas9 gene editing vector containing the sgRNA2 target sequence, and the △lnt39-P containing the upstream and downstream homology arm fragments of the lnt39 gene and the expression cassette fragment of the FKBP22 gene are added. AOX1 -FKBP22-Donor fragments were mixed and transformed by electroporation to obtain recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22;

[0035] The nucleotide sequence of the sgRNA2 target sequence is shown in SEQ ID NO: 41;

[0036] The Δlnt39-P AOX1 The nucleotide sequence of the -FKBP22-Donor fragment is shown in SEQ ID NO: 54.

[0037] Furthermore, during the electroporation transformation process, the recombinant Pichia pastoris genetic engineering bacteria X33△ku70 was combined with the CRISPR / Cas9 gene editing vector containing the sgRNA2 target sequence and △lnt39-P AOX1 The ratio of -FKBP22-Donor fragment is 50~100μL: 2~4μg: 2~4μg.

[0038] In a specific embodiment of the present invention, the recombinant Pichia pastoris genetically engineered bacteria X33△ku70 is combined with a CRISPR / Cas9 gene editing vector containing an sgRNA2 target sequence and a △lnt39-P AOX1 The ratio of -FKBP22-Donor fragment was 50 μL: 2 μg: 2 μg, and the OD of recombinant Pichia pastoris genetically engineered bacteria X33△ku70 was 0. 600 It is 40~100.

[0039] Furthermore, in step S3, the recombinant Pichia pastoris genetically engineered bacteria X33Δku70:FKBP22 described in step S2 and the recombinant vector containing the recombinant 4-proline hydroxylase encoding gene are mixed and transformed by electroporation to obtain the recombinant Pichia pastoris genetically engineered bacteria X33Δku70:FKBP22 / α1β.

[0040] Furthermore, the recombinant vector containing the recombinant 4-proline hydroxylase encoding gene is a pPIC9K-P4Hα1β recombinant vector containing the recombinant 4-proline hydroxylase α1 subunit encoding gene and a P4Hβ subunit expression cassette; the nucleotide sequence of the P4Hβ subunit expression cassette is shown in SEQ ID NO: 7; and the backbone vector of the pPIC9K-P4Hα1β recombinant vector is a pPIC9K vector.

[0041] Furthermore, the pPIC9K plasmid was double-digested with BamH I and EcoR I enzymes to obtain a linearized pPIC9K vector; the recombinant 4-proline hydroxylase α1 subunit encoding gene was connected between the BamH I and EcoR I restriction sites of the linearized pPIC9K vector to obtain a pPIC9K-P4Hα1 recombinant vector; the pPIC9K-P4Hα1 recombinant vector was digested with AatII endonuclease to obtain a linearized pPIC9K-P4Hα1 recombinant vector; the P4Hβ subunit expression cassette was connected to the linearized pPIC9K-P4Hα1 recombinant vector to obtain a pPIC9K-P4Hα1β recombinant vector.

[0042] Furthermore, during the electroporation transformation process, the ratio of the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 to the recombinant vector containing the recombinant 4-proline hydroxylase encoding gene was 50~100 μL: 2~4 μg.

[0043] In a specific embodiment of the present invention, the ratio of the recombinant Pichia pastoris genetic engineering strain X33△ku70:FKBP22 to the recombinant vector containing the recombinant 4-proline hydroxylase encoding gene is 100 μL: 4 μg, and the OD of the recombinant Pichia pastoris genetic engineering strain X33△ku70:FKBP22 is 0. 600 It is 40~100.

[0044] Furthermore, in step S4, the recombinant Pichia pastoris genetically engineered bacteria X33Δku70:FKBP22 / α1β described in step S3 is mixed with a recombinant vector containing a recombinant type III collagen gene encoding a full-chain-length triple-helical structure, and transformed by electroporation to obtain the recombinant Pichia pastoris genetically engineered bacteria X33Δku70:FKBP22 / α1β-3A1ncF. The backbone vector of the recombinant vector containing a recombinant type III collagen gene encoding a full-chain-length triple-helical structure is a pPICZαA vector.

[0045] Furthermore, during the electroporation transformation process, the ratio of the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β to the recombinant vector containing the recombinant type III human collagen encoding gene with full chain length and triple helix structure was 50~100 μL: 2~4 μg.

[0046] In a specific embodiment of the present invention, the ratio of the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β to the recombinant type III human collagen encoding gene with full chain length and triple helical structure is 100 μL: 4 μg, and the OD of the recombinant Pichia pastoris genetically engineered bacteria X33△ku70:FKBP22 / α1β is 0. 600 It is 40~100.

[0047] In a fourth aspect, the present invention provides a method for preparing collagen, comprising culturing the recombinant Pichia pastoris genetically engineered bacteria to express collagen, wherein the collagen is a recombinant type III human collagen having a full chain length and a triple helical structure, and the amino acid sequence of the recombinant type III human collagen having a full chain length and a triple helical structure is shown in SEQ ID NO: 3. Due to modification by 4-proline hydroxylase, the proline in the recombinant full-chain humanized collagen is 4-hydroxyproline.

[0048] Furthermore, the method for culturing the recombinant Pichia pastoris genetically engineered bacteria to express collagen comprises the following steps:

[0049] S41: Cultivate the recombinant Pichia pastoris genetically engineered bacteria using YPD medium until the OD value of the recombinant Pichia pastoris genetically engineered bacteria is 600 When the temperature reaches 3~5, the first-grade seed solution mother liquor is obtained;

[0050] S42: Inoculate the primary seed solution mother solution in step S41 into BMGY culture medium and culture until the OD value of the recombinant Pichia pastoris genetically engineered bacteria reaches 600 When the temperature reaches 4-6, the secondary seed solution is obtained;

[0051] S43: inoculating the secondary seed solution described in step S42 into an inorganic salt culture medium and culturing until the dissolved oxygen content of the culture system reaches 70% or more, adding feed solution 1 for fed-batch culture until the wet weight of the bacteria reaches 300-350 g / L, wherein the feed solution 1 contains PTM1 and glycerol;

[0052] S44: After the culture in step S43, the dissolved oxygen content of the culture system reaches more than 80%, and feeding solution 2 and feeding solution 3 are added for fed-batch culture. The feeding solution 2 contains PTM1 and methanol; the feeding solution 3 contains casein peptone, L-ascorbic acid and α-ketoglutaric acid.

[0053] Furthermore, in step S42, the inoculation amount of the first-stage seed solution mother solution is 1-10%, and the culture conditions are: 25-30°C, 200-300 rpm, and 16-20 hours. Preferably, the inoculation amount of the first-stage seed solution mother solution is 1%, and the culture conditions are: 29°C, 250 rpm, and 16-20 hours.

[0054] Furthermore, in step S43, the inoculum size of the secondary seed solution is 2-10%, and the culture conditions are: 23-30°C, pH 5-6, 300-1000 rpm, aeration 0.5-2 vvm, and dissolved oxygen 20%-30%. Preferably, the inoculum size of the secondary seed solution is 10%, 28°C, pH 5.5, 300 rpm, aeration 1 vvm, and dissolved oxygen 20%-30%.

[0055] Furthermore, in step S43, the inorganic salt culture medium contains KH2PO4, K2HPO4, NH4H2PO4, CaSO4, MgSO4·7H2O, glycerol and PTM1 (trace element complex mother solution).

[0056] Furthermore, in step S43, the inorganic salt culture medium contains 17-22 g / L KH2PO4, 5-10 g / L K2HPO4, 5-10 g / L NH4H2PO4, 1-5 g / L CaSO4, 5-10 g / L MgSO4·7H2O, 10-40 g / L glycerol, and 4-12 mL / L PTM1. Preferably, the inorganic salt culture medium contains 20 g / L KH2PO4, 6.85 g / L K2HPO4, 6.9 g / L NH4H2PO4, 1.2 g / L CaSO4, 12.5 g / L MgSO4·7H2O, 40 g / L glycerol, and 4.5 mL / L PTM1.

[0057] Furthermore, in step S43, the PTM1 concentration in the feed solution 1 is 4-12 mL / L, and the glycerol concentration is 10-50% v / v. Preferably, the PTM1 concentration in the feed solution 1 is 12 mL / L, and the glycerol concentration is 50% v / v.

[0058] Furthermore, in step S43, during the fed-batch culture, the feeding rate is 6.5-10.2 g / min, the pH is maintained at 5-6, and the dissolved oxygen is 20%-30%. Preferably, the feeding rate is 8.4 g / min, the pH is maintained at 5, and the dissolved oxygen is 30%.

[0059] Furthermore, in step S44, the feeding solution 3 is added at a constant rate of 1 to 12 mL / h throughout the culturing process in step S43. Preferably, the feeding solution 3 is added at a constant rate of 10 mL / h throughout the culturing process in step S43.

[0060] Furthermore, in step S44, the concentration of PTM1 in feed solution 2 is 5-20 mL / L, and feed solution 3 contains 10-30 g / L casein peptone, 2-20 g / L L-ascorbic acid, and 1-5 g / L α-ketoglutaric acid. Preferably, the concentration of PTM1 in feed solution 2 is 12 mL / L, and feed solution 3 contains 20 g / L casein peptone, 2 g / L L-ascorbic acid, and 1 g / L α-ketoglutaric acid.

[0061] Furthermore, in step S44, the initial feed rate of feeding solution 2 is 1-4 g / h, the feeding rate is adjusted to 2-8 g / h after 1-2 hours of fed-batch culture, the feeding rate is adjusted to 3-9 g / h after 1-2 hours of fed-batch culture, and the culture is continued for 60-72 hours. Preferably, the initial feed rate of feeding solution 2 is 3.6 g / h, the feeding rate is adjusted to 7.2 g / h after 1 hour of fed-batch culture, the feeding rate is adjusted to 8.4 g / h after 1 hour of fed-batch culture, and the culture is continued for 60-72 hours.

[0062] Furthermore, the collagen is separated and purified, including lysing the recombinant Pichia pastoris genetically engineered bacteria cultured in step S44, acidic resolubilization, enzymatic hydrolysis, protein chromatography and ultrafiltration desalination.

[0063] Furthermore, the lysis includes the following steps: centrifuging the fermentation broth after culturing in step S44, collecting the bacterial sludge, mixing the bacterial sludge with a homogenized lysis solution, and fully resuspending the sludge. The ratio of bacterial sludge to homogenized lysis solution is 10~30g:100mL, 800~1000bar homogenization cycle is broken 2~5 times, centrifuged, and the precipitate is collected, which is the precipitate after lysis; the homogenized lysis solution includes Tris / HCl buffer, NaCl and glycerol.

[0064] Furthermore, the centrifugation conditions of the fermentation liquid after the culture in step S44 are: below 10° C., 5000-8000 rpm, 5-10 min. Preferably, the centrifugation conditions of the fermentation liquid after the culture in step S44 are: 8000 rpm, 10 min.

[0065] Preferably, the ratio of bacterial sludge to homogenized lysis solution is 20g:100mL.

[0066] Preferably, the homogenization cycle at 900 bar is repeated for 3 times.

[0067] Furthermore, the centrifugation conditions after the homogenization cycle are: below 10°C, 8000-12000 rpm, 5-30 min. Preferably, the centrifugation conditions after the homogenization cycle are: 4°C, 10000 rpm, 30 min.

[0068] Furthermore, the homogenized lysate comprises 10-50 mM Tris / HCl buffer, 0.1-0.3 M NaCl and 5-20% v / v glycerol, with a pH of 7. Preferably, the homogenized lysate comprises 50 mM Tris / HCl buffer, 0.2 M NaCl and 15% v / v glycerol.

[0069] Furthermore, the acidic reconstitution comprises the following steps: mixing the lysed precipitate and the acidic reconstitution solution, and fully resuspending them to obtain an acidic resuspension, wherein the ratio of the precipitate to the acidic reconstitution solution is 0.1-1 g: 10-50 mL, and the acidic reconstitution solution contains 0.1-0.5 M acetic acid and 0.1-0.3 M NaCl.

[0070] Preferably, the ratio of precipitate to acidic reconstitution solution is 1 g:50 mL, and the acidic reconstitution solution contains 0.1 M acetic acid and 0.15 M NaCl.

[0071] Furthermore, the enzymatic hydrolysis comprises the following steps: mixing the acidic resuspension with pepsin, wherein the final concentration of the pepsin is 0.1-1 g / 100 mL, digesting at below 10° C. for 12-16 hours to obtain an enzymatic hydrolyzate.

[0072] Preferably, the final concentration of pepsin is 0.2 g / 100 mL, and the enzymatic solution is digested at 4°C.

[0073] Furthermore, the protein chromatography includes the following steps: centrifuging the enzymatic hydrolysate at room temperature, collecting the supernatant, adjusting the pH of the supernatant to 3-5, adding NaCl to a final concentration of 0.35-0.5 M, and obtaining a chromatography loading clear solution; flushing and balancing the chromatography column with buffer A, eluting with eluent B, and collecting the eluate; the buffer A contains KH2PO4 and NaCl, and the pH is adjusted to 3-5 with acetic acid; the eluent B contains KH2PO4, and the pH is adjusted to 3-5 with acetic acid; the elution volume is 10-20CV.

[0074] Furthermore, the enzymatic hydrolysate is centrifuged at room temperature at 3000-8000 rpm for 5-10 min, preferably 5000 rpm for 5 min.

[0075] Furthermore, the pH of the supernatant was adjusted with 0.1 M KOH, the buffer A contained 10-50 mM KH2PO4 and 0.5-1 M NaCl; and the eluent B contained 10-50 mM KH2PO4.

[0076] Preferably, the supernatant is adjusted to pH 5 with 0.1 M KOH, and NaCl is added to a final concentration of 0.5 M to obtain a chromatographic loading clear solution; the chromatography column is rinsed and balanced with buffer A, eluted with eluent B, and the eluate is collected; the buffer A contains 20 mM KH2PO4 and 0.5 M NaCl, and the pH is adjusted to 5 with acetic acid, and the eluent B contains 20 mM KH2PO4, and the pH is adjusted to 5 with acetic acid, and the elution volume is 20CV.

[0077] Furthermore, the molecular weight of the ultrafiltration membrane used for ultrafiltration desalination is 30-100 KDa, preferably 50 KDa.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] (1) The present invention fuses a special signal peptide sequence sPC1 and a folding domain sequence Foldon at the N-terminus and C-terminus of the full-chain length type III human collagen α1 chain sequence, respectively, to promote the formation of a triple helical structure, thereby obtaining a recombinant full-chain length triple helical recombinant type III human collagen and its encoding gene fragment.

[0080] (2) The present invention has modified the chassis host bacteria, including introducing the key molecular chaperones in the biosynthesis of human collagen, the peptidyl prolyl cis-trans isomerase FKBP22 gene and the human prolyl-4-hydroxylase gene P4Hα1β, to enhance the formation of the triple helical structure.

[0081] (3) The present invention transforms the recombinant full-chain triple-helix recombinant type III human collagen encoding gene fragment into the modified chassis strain, and constructs a recombinant Pichia pastoris genetically engineered bacterium that co-expresses molecular chaperones, hydroxylases and recombinant full-chain collagen.

[0082] (4) The recombinant Pichia pastoris provided by the present invention can obtain a high expression level (>0.5 g / L) of recombinant full-chain triple-helical recombinant type III human collagen through high-density fermentation; the purification process provided by the present invention is relatively simple, and the triple-helical type III recombinant human collagen obtained after enzymatic hydrolysis and purification does not contain heterologous sequences, and has a high hydroxyproline content and thermal stability, which is close to that of natural human collagen.

[0083] (5) The recombinant full-chain triple-helix recombinant type III human collagen provided by the present invention has a 100% identical amino acid sequence to the natural human type III collagen, and has a stable triple-helix structure and high thermal stability. It can meet the application in medical health and medical aesthetics fields such as medical filling and tissue repair, and is more suitable for development into related Class III medical device products. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Figure 2 is a map of the HHP-sgRNA plasmid.

[0085] Figure 2 Map of the recombination editing plasmid pHTXp-sgRNA-HsCas9-CENARS-△ku70.

[0086] Figure 3 Map of the recombineering editing plasmid pHTXp-sgRNA-HsCas9-CENARS-lnt39.

[0087] Figure 4The expression of the target protein in shake flask fermentation of the recombinant strain X33△ku70:FKBP22 / α1β-3A1ncF-68# is shown. A is the result of non-reducing SDS-PAGE; B is the result of non-reducing SDS-PAGE after pepsin treatment; C is the result of reducing SDS-PAGE.

[0088] Figure 5 The expression of the target protein in the recombinant strain X33△ku70:FKBP22 / α1β-3A1ncF-68# under high-density fermentation conditions in a 5L fermenter and after isolation and purification. A is the electrophoresis of the target protein under high-density fermentation conditions in a 5L fermenter; B is the electrophoresis of the target protein after isolation and purification.

[0089] Figure 6 CD detection spectrum of the target protein.

[0090] Figure 7 Thermal stability test profile of the target protein.

[0091] Figure 8 Transmission electron microscopy detection map of the target protein.

[0092] Caption: Figures 1 to 3 In the vector, ori: origin of replication; PARS1: protease-activated receptor 1; CYC TT: cytochrome C protein terminator; HygB: hygromycin resistance gene; TEF promoter: transcription enhancer factor promoter; AOX1TT: alcohol oxidase 1 terminator; HDV: 3'-cleaved hepatitis delta virus nuclease sequence; gRNA scaffold: expressed together with N20 (recognition sequence of editing site) to form sgRNA (guide RNA) structure, which can bind to cas9 protein (Ishino nuclease 9) and target the genome; N20-gRNA1: recognition sequence of editing site required for ku70 gene knockout; N20-gRNA2: recognition sequence of editing site required for lnt39 gene knockout; HH: 5'-cleaved hammerhead ribozyme sequence; HTXp: a bidirectional promoter for yeast expression; Cas: human codon-optimized Cas9 protein (Ishino nuclease 9) gene; SV40 NLS: a nuclear localization signal derived from simian virus 40 tumor antigen; DAS1t: yeast-derived terminator; AmpR promoter: ampicillin resistance gene promoter; AmpR: ampicillin resistance gene; SUP4 terminator: suppressor of ochre mutation gene terminator; Factor Xa site: coagulation factor Xa cleavage site; pSER: phosphorylated serine. DETAILED DESCRIPTION

[0093] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0094] Example 1 Construction of a recombinant expression vector expressing recombinant full-length collagen α1 chain

[0095] 1. Based on the human type III collagen α1 chain preproprotein sequence (NCBI Reference Sequence: NP_000081.2) included in the GenBank database, the mature collagen α1 chain sequence (amino acids 149-1221) containing the N-terminal telopeptide, the classic triple-helical domain, and the C-terminal telopeptide was selected as the amino acid sequence of the full-chain human type III collagen. A mammalian secretory signal peptide sequence sPC1 (SEQ ID NO: 1) was attached to its N-terminus, and a phage T4 fibrin-derived foldon sequence (SEQ ID NO: 2) was fused to its C-terminus to generate a fusion protein (SEQ ID NO: 3). The fusion protein sequence was then codon-optimized according to the codon usage preference of Pichia pastoris to synthesize the gene fragment encoding the recombinant full-chain triple-helical recombinant type III collagen sPC1-3A1ncF (SEQ ID NO: 4).

[0096] 2. Double-digest the pPICZαA plasmid with BstBI and Sal I enzymes to obtain linearized pPICZαA vector 1. Connect the sPC1-3A1ncF fragment to the linearized pPICZαA vector 1 using seamless cloning technology to obtain the pPICZ-sPC1-3A1ncF recombinant vector.

[0097] Example 2 Construction of recombinant prolyl-4-hydroxylase recombinant expression vector

[0098] 1. Based on the codon usage preference of Pichia pastoris, the codons of the human prolyl-4-hydroxylase α1 subunit (P4Hα1, NCBI Reference Sequence: NP_000908.2) and β subunit (P4Hβ, NCBI Reference Sequence: NP_000909.2) were optimized to obtain the optimized P4Hα1 subunit encoding gene fragment (SEQ ID NO: 5) and the optimized P4Hβ subunit encoding gene fragment (SEQ ID NO: 6).

[0099] 2. The pPIC9K plasmid was double-digested with BamH I and EcoR I enzymes to obtain linearized pPIC9K vector 1; the pPICZαA plasmid was double-digested with EcoR I and Sal I enzymes to obtain linearized pPICZαA vector 2.

[0100] 3. Using seamless cloning technology, the optimized P4Hα1 subunit encoding gene fragment was ligated between the BamH I and EcoR I restriction sites of the linearized pPIC9K vector 1 to obtain the pPIC9K-P4Hα1 recombinant vector; the optimized P4Hβ subunit encoding gene fragment was ligated between the EcoR I and Sal I restriction sites of the linearized pPICZαA vector 2 to obtain the pPICZαA-P4Hβ recombinant vector.

[0101] 4. Use AatII endonuclease to digest the pPIC9K-P4Hα1 recombinant vector to obtain the linearized pPIC9K-P4Hα1 recombinant vector.

[0102] 5. Amplification of the P4Hβ subunit expression cassette (SEQ ID NO: 7): Using the pPICZαA-P4Hβ recombinant vector as a template, PCR amplification was performed using primers P4Hβ-F (SEQ ID NO: 8) and P4Hβ-R (SEQ ID NO: 9). The PCR amplification system consisted of 12.5 μL of 2× Phanta Max Buffer, 1 μL of dNTP Mix, 1 μL of P4Hβ-F (10 μM), 1 μL of P4Hβ-R (10 μM), 1 μL of the pPICZαA-P4Hβ recombinant vector, and 1 μL of Phanta Max Super-Friendly DNA polymerase. The total PCR amplification system was made up to 25 μL with ddH2O. The PCR amplification program was as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 100 s, and 72°C for 5 min.

[0103] 6. Using seamless cloning technology, the P4Hβ subunit expression cassette fragment was connected to the linearized pPIC9K-P4Hα1 recombinant vector to obtain the pPIC9K-P4Hα1β recombinant vector carrying both the P4Hα1 subunit and the P4Hβ subunit expression cassettes.

[0104] Example 3 Construction of a recombinant expression vector expressing the molecular chaperone peptidylprolyl cis-trans isomerase FKBP22

[0105] 1. Based on the codon usage preference of Pichia pastoris, the molecular chaperone peptidylprolyl cis-trans isomerase FKBP22 (NCBI Reference Sequence: NP_060416.1) was codon optimized to obtain the optimized FKBP22 encoding gene fragment (SEQ ID NO: 10).

[0106] 2. Double-digest the pPIC9K plasmid with BamHI and Not I enzymes to obtain linearized pPIC9K vector 2. Use seamless cloning technology to ligate the optimized FKBP22 encoding gene fragment between the BamHI and Not I restriction sites of the linearized pPIC9K vector 2 to obtain the pPIC9K-FKBP22 recombinant vector.

[0107] Example 4 Construction of Pichia pastoris X33 with ku70 gene knockout (X33Δku70)

[0108] 1. Construction of pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector

[0109] (1) Synthesize an sgRNA expression cassette with ribozyme flanks (HTXp-N20-gRNA expression cassette, SEQ ID NO: 11). Ligate the sgRNA expression cassette with ribozyme flanks between the EcoRI and HindIII sites of the pUC19 vector to obtain the pUC19-HTXp-N20-sgRNA recombinant vector.

[0110] (2) Amplification of the HTXp-N20-gRNA expression cassette: The template of the PCR amplification system in Example 2 was replaced with the pUC19-HTXp-N20-sgRNA recombinant vector, and the primers were replaced with HTXp-sgRNA-F (SEQ ID NO: 12) and AOX1t-sgRNA-R (SEQ ID NO: 13). Other components and amounts remained unchanged. The PCR amplification program was as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 60 s, and 72°C for 5 min, for 30 cycles.

[0111] Amplification Figure 1The pHHP-sgRNA backbone vector shown (the fragment between the AmpR promoter and HygB in the pHHP-sgRNA plasmid is obtained, excluding the entire sgRNA expression cassette sequence) was prepared by replacing the template in the PCR amplification system in Example 2 with the pHHP-sgRNA plasmid and replacing the primers with pHHP-F (SEQ ID NO: 14) and pHHP-R (SEQ ID NO: 15). All other components and amounts remained unchanged. PCR amplification protocol: 95°C for 3 minutes, followed by 30 cycles of 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 80 seconds, and 72°C for 5 minutes.

[0112] The HTXp-N20-gRNA expression cassette was connected between the AmpR promoter and TEF promoter of the pHHP-sgRNA backbone vector using seamless cloning technology to obtain the pHTXp-sgRNA recombinant vector.

[0113] (3) Amplification of the yeast-derived terminator DAS1t fragment (SEQ ID NO: 16): The template in the PCR amplification system described in Example 2 was replaced with Pichia pastoris genomic DNA, and the primers were replaced with DAS1t-F (SEQ ID NO: 17) and DAS1t-R (SEQ ID NO: 18). Other components and amounts remained unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 55°C for 15 s, 72°C for 20 s, and 72°C for 5 min, for 30 cycles.

[0114] Amplify the HsCas9 fragment (SEQ ID NO: 19): Replace the template in the PCR amplification system in Example 2 with the pGAP-Cas9 plasmid and the primers with HsCas9-F (SEQ ID NO: 20) and HsCas9-R (SEQ ID NO: 21). All other components and amounts remain unchanged. PCR amplification protocol: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 90 s, and 72°C for 5 min, for 30 cycles.

[0115] Amplify the pHTXp-sgRNA backbone vector: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA plasmid and the primers with pHTXp-sgRNA-F (SEQ ID NO: 22) and pHTXp-sgRNA-R (SEQ ID NO: 23). All other components and amounts remain unchanged. PCR amplification protocol: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 90 s, and 72°C for 5 min, for 30 cycles.

[0116] Using seamless cloning technology, the DAS1t fragment, HsCas9 fragment and pHTXp-sgRNA backbone vector were connected. The HsCas9 fragment was located counterclockwise downstream of the HTXp promoter of the pHTXp-sgRNA backbone vector, and the DAS1t fragment followed it to obtain the pHTXp-sgRNA-HsCas9 recombinant vector.

[0117] (4) Amplification of the linearized pHTXp-sgRNA-HsCas9 vector: The template of the PCR amplification system in Example 2 was replaced with the pHTXp-sgRNA-HsCas9 recombinant vector, and the primers were replaced with pHTXp-sgRNA-HsCas9-F (SEQ ID NO: 24) and pHTXp-sgRNA-HsCas9-R (SEQ ID NO: 25). Other components and contents remained unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 100 s, and 72°C for 5 min, for 30 cycles.

[0118] The CEN / ARS sequence (SEQ ID NO: 26) of the replicon derived from Saccharomyces cerevisiae was synthesized and ligated between the EcoRI and HindIII sites of the pUC19 vector to obtain the pUC19-CEN / ARS recombinant vector.

[0119] Amplify the CEN / ARS fragment: Replace the template in the PCR amplification system in Example 2 with the pUC19-CENARS recombinant vector and the primers with CENARS-F (SEQ ID NO: 27) and CENARS-R (SEQ ID NO: 28). All other components and amounts remain unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 10 s, and 72°C for 5 min, for 30 cycles.

[0120] Using seamless cloning technology, the linearized pHTXp-sgRNA-HsCas9 vector was connected to the CEN / ARS fragment to obtain a gene editing tool plasmid that can target and edit the N20 sequence on the X gene (i.e., the target gene): the pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector.

[0121] 2. Construct a knockout plasmid for the ku70 gene (Gene ID: 8199462) in the Pichia pastoris genome.

[0122] The recognition sequence N20-gRNA1 sequence (SEQ ID NO: 29) of the editing site required for ku70 gene knockout was designed using the https: / / chopchop.cbu.uib.no / online database.

[0123] Amplify the Δku70-N20-sgRNA fragment (SEQ ID NO: 30) that targets a specific site on the ku70 gene: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector, and replace the primers with ku70-N20-F (SEQ ID NO: 31) and Δku70-N20-R (SEQ ID NO: 32). All other components and amounts remain unchanged. PCR amplification protocol: 95°C for 3 min; 30 cycles of 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, and 72°C for 3 min.

[0124] The pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector was digested with Not I and Pem I endonucleases to obtain a linearized pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector. The △ku70-N20-sgRNA fragment was ligated between the Not I and Pem I restriction sites of the linearized pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector using seamless cloning technology to obtain the pHTXp-sgRNA-HsCas9-CENARS-△ku70 recombinant vector with the function of targeted editing of the ku70 gene ( Figure 2 ).

[0125] Amplify the homology arm fragment Δku70-up (SEQ ID NO: 33) upstream of the ku70 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with Δku70-up-F (SEQ ID NO: 34) and Δku70-up-R (SEQ ID NO: 35). All other components and amounts remain unchanged. PCR amplification program: 95°C for 3 min; 30 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 20 s, and 72°C for 3 min.

[0126] Amplify the Δku70-dw (SEQ ID NO: 36) homology arm fragment downstream of the ku70 gene editing site: Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with Δku70-dw-F (SEQ ID NO: 37) and Δku70-dw-R (SEQ ID NO: 38). All other components and amounts remain unchanged. The PCR amplification procedure is the same as for amplifying the homology arm fragment upstream of the ku70 gene editing site.

[0127] Amplify the Δku70-Donor fragment (SEQ ID NO: 39) containing the upstream and downstream homology arms of the ku70 gene editing site: Replace the templates in the PCR amplification system in Example 2 with the upstream and downstream homology arms of the ku70 gene editing site (1 μL each), and replace the primers with Δku70-up-F and Δku70-dw-R. Perform overlap PCR amplification, while keeping all other components and amounts unchanged. PCR amplification program: 95°C for 3 min; 30 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 30 s, and 72°C for 3 min.

[0128] 3. Prepare competent cells of Pichia pastoris X33: Pick a single colony of Pichia pastoris X33 and inoculate it into 5 mL of YPD liquid medium. Incubate it at 29°C, 250 rpm and shake overnight (16-24 hours) to obtain a culture. Take 50 μL of the culture and inoculate it into 50 mL of YPD liquid medium. Incubate it at 29°C, 250 rpm and shake until the OD of Pichia pastoris X33 reaches 0. 600 When the pH value reaches 1-1.5, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 50 mL of ice-cold sterile double-distilled water, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 25 mL of ice-cold sterile double-distilled water, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 20 mL of ice-cold 1 M sorbitol solution, centrifuge at 5000 rpm for 5 minutes, discard the supernatant, and collect the cells. Resuspend the cells in 0.3 mL of ice-cold 1 M sorbitol solution to obtain a Pichia pastoris X33 competent cell suspension. Aliquot into 80 μL portions and store at -80°C until needed.

[0129] 4. Electroporation: Take 50 μL of a tube of Pichia pastoris X33 competent cell suspension (OD 600 Thaw a 40-100 μL (40-100 μg) tube on ice, add 2 μg of the △ku70-Donor fragment and 2 μg of the pHTXp-sgRNA-HsCas9-CENARS-△ku70 recombinant vector, gently pipette to mix, transfer to a pre-chilled electroporation cuvette, and continue to chill on ice for 5 minutes. Set the electroporation voltage to 1.5 kV for 4-6 msec. After the electroporation, quickly add 1 mL of ice-cold 1 M sorbitol solution, gently pipette to mix, and transfer to a 1.5 mL sterile centrifuge tube. Incubate in a 30°C incubator for 1-2 hours to obtain the electroporated bacterial suspension.

[0130] 5. Spreading Culture: Spread 200 μL of the electroporated bacterial suspension onto a YPD plate supplemented with hygromycin at a final concentration of 0.3 mg / mL. Incubate in an incubator at 30°C for 2-3 days, inverted, until single colonies appear. Identify the ku70 gene knockout strain, Pichia pastoris X33Δku70, by colony PCR.

[0131] Example 5 Construction of Pichia pastoris X33 with knock-in of molecular chaperone FKBP22 gene and knock-out of ku70 gene

[0132] 1. Using the online database https: / / chopchop.cbu.uib.no / , the recognition site N20-gRNA2 sequence (SEQ ID NO: 41) required for gene editing at the lnt39 gene sequence (PAS_chr1-4_0294...PAS_chr1-4_0295, SEQ ID NO: 40) on the Pichia pastoris genome was designed.

[0133] Amplify the Δlnt39-N20-sgRNA fragment (SEQ ID NO: 42) that targets a specific site in the lnt39 gene sequence: Replace the template in the PCR amplification system in Example 2 with the pHTXp-sgRNA-HsCas9-CENARS-ΔX recombinant vector, and replace the primers with lnt39-N20-F (SEQ ID NO: 43) and lnt39-N20-R (SEQ ID NO: 44). All other components and amounts remain unchanged. PCR amplification protocol: 95°C for 3 min; 30 cycles of 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, and 72°C for 3 min.

[0134] The lnt39-N20-sgRNA fragment was connected between the Not I and Pem I restriction sites of the linearized pHTXp-sgRNA-HsCas9-CENARS-△X recombinant vector using seamless cloning technology to obtain the pHTXp-sgRNA-HsCas9-CENARS-lnt39 recombinant vector with the function of targeted editing of the lnt39 gene sequence ( Figure 3 ).

[0135] 2. Amplify the homology arm fragment upstream of the lnt39 gene editing site (SEQ ID NO: 45): Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with lnt39-up-F (SEQ ID NO: 46) and lnt39-up-R (SEQ ID NO: 47). Other components and amounts remain unchanged. PCR amplification program: 95°C for 3 min; 95°C for 15 s, 65°C for 20 s, 72°C for 15 s, and 72°C for 5 min, for 30 cycles.

[0136] Amplify the homology arm fragment downstream of the lnt39 gene editing site (SEQ ID NO: 48): Replace the template in the PCR amplification system in Example 2 with the Pichia pastoris X33 genome, and replace the primers with lnt39-dw-F (SEQ ID NO: 49) and lnt39-dw-R (SEQ ID NO: 50). Other components and amounts remain unchanged. The PCR amplification procedure is the same as for amplifying the homology arm fragment upstream of the lnt39 gene editing site.

[0137] 3. Amplify the expression cassette fragment of the molecular chaperone FKBP22 gene (P AOX1 -FKBP22, SEQ ID NO: 51): The template of the PCR amplification system in Example 2 was replaced with the pPIC9K-FKBP22 recombinant expression vector constructed in Example 3, and the primers were replaced with P AOX1 -FKBP22-F (SEQ ID NO: 52) and P AOX1 -FKBP22-R (SEQ ID NO: 53), with other components and contents unchanged. PCR amplification program: 95°C for 3 min, followed by 30 cycles of 95°C for 15 s, 55°C for 20 s, 72°C for 75 s, and 72°C for 5 min.

[0138] Amplification of Δlnt39-P AOX1 -FKBP22-Donor fragment (SEQ ID NO: 54): Replace the template in the PCR amplification system in Example 2 with the upstream and downstream homology arms of the lnt39 gene editing site and the FKBP22 gene expression cassette fragment (1 μL each). Replace the primers with lnt39-up-F and lnt39-dw-R. All other components and amounts remain unchanged. PCR amplification program: 95°C for 3 min; 30 cycles of 95°C for 15 s, 58°C for 30 s, 72°C for 90 s, and 72°C for 5 min.

[0139] 4. According to the method of Example 4, Pichia pastoris X33 was replaced with Pichia pastoris X33Δku70 to prepare Pichia pastoris X33Δku70 competent cells. Take 50 μL of a tube of Pichia pastoris X33Δku70 competent cell suspension (OD 600 =40~100) was thawed on ice and 2 μg △ lnt39-P was added AOX1 -FKBP22-Donor fragment and 2 μg pHTXp-sgRNA-HsCas9-CENARS-lnt39 recombinant vector were gently pipetted to mix, and electroporation, plating culture and identification were performed according to the method of Example 4 to obtain Pichia pastoris X33△ku70:FKBP22 with the expression cassette of the FKBP22 gene successfully knocked in.

[0140] Example 6 Construction of a recombinant strain co-expressing prolyl-4-hydroxylase and full-chain collagen and its culture

[0141] 1. Construction of a recombinant strain co-expressing prolyl-4-hydroxylase and full-chain collagen

[0142] (1) The pPIC9K-P4Hα1β recombinant vector of Example 2 was digested with BspE I endonuclease to obtain a linearized pPIC9K-P4Hα1β recombinant vector. According to the method of Example 4, Pichia pastoris X33 was replaced with Pichia pastoris X33△ku70:FKBP22 to prepare Pichia pastoris X33△ku70:FKBP22 competent cells. Take 100 μL of a tube of Pichia pastoris X33△ku70:FKBP22 competent cell suspension (OD 600 =40-100) was thawed on ice, 4 μg of the linearized pPIC9K-P4Hα1β recombinant vector was added, and the mixture was gently pipetted. Electroporation, plating culture, and identification were performed according to the method of Example 4 to obtain Pichia pastoris X33Δku70:FKBP22 / α1β with single copies of the P4Hα1 subunit and P4Hβ subunit expression cassettes inserted. The 0.3 mg / mL hygromycin in the plating culture medium was replaced with 0.5 mg / mL neomycin G418.

[0143] (2) The pPICZ-sPC1-3A1ncF recombinant vector of Example 1 was digested with Sac I endonuclease to obtain a linearized pPICZ-sPC1-3A1ncF recombinant vector. According to the method of Example 4, Pichia pastoris X33 was replaced with Pichia pastoris X33△ku70:FKBP22 / α1β competent cells. Take 100 μL of a tube of Pichia pastoris X33△ku70:FKBP22 / α1β competent cell suspension (OD 600 =40~100) was thawed on ice, 4 μg of linearized pPICZ-sPC1-3A1ncF recombinant vector was added, and the mixture was mixed by gently pipetting. Electroporation and plating were performed according to the method of Example 4 until single colonies appeared. The plating culture medium was supplemented with a final concentration of 0.1 mg / mL Zeocin. TM YPDS plates.

[0144] (3) Use a 10 μL pipette to carefully pick up the single colonies with good growth in step (2) and resuspend and mix them in 5 μL of sterile water to obtain a resuspended bacterial solution; then take 1 μL of the resuspended bacterial solution and copy it onto the solution with different final concentrations (0.5, 1 and 1.5 mg / mL) of Zeocin. TMThe plate was placed on a YPDS plate and incubated upside down at 30°C for 2-3 days until a single colony appeared. Colony PCR was performed to confirm that the full-length collagen gene was successfully integrated into the Pichia pastoris genome. This allowed the selection of Pichia pastoris X33△ku70:FKBP22 / α1β-3A1ncF that co-expressed prolyl-4-hydroxylase and full-length collagen genes. 5 mL of overnight culture was aspirated to detect its copy number and OD 600 .

[0145] The results are shown in Table 1. A total of 5 recombinant strains were obtained, among which the X33△ku70:FKBP22 / α1β-3A1ncF-68# recombinant strain (No. 68) had the best expression effect.

[0146] Table 1

[0147]

[0148] 2. Shake flask fermentation and expression identification

[0149] (1) Pick a single colony of X33△ku70:FKBP22 / α1β-3A1ncF that has been identified and inoculated into 10 mL of YPD medium and culture overnight at 29°C and 250 rpm until the OD 600 =3~5, and you get the overnight culture.

[0150] (2) Inoculate the overnight culture into 30 mL of BMGY medium at a 1% inoculation ratio and culture at 29°C and 250 rpm until the OD 600 =4~6, centrifuge at 5000 rpm for 5 min at room temperature to collect the bacteria, resuspend the bacteria in BMMY medium to OD600=1 to obtain a resuspended bacterial solution, transfer 30 mL of the resuspended bacterial solution to a 250 mL conical flask, and add L-ascorbic acid at a final concentration of 100 mg / L and 0.6 mM FeSO4 as a cofactor for hydroxylase. Methanol at a final concentration of 1% (v / v) and 100 mg / mL of L-ascorbic acid were added to the culture system every 24 h, and the induction culture was continued. After the induction culture was completed, the bacterial solution was collected and the expression of the target protein was detected by reducing and non-reducing SDS-PAGE. The bacterial solution was digested with pepsin and its pepsin resistance was detected by non-reducing SDS-PAGE.

[0151] The electrophoresis results are as follows Figure 4 A in Figure 4 B and Figure 4As shown in Figure C, the molecular weight of the resulting recombinant collagen is close to 300 kDa, consistent with the molecular weight of a placenta-derived human type III collagen standard (Sigma-Aldrich, Product No. CC054). The protein also exhibits good pepsin resistance. Under reducing conditions, the triple helix melts into single collagen chains, resulting in a molecular weight approximately one-third that of the triple helix. This demonstrates the successful expression of the full-length triple helix recombinant human type III collagen protein of the present invention.

[0152] The obtained X33△ku70:FKBP22 / α1β-3A1ncF-68# was named: Pichia pastoris komagataella phaffiiXF / 3A1ncF-68#, and was deposited in Guangdong Provincial Microbiological Culture Collection on April 21, 2025. Its deposit number is: GDMCC No: 66181, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0153] Example 7 High-density fermentation of co-expression engineered bacteria

[0154] (1) The recombinant strain X33Δku70:FKBP22 / α1β-3A1ncF-68# of Example 6 was streaked onto a YPD slant medium and cultured in a constant temperature incubator at 29°C for 3 days;

[0155] (2) Preparation of the primary seed solution: Scrape the strain cultured in the slant culture medium in step (1) and transfer it to 10 mL YPD liquid medium. Cultivate at 29°C and 250 rpm for 16-20 h until the recombinant strain OD reaches 600 When it reaches 3~5, the first-level seed liquid mother liquor is obtained.

[0156] (3) Preparation of secondary seed solution: transfer the primary seed solution to 200 mL of BMGY liquid medium at a 1% (v / v) inoculation volume (i.e., the volume concentration of the primary seed solution in the secondary seed solution), and culture at 29°C and 250 rpm for 16-24 h until the OD value of the recombinant strain reaches 0. 600 Up to 4~6.

[0157] (4) Transplantation and glycerol culture stage: 200 mL of the secondary seed solution was added to a 5 L fermenter containing 1.8 L of inorganic salt medium at a 10% (v / v) inoculation volume (i.e., the volume concentration of the secondary seed solution in 1.8 L of inorganic salt medium). The culture was carried out at 28°C, pH 5.5, an initial rotation speed of 300 rpm, and an aeration volume of 1 vvm. The dissolved oxygen content was controlled at 20%-30%. The inorganic salt medium consisted of 20 g / L KH2PO4, 6.85 g / L K2HPO4, 6.9 g / L NH4H2PO4, 1.2 g / L CaSO4, 12.5 g / L MgSO4·7H2O, 40 g / L glycerol, and 4.5 ml / L PTM1 (trace element complex mother solution).

[0158] (5) Glycerol-fed batch culture stage: After the dissolved oxygen content rises to 70% in step (4), start feeding with glycerol feeding solution at a feeding rate of 8.4 g / min, maintain the pH at 5, and the dissolved oxygen content at 30%; culture until the wet weight of the bacteria reaches 300-350 g / L and stop feeding; the glycerol feeding solution consists of 12 mL / L PTM1 and 50% (v / v) sterile glycerol.

[0159] (6) Methanol feed induction culture stage: After the dissolved oxygen content rises to 80% in step (5), the methanol feed solution and the auxiliary feed solution are opened for fed-batch culture. The initial methanol feed solution rate is set to 3.6 g / h. After 1 hour of fed-batch culture, the feed rate is adjusted to 7.2 g / h. After another hour of fed-batch culture, the feed rate is adjusted to 8.4 g / h. The fermentation is terminated after 60-72 hours of methanol fed-batch induction culture. The methanol feed solution consists of PTM1 and methanol, and the PTM1 concentration is 12 mL / L. The auxiliary feed solution consists of 20 g / L casein peptone, 2 g / L L-ascorbic acid, and 1 g / L α-ketoglutaric acid. It is added at a constant rate of 10 mL / h throughout the fed-batch culture. The changes in dissolved oxygen throughout the process are observed, and the dissolved oxygen content is controlled at 20-40%.

[0160] (7) The SDS-PAGE electrophoresis results of the target protein after culture in a 5L fermenter are as follows Figure 5 As shown in Figure A, the expression level of the obtained recombinant type III human collagen with a full-chain triple-helix structure exceeded 0.5 g / L.

[0161] Example 8 Isolation, Purification and Preparation of Recombinant Triple Helical Collagen

[0162] (1) After the culture of Example 7 was completed, the fermentation broth was cooled (below 10°C) and centrifuged (8000 rpm, 10 min) to collect the bacterial sludge.

[0163] (2) The bacterial sludge from step (1) was mixed with homogenized lysis buffer (50 mM Tris / HCl buffer, pH 7, containing 0.2 M NaCl and 15% v / v glycerol), and the mixture was fully resuspended. The ratio of bacterial sludge to homogenized lysis buffer was 20 g:100 mL. The mixture was then crushed three times under homogenization conditions at 4°C and 900 bar. After the crushing, the mixture was centrifuged at 10,000 rpm and 4°C for 30 min, and the precipitate was collected.

[0164] (3) The precipitate from step (2) was mixed with an acidic resolution solution (0.1 M acetic acid and 0.15 M NaCl) and fully resuspended. The ratio of precipitate to acidic resolution solution was 1 g:50 mL. Pepsin was then added at a final concentration of 0.2 mg / mL and digested at 4°C for 12-16 h to obtain an enzymatic hydrolysate.

[0165] (4) Centrifuge the enzymatic hydrolyzate from step (3) at 5000 rpm for 5 min at room temperature, collect the supernatant, adjust the pH of the supernatant to 5 with 0.1 M KOH, and add NaCl to a final concentration of 0.5 M to obtain the chromatographic loading clear solution;

[0166] (5) Protein chromatography: First, equilibrate the hydrophobic chromatography column (brand: Bio-LINK, model: Maxtar Butyl HR) with 5CV buffer A (20mM KH2PO4, 0.5M NaCl, acetic acid adjusted to pH 5); after the equilibration, load the chromatographic sample clarification solution of step (4), and after the loading, rinse and equilibrate the column with buffer A until A220 drops to the baseline and the conductivity is stable; set the linear gradient from 0 to 100% eluent B (20mM KH2PO4, acetic acid adjusted to pH 5) for elution, the elution volume is 20CV, and the eluate is collected during the process; the eluate is detected by SDS-PAGE electrophoresis, and the results are as follows: Figure 5 As shown in B, the purity of the target protein was significantly improved after chromatography purification.

[0167] (6) Desalting the target protein in step (5) using an ultrafiltration membrane with a molecular weight of 50 KDa, collecting the concentrate, and freeze-drying it using a freeze dryer to obtain a recombinant type III human collagen freeze-dried powder with a full-chain triple helical structure and a purity greater than 95%.

[0168] Example 9 Structural Characterization of Recombinant Triple Helical Collagen

[0169] (1) Mass spectrometry: The amino acid composition of the purified recombinant type III human collagen with a full-chain triple-helical structure obtained in Example 8 was analyzed. The results showed that the amino acid coverage of the recombinant type III human collagen with a full-chain triple-helical structure reached 100%, and it contained 9.55% 4-hydroxyproline.

[0170] (2) Circular dichroism detection: Set the parameters as starting wavelength = 190nm, ending wavelength = 260nm, step size = 1nm, repeat 1 time, collect at room temperature, record time = 1s / point, cuvette width 0.1cm. The blank control solution (0.2M acetic acid solution) and sample solution (recombinant type III human collagen with full chain length triple helix structure of different concentrations prepared with 0.2M acetic acid solution) were loaded in sequence. Each sample was measured three times, and the average value of the three scans was calculated. The results are as follows. Figure 6 As shown, the recombinant type III human collagen with a full-chain triple-helix structure prepared in the present invention has a negative peak at 195 nm and a maximum positive absorption peak at 221 nm, which is consistent with the CD characteristics of triple-helix collagen. Therefore, it is judged that the recombinant type III human collagen with a full-chain triple-helix structure prepared in Example 8 has a triple-helix structure.

[0171] (3) Thermal stability test: The recombinant type III human collagen with a full-chain triple-helix structure prepared in Example 8 was dissolved in 20 mM PB buffer (pH 7.4) with a final protein concentration of 2 mg / mL. The samples were placed in a metal bath at different temperatures (25°C, 27°C, 30°C, 32°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 42°C and 45°C) and incubated for 5 min. Samples were taken for circular dichroism (CD) spectroscopy test. The results were as follows: Figure 7 As shown in the figure, there is no obvious denaturation trend in the range of 25℃~36℃. The protein has good thermal stability, which is close to the thermal stability of natural human type III collagen (37~40℃).

[0172] (4) Fibrogenesis test: The recombinant type III human collagen with a full-chain triple-helix structure of Example 8 was redissolved in 20 mM phosphate buffer (pH 7.4, containing 0.15 M NaCl and 1 mM CaCl2) to obtain a 1 mg / mL sample solution, and incubated at 4°C for 24 h; 20 μL of the sample solution was dropped onto a clean copper mesh, allowed to stand for 5 min, and the sample was carefully removed with a clean filter paper. The mesh was then negatively stained with 2 g / 100 mL uranyl acetate for 5 min, and excess dye was removed with a filter paper. The mesh was then rinsed three times with pure water and allowed to air dry naturally. TEM samples were then prepared and tested using a transmission electron microscope. The results are shown in the figure below. Figure 8The black arrows indicate that the prepared recombinant type III human collagen can form collagen microfibrils with typical structural characteristics of alternating light and dark patterns, which is consistent with the collagen characteristics reported in mainstream literature (Holmes DF, Graham HK, Trotter JA, et al. STEM / TEM studies of collagenfibril assembly[J]. Micron, 2001, 32(3):273-285. DOI:10.1016 / S0968-4328(00)00040-8.). The collagen fibers can be longer than 1 μm and have a width between 100 nm and 300 nm. The collagen fibers have alternating light and dark stripes, which are produced by the lateral aggregation of collagen molecules with a triple helical structure. Transmission electron microscopy results show that the collagen of the present invention has a triple helical structure and has the ability to self-assemble into collagen fibers.

[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A recombinant Pichia pastoris genetically engineered bacterium, characterized in that: The recombinant Pichia pastoris genetically engineered bacteria expresses recombinant type III human collagen with a full chain length and a triple helical structure, and the ku70 gene of the recombinant Pichia pastoris genetically engineered bacteria is knocked out and recombinant 4-proline hydroxylase encoding genes and FKBP22 encoding genes are knocked in; The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO: 3; The ku70 gene is numbered in NCBI as Gene ID: 8199462; The recombinant 4-proline hydroxylase is a recombinant 4-proline hydroxylase α1 subunit with a nucleotide sequence as shown in SEQ ID NO: 5 and a recombinant 4-proline hydroxylase β subunit with a nucleotide sequence as shown in SEQ ID NO: 6; The nucleotide sequence of the FKBP22 encoding gene is shown in SEQ ID NO: 10; The FKBP22 encoding gene was knocked into the lnt39 gene locus of the Pichia pastoris chassis strain X33, wherein the nucleotide sequence of the lnt39 gene is shown in SEQ ID NO: 40; The recombinant Pichia pastoris genetically engineered bacterium is Pichia pastoris komagataella phaffiiXF / 3A1ncF-68#, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 21, 2025, with a deposit number of GDMCC No: 66181.

2. Use of the recombinant Pichia pastoris genetically engineered bacteria according to claim 1 in the preparation of collagen.

3. A method for preparing collagen, characterized in that: The recombinant Pichia pastoris genetically engineered bacteria according to claim 1 are cultured to express collagen, wherein the collagen is a recombinant type III human collagen with a full chain length and a triple helical structure, and the amino acid sequence of the recombinant type III human collagen with a full chain length and a triple helical structure is shown in SEQ ID NO:

3.

4. The preparation method according to claim 3, characterized in that The method for culturing the recombinant Pichia pastoris genetically engineered bacteria to express collagen comprises the following steps: S41: Cultivate the recombinant Pichia pastoris genetically engineered bacteria using YPD medium until the OD value of the recombinant Pichia pastoris genetically engineered bacteria is 600 When the temperature reaches 3~5, the first-grade seed solution mother liquor is obtained; S42: Inoculate the primary seed solution mother solution in step S41 into BMGY culture medium and culture until the OD value of the recombinant Pichia pastoris genetically engineered bacteria reaches 600 When the temperature reaches 4-6, the secondary seed solution is obtained; S43: inoculating the secondary seed solution described in step S42 into an inorganic salt culture medium and culturing until the dissolved oxygen content of the culture system reaches 70% or more, adding feed solution 1 for fed-batch culture until the wet weight of the bacteria reaches 300-350 g / L, wherein the feed solution 1 contains PTM1 and glycerol; S44: After the culture in step S43, the dissolved oxygen content of the culture system reaches more than 80%, and feeding solution 2 and feeding solution 3 are added for fed-batch culture. The feeding solution 2 contains PTM1 and methanol; the feeding solution 3 contains casein peptone, L-ascorbic acid and α-ketoglutaric acid.

5. The preparation method according to claim 4, characterized in that In step S43, the PTM1 concentration in the feeding solution 1 is 4-12 mL / L, and the glycerol concentration is 10-50% v / v.

6. The preparation method according to claim 4, characterized in that In step S44 , the concentration of PTM1 in feed solution 2 is 5-20 mL / L, and feed solution 3 contains 10-30 g / L casein peptone, 2-20 g / L L-ascorbic acid, and 1-5 g / L α-ketoglutaric acid.

7. The preparation method according to claim 4, characterized in that The collagen is separated and purified, including lysing the recombinant Pichia pastoris genetically engineered bacteria cultured in step S44, acidic resolubilization, enzymatic hydrolysis, protein chromatography and ultrafiltration desalination.

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