A recombinant humanized collagen capable of low-temperature self-assembly and long-term heat resistance and stability, and preparation method and application thereof

Abstract

The present application discloses a recombinant humanized collagen that can self-assemble at low temperature and is long-lasting heat-resistant and stable, and its preparation method and application, and relates to the field of genetic engineering technology. A recombinant humanized collagen that can self-assemble at low temperature and is long-lasting heat-resistant and stable, the gene sequence of the recombinant humanized collagen is shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.26. The recombinant humanized collagen of the present application has a low-temperature self-assembly property similar to that of natural collagen, and can form a stable collagen gel without the addition of a cross-linking agent or a gel excipient, and it has high stability and biological activity, is not easily denatured or degraded by high temperature, and has good in vitro resistance to collagenase hydrolysis and long-term effect in vivo.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability, and a preparation method and application thereof. Background Art

[0002] Collagen is one of the main components of the extracellular matrix, responsible for maintaining the stability of tissue structure and providing biomechanical support. Natural collagen has a large molecular weight, complex structure, and physical properties of low-temperature self-assembly. Recombinant collagen is generally expressed by truncating a section of the amino acid sequence of natural collagen. At present, recombinant collagen has shown application potential in many fields due to its excellent biocompatibility and biological activity. However, recombinant collagen also has some defects. On the one hand, its structure and function are different from those of natural collagen. Due to the structural differences between recombinant collagen and natural collagen, as well as the changes in the amino acid sequence of recombinant collagen and the differences in protein processing and modification, most of the recombinant collagen on the market do not have the low-temperature self-assembly properties unique to natural collagen; on the other hand, recombinant collagen has poor thermal stability and is easily degraded. Most recombinant collagen products on the market are not stable enough under high temperature conditions. The melting point temperature (Tm value) of recombinant collagen reported in general literature is between 40℃ and 60℃, so it cannot withstand high-temperature emulsification and sterilization temperatures, which limits its application in creams and cosmetics. Moreover, in vivo or in high-temperature environments, recombinant collagen with poor stability is more easily degraded and does not have long-term effectiveness. Based on this, the present application proposes a recombinant humanized collagen that can self-assemble at low temperature and is long-lasting and heat-resistant and stable, as well as its preparation method and application. Summary of the invention

[0003] The main purpose of this application is to provide a recombinant humanized collagen that can self-assemble at low temperature and has long-term heat stability and a preparation method and application thereof, aiming to solve the technical problem that the existing recombinant collagen does not have low-temperature self-assembly properties and heat stability.

[0004] To achieve the above objectives, the present application proposes a recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability. The gene sequence of the recombinant humanized collagen is shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.26.

[0005] The present application also proposes a method for preparing a recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability, comprising the following steps:

[0006] Based on human type I collagen peptide fragments and human type III collagen peptide fragments, a recombinant humanized collagen amino acid sequence that does not contain the main restriction site sequence of MMPs is designed;

[0007] Based on the recombinant humanized collagen amino acid sequence, after optimizing the codon preference of Pichia pastoris, a recombinant humanized collagen gene sequence is obtained;

[0008] Based on the recombinant humanized collagen gene sequence and the Pichia pastoris expression plasmid pGAPZαA, a recombinant humanized collagen plasmid is constructed;

[0009] The human gene P4H was used as the amino acid sequence of proline hydroxylase, and the gene sequence of recombinant proline hydroxylase was obtained after optimizing the design according to the codon preference of the Pichia pastoris host.

[0010] Based on the gene sequence of the recombinant proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K, construct a recombinant proline hydroxylase plasmid;

[0011] The recombinant humanized collagen protein plasmid is subjected to enzyme cutting and linearization to obtain a linearized recombinant human collagen protein plasmid; the linearized recombinant human collagen protein plasmid is electro-transformed into Pichia pastoris GS115 competent cells, and after culture and positive transformant screening, a recombinant human collagen yeast engineering bacterium is obtained; the recombinant proline hydroxylase plasmid is subjected to enzyme cutting and linearization to obtain a linearized recombinant proline hydroxylase plasmid; the recombinant human collagen yeast engineering bacterium is used to prepare a recombinant engineering bacterium competent cell, the linearized recombinant proline hydroxylase plasmid is electro-transformed into the recombinant engineering bacterium competent cells, and after culture and co-expression positive transformant screening, a recombinant human collagen co-expressing proline hydroxylase yeast engineering bacterium is obtained;

[0012] Inducing the expression of the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria, and collecting the fermentation broth supernatant by centrifugation;

[0013] After the fermentation broth supernatant is purified, a recombinant humanized collagen stock solution is obtained.

[0014] Optionally, the step of designing a recombinant humanized collagen amino acid sequence that does not contain a major MMPs cleavage site sequence based on human type I collagen peptide fragments and human type III collagen peptide fragments comprises:

[0015] Connecting a human type I collagen peptide fragment and a human type III collagen peptide fragment end to end to form a wild-type collagen peptide, wherein the wild-type collagen peptide does not contain a major MMPs cleavage site;

[0016] Based on the wild-type collagen peptide, computer-aided protein design was used to perform structural simulation and energy calculation to obtain 7 collagen peptide mutants;

[0017] The wild-type collagen peptide and the seven collagen peptide mutants were repeated in series four times as basic units to obtain a recombinant humanized collagen amino acid sequence as shown in Seq ID NO.11, Seq ID NO.12, Seq ID NO.13, Seq ID NO.14, Seq ID NO.15, Seq ID NO.16, Seq ID NO.17 or Seq ID NO.18.

[0018] Optionally, the main cleavage sites of the MMPs include -Gly-|-Ile-, -Pro-Gln-Gly-|-Ile-Ala-Gly-Gln-, -Asn / Asp-|-Leu-Gly-|-Ile-Asn-|-Phe-Gln-|-Thr, -Ala-|-Gln-, -Ala-|-Leu- and -Tyr-|-Leu-.

[0019] Optionally, the step of obtaining a recombinant humanized collagen gene sequence after optimizing the codon preference of Pichia pastoris based on the recombinant humanized collagen amino acid sequence comprises:

[0020] According to the recombinant humanized collagen amino acid sequence shown in Seq ID NO.11, Seq ID NO.12, Seq ID NO.13, Seq ID NO.14, Seq ID NO.15, Seq ID NO.16, Seq ID NO.17 or Seq ID NO.18, a terminator was added to the C-terminus, and the Pichia pastoris codon preference was optimized to obtain the recombinant humanized collagen gene sequence shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.26.

[0021] Optionally, the step of constructing a recombinant humanized collagen plasmid based on the recombinant humanized collagen gene sequence and the Pichia pastoris expression plasmid pGAPZαA comprises:

[0022] Gene fragments were synthesized according to the recombinant humanized collagen gene sequences shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.26, and the synthesized gene fragments were inserted between the restriction sites Xho Ⅰ and Not Ⅰ on the Pichia pastoris expression plasmid pGAPZαA to obtain recombinant humanized collagen plasmids.

[0023] Optionally, in the step of obtaining the gene sequence of recombinant proline hydroxylase by using the human gene P4H as the amino acid sequence of proline hydroxylase and optimizing the design according to the codon preference of the Pichia pastoris host, the gene sequence of the recombinant proline hydroxylase is as shown in Seq ID NO.28.

[0024] Optionally, the step of constructing a recombinant proline hydroxylase plasmid based on the gene sequence of the recombinant proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K comprises:

[0025] According to the gene sequence of the recombinant proline hydroxylase as shown in Seq ID NO.28, the gene fragment was synthesized, and the synthesized gene fragment was inserted between the restriction sites EcoR Ⅰ and Not Ⅰ on the Pichia pastoris expression plasmid pPIC3.5K to obtain the recombinant proline hydroxylase plasmid.

[0026] Optionally, the step of linearizing the recombinant humanized collagen protein by enzyme digestion to obtain a linearized recombinant human collagen protein protein particle comprises:

[0027] The recombinant humanized collagen protein particle was subjected to enzymatic linearization treatment using QuickCut SacⅠ, the enzymatic cutting temperature was 37°C, the enzymatic cutting time was 5h, and then 3M NaAc and anhydrous ethanol were added. After being placed at -20°C overnight, the particle was centrifuged at 4°C and 13000 rpm for 20min, the supernatant was discarded, the precipitate was collected and rinsed with 75% ethanol, centrifuged again and the supernatant was discarded, the water and residual ethanol in the collected precipitate were removed, and ddH2O was used to dissolve it to obtain a linearized recombinant human collagen protein particle.

[0028] Optionally, the step of electro-transforming the linearized recombinant human collagen protein plasmid into Pichia pastoris GS115 competent cells, culturing and screening positive transformants to obtain recombinant human collagen yeast engineering bacteria comprises:

[0029] The linearized recombinant human collagen protein particles were mixed with Pichia pastoris GS115 competent cells, and after ice bath, they were electroporated, and then sorbitol solution was added, mixed, and transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, they were spread on a YPD plate containing Zeocin, and allowed to stand at room temperature for 10min, and then inverted and cultured at 30°C for 2d-5d until a single colony grew;

[0030] A single colony on the YPD plate was picked up, transferred to a 96-well culture plate containing a YPD medium containing Zeocin, and cultured at 30° C. to screen and obtain recombinant human collagen yeast engineering bacteria.

[0031] Optionally, the step of linearizing the recombinant proline hydroxylase plasmid by enzyme digestion to obtain a linearized recombinant proline hydroxylase plasmid comprises:

[0032] The recombinant proline hydroxylase plasmid was linearized by enzyme digestion with SacⅠ, the enzyme digestion temperature was 37°C, the enzyme digestion time was 5h, and then 3M NaAc and anhydrous ethanol were added. After being placed at -20°C overnight, the mixture was centrifuged at 4°C and 13,000 rpm for 20min, the supernatant was discarded, the precipitate was collected and rinsed with 75% ethanol, centrifuged again and the supernatant was discarded. After removing the water and residual ethanol in the collected precipitate, the mixture was dissolved with ddH2O to obtain a linearized recombinant proline hydroxylase plasmid.

[0033] Optionally, the step of preparing competent cells of recombinant engineered bacteria using the recombinant human collagen yeast engineered bacteria comprises:

[0034] After streaking the recombinant human collagen yeast engineering bacteria plate, a single colony was picked and inoculated into YPD solid medium, and cultured at 30°C and 225 rpm for 24 h; then transferred to YPD liquid medium at an inoculation ratio of 1:1000, and cultured at 30°C and 225 rpm until OD 600nm The absorbance value is 1.3-1.5; then transfer to a sterile centrifuge tube, centrifuge at 4°C and 1500rpm for 5 minutes, discard the supernatant and collect the engineered bacteria;

[0035] The engineered bacteria were resuspended with sterile ultrapure water, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the engineered bacteria were resuspended with sterile ultrapure water, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the cell pellet was resuspended with sterile 1 M sorbitol, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the cell pellet was resuspended with sterile 1 M sorbitol, and the mixture was rotated to mix, and then placed on ice to obtain recombinant engineered bacteria competent cells.

[0036] Optionally, the step of electroporating the linearized recombinant proline hydroxylase plasmid into the competent cells of the recombinant engineering bacteria, culturing and screening co-expression positive transformants to obtain the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria comprises:

[0037] The linearized recombinant proline hydroxylase plasmid and the competent cells of the recombinant engineering bacteria were mixed, and after ice bath, electric shock was performed, and then sorbitol solution was added, mixed, and transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, it was spread on an MD solid plate, and allowed to stand at room temperature for 10min, and then inverted and cultured at 30°C for 2d-5d, until a single colony grew;

[0038] Scrape off the His on the MD solid plate + After the transformant is diluted, it is spread on a YPD plate containing G418, and cultured at 30°C upside down until a single colony appears. Then, the single colony on the YPD plate is picked and transferred to a 96-well culture plate containing YPD medium, and cultured at 30°C. The co-expression positive transformants that can grow on a plate containing 4 mg / mL of G418 are screened to obtain the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria.

[0039] Optionally, the step of inducing expression of the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria and collecting the fermentation broth supernatant by centrifugation comprises:

[0040] A single colony growing on a plate containing 4 mg / mL G418 was picked and inoculated into BMGY yeast growth medium and cultured at 30°C and 220 rpm until OD 600nm The absorbance value was 2-6, and then centrifuged at 3000 rpm for 10 min, the cells were collected and resuspended in BMMY yeast induction medium with an equal volume of the BMGY yeast growth medium to make the initial OD 600nm When the absorbance value was 2, the culture was continued at 30°C and 220 rpm, and 0.5% methanol, 1% trace elements and ascorbic acid were added every 24 hours. At 72 hours after induction, the expression bacterial precipitate, the supernatant after the bacterial precipitate was broken, and the supernatant of the fermentation liquid were taken respectively.

[0041] Optionally, the step of obtaining a recombinant humanized collagen stock solution after purifying the fermentation broth supernatant comprises:

[0042] A cation exchange medium was used, and the chromatography column was equilibrated with a phosphate buffer until the conductivity value and the A280 absorbance value remained unchanged. The fermentation broth supernatant was loaded, and the loading flow rate was set to 20 cm / h, and the ultraviolet A280 absorbance value was detected. After the loading was completed, the chromatography column was re-equilibrated with a phosphate buffer until the conductivity and the ultraviolet A280 absorbance values ​​were the lowest and no longer changed, and the sampling was stopped; and then the corresponding protein was eluted with a phosphate buffer containing NaCl and collected to obtain a recombinant humanized collagen stock solution.

[0043] The present application also proposes an application of a recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability, and the recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability is applied to cream-type cosmetics.

[0044] The present application fuses human type I collagen with type III collagen and avoids the main cleavage sites recognized by matrix metalloproteinases to obtain wild-type collagen peptides, and then uses a computer-aided protein design method to obtain 7 collagen peptide mutants with lower binding free energy and more conducive to cross-linking between protein molecules through structural simulation and energy calculation, so as to avoid or reduce the hydrolysis of recombinant humanized collagen by proteases in vivo, thereby ensuring its long-term effectiveness in vivo; and then, after the wild-type collagen peptide and the 7 collagen peptide mutants are repeated in series 4 times as basic units, 8 recombinant humanized collagens are obtained, whose gene sequences are as follows: Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 and Seq ID As shown in NO.26; the Pichia pastoris eukaryotic expression system was then used to co-express hydroxyproline and recombinant humanized collagen. Hydroxyproline can further enhance the cross-linking and stability between collagen molecules, thereby successfully obtaining hydroxyproline-modified recombinant humanized collagen. The recombinant humanized collagen has similar low-temperature self-assembly properties to natural collagen. It can form a stable collagen gel without the need for the addition of additional cross-linking agents or gel excipients, and avoids the risk of xenobiotic allergic reactions. It has good stability and biological activity, is not easily denatured or degraded by high temperatures, and has good resistance to collagenase hydrolysis in vitro and long-term efficacy in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0046] Figure 1 This is a graph showing the difference in non-covalent forces between collagen peptide mutant 1 and wild-type collagen peptide;

[0047] Figure 2 This is a graph showing the difference in non-covalent interaction between collagen peptide mutant 2 and wild-type collagen peptide;

[0048] Figure 3 This is a graph showing the difference in non-covalent interaction between collagen peptide mutant 3 and wild-type collagen peptide;

[0049] Figure 4 This is a graph showing the difference in non-covalent forces between collagen peptide mutant 4 and wild-type collagen peptide;

[0050] Figure 5 This is a graph showing the difference in non-covalent interaction between collagen peptide mutant 5 and wild-type collagen peptide;

[0051] Figure 6 This is a graph showing the difference in non-covalent interaction between collagen peptide mutant 6 and wild-type collagen peptide;

[0052] Figure 7 This is a graph showing the difference in non-covalent forces between collagen peptide mutant 7 and wild-type collagen peptide;

[0053] Figure 8 The figure shows the PCR electrophoresis results of 8 recombinant humanized collagen transformants;

[0054] Fig. 9 The figure is the SDS-PAGE electrophoresis result of the expression of 8 kinds of recombinant humanized collagen;

[0055] Fig.10 This is the result of PCR electrophoresis of P4H transformant;

[0056] Fig.11 This is a diagram showing the Western-bolt identification results of the fermentation cell precipitate and cell supernatant of the yeast engineering bacteria that co-expressed proline hydroxylase with recombinant humanized collagen;

[0057] Fig.12 This is the result of SDS-PAGE electrophoresis analysis of the fermentation supernatant of yeast engineering bacteria with recombinant humanized collagen co-expressing proline hydroxylase;

[0058] Fig.13 The figure is the SDS-PAGE test result of the purity of 8 kinds of recombinant humanized collagen stock solutions;

[0059] Fig.14 This is the appearance change of recombinant humanized collagen mutant 7 after heating at 100°C for 30 minutes;

[0060] Fig.15 This is the result of protein degradation analysis of recombinant humanized collagen mutant 7 after heating at 100°C for 30 minutes;

[0061] Fig.16 This is a diagram showing the appearance changes of recombinant humanized collagen mutant 7 after heating and refrigeration;

[0062] Fig.17 This is the electrophoresis result of the in vitro enzymatic resistance test of the recombinant humanized collagen mutant 7 and commercially available collagen;

[0063] Fig.18 These are the results of immunohistochemistry detection after collagen is applied to the skin for 4 hours; Figure A is the result of immunohistochemistry detection after recombinant human collagen mutant 7 is applied to the skin for 4 hours; Figure B is the result of immunohistochemistry detection after commercially available collagen peptide is applied to the skin for 4 hours; Figure C is the detection picture of the normal saline control group.

[0064] Fig.19 These are the results of immunohistochemistry after collagen was applied to the skin for 24 hours; Figure A is the result of immunohistochemistry after recombinant human collagen mutant 7 was applied to the skin for 24 hours; Figure B is the result of immunohistochemistry after commercially available collagen peptide was applied to the skin for 24 hours; Figure C is the test result of the normal saline control group.

[0065] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0066] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0067] Description of sequence listing (sequence listing content is provided separately):

[0068] Seq ID NO.1 shows the amino acid sequence of the human type I collagen peptide fragment in the examples of this application;

[0069] Seq ID NO.2 shows the amino acid sequence of the human type III collagen peptide fragment in the examples of this application;

[0070] Seq ID NO.3 shows the amino acid sequence of the wild-type collagen peptide in the examples of this application;

[0071] Seq ID NO.4 shows the amino acid sequence of collagen peptide mutant 1 in the examples of this application;

[0072] Seq ID NO.5 shows the amino acid sequence of collagen peptide mutant 2 in the examples of this application;

[0073] Seq ID NO.6 shows the amino acid sequence of collagen peptide mutant 3 in the examples of this application;

[0074] Seq ID NO.7 shows the amino acid sequence of collagen peptide mutant 4 in the examples of this application;

[0075] Seq ID NO.8 shows the amino acid sequence of collagen peptide mutant 5 in the examples of this application;

[0076] Seq ID NO.9 shows the amino acid sequence of collagen peptide mutant 6 in the examples of this application;

[0077] Seq ID NO.10 shows the amino acid sequence of collagen peptide mutant 7 in the examples of this application;

[0078] Seq ID NO.11 shows the amino acid sequence of wild-type recombinant human collagen in the examples of this application;

[0079] Seq ID NO.12 shows the amino acid sequence of recombinant human collagen mutant 1 in the examples of this application;

[0080] Seq ID NO.13 shows the amino acid sequence of recombinant human collagen mutant 2 in the examples of this application;

[0081] Seq ID NO.14 shows the amino acid sequence of recombinant human collagen mutant 3 in the examples of this application;

[0082] Seq ID NO.15 shows the amino acid sequence of recombinant human collagen mutant 4 in the examples of this application;

[0083] Seq ID NO.16 shows the amino acid sequence of recombinant human collagen mutant 5 in the examples of this application;

[0084] Seq ID NO.17 shows the amino acid sequence of recombinant human collagen mutant 6 in the examples of this application;

[0085] Seq ID NO.18 shows the amino acid sequence of recombinant human collagen mutant 7 in the examples of this application;

[0086] Seq ID NO.19 shows the wild-type recombinant human collagen gene sequence in the present application example;

[0087] Seq ID NO.20 shows the gene sequence of recombinant human collagen mutant 1 in the examples of this application;

[0088] Seq ID NO.21 shows the gene sequence of recombinant human collagen mutant 2 in the examples of this application;

[0089] Seq ID NO.22 shows the gene sequence of recombinant human collagen mutant 3 in the examples of this application;

[0090] Seq ID NO.23 shows the gene sequence of recombinant human collagen mutant 4 in the examples of this application;

[0091] Seq ID NO.24 shows the gene sequence of recombinant human collagen mutant 5 in the examples of this application;

[0092] Seq ID NO.25 shows the gene sequence of recombinant human collagen mutant 6 in the examples of this application;

[0093] Seq ID NO.26 shows the gene sequence of recombinant human collagen mutant 7 in the examples of this application;

[0094] Seq ID NO.27 shows the amino acid sequence of proline hydroxylase in the examples of this application;

[0095] Seq ID NO.28 shows the gene sequence of proline hydroxylase in the examples of this application.

[0096] Type I collagen is one of the most abundant proteins in the human body and is essential for the structure and function of tissues such as skin, bones, tendons and blood vessels. Type III collagen plays a key role in many biological processes, including angiogenesis, wound healing and tissue remodeling. Recombinant type I collagen fused with type III collagen has good biocompatibility and will not cause immune or rejection reactions. Due to the addition of type III collagen, the mechanical strength of recombinant type I collagen fused with type III collagen has been significantly improved, and it can withstand greater external forces. The addition of type III collagen can improve the stability of recombinant type I collagen fused with type III collagen, reduce the possibility of enzymatic degradation or oxidation, and thus extend its service life.

[0097] Proline hydroxylation is an important post-translational modification of collagen, which can affect the structure and function of collagen. Prolyl 4-Hydroxylase (P4H) is a key enzyme that plays an important role in the biosynthesis of collagen. The main function of hydroxyprolinease P4H is to catalyze the hydroxylation reaction of proline residues in collagen. P4H adds the hydroxyl group of proline residues to its side chain to form 3-hydroxyproline. This process is crucial for the stability and cross-linking of collagen, because 3-hydroxyproline residues can form hydrogen bonds with other proline residues, thereby enhancing the interaction and stability between collagen molecules.

[0098] Computer-Aided Protein Design (CAPD) is a scientific method that uses computer technology and algorithms to design and optimize protein structure and function. It combines technologies from fields such as bioinformatics, computational biology, molecular modeling, and artificial intelligence to achieve goals such as designing new functional proteins, optimizing existing proteins, and understanding protein-ligand interactions. In recent years, new methods for protein design based on structural simulation and energy calculation have emerged in computer-aided protein design, as well as new ideas for using artificial intelligence (AI) technology to guide protein modification. Compared with directed evolution, the modification scheme provided by computer-aided protein design is more clear, which greatly reduces the workload of establishing and screening mutant libraries, improves experimental efficiency and success rate, and reduces costs.

[0099] In order to facilitate those skilled in the art to understand the technical solution of the present application, the technical solution of the present application is described in detail below in conjunction with specific implementation methods.

[0100] Example 1 Construction and identification of yeast engineering bacteria co-expressing proline hydroxylase and preparation of recombinant human collagen

[0101] 1 Protein sequence selection and computer-aided protein design

[0102] Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent endopeptidases that can target and cleave collagen in the extracellular matrix and are also the main collagen hydrolases in the extracellular matrix. The main cleavage sites when MMPs hydrolyze collagen (including gelatin) include -Gly-|-Ile-, -Pro-Gln-Gly-|-Ile-Ala-Gly-Gln-, -Asn / Asp-|-Leu-Gly-|-Ile-Asn-|-Phe-Gln-|-Thr, -Ala-|-Gln-, -Ala-|-Leu- and -Tyr-|-Leu-. Avoiding these cleavage sites as much as possible when selecting the amino acid sequence of recombinant humanized collagen can reduce the hydrolysis of recombinant humanized collagen by MMPs, thereby exerting its long-term effect in the extracellular matrix.

[0103] The amino acid sequence of the recombinant humanized collagen of the present application is derived from the sequence of human type I collagen (reference UniProt: P02452) and human type III collagen (reference UniProt: P02461). The human type I collagen peptide fragment as shown in Seq ID NO.1 and the human type III collagen peptide fragment as shown in Seq ID NO.2 are selected respectively, and the two collagen peptide fragments are connected end to end to form a wild-type collagen peptide as shown in Seq ID NO.3. The amino acid sequence of the wild-type collagen peptide does not contain the main cleavage site sequence of MMPs, so as to avoid or reduce the hydrolysis of the recombinant humanized collagen by proteases in the body, thereby ensuring its long-term effect in the body.

[0104] Furthermore, based on the wild-type collagen peptide, a computer-aided protein design method was used to perform structural simulation and energy calculation, specifically: the Alphafold tool was used to predict the protein structure of the wild-type collagen peptide, and then the Discovery studio software was used to perform a full amino acid scan and a difference analysis of the binding free energy between protein molecules on the wild-type collagen peptide, and a total of 7 collagen peptide mutants (referred to as collagen peptide mutants 1-7) with lower binding free energy and more conducive to cross-linking between protein molecules were obtained. The amino acid sequences of collagen peptide mutants 1-7 are shown in Seq ID NO.4, Seq ID NO.5, Seq ID NO.6, Seq ID NO.7, Seq ID NO.8, Seq ID NO.9, and Seq ID NO.10, respectively. The amino acid sequences of all collagen peptide mutants do not contain the above-mentioned MMPs main enzyme cleavage site sequences.

[0105] Among them, collagen peptide mutant 1 is a point mutant in which alanine at position 66 in the wild-type collagen peptide molecule is replaced by arginine;

[0106] Collagen peptide mutant 2 is a point mutant in which the lysine at position 75 in the wild-type collagen peptide molecule is replaced by arginine;

[0107] Collagen peptide mutant 3 is a point mutant in which alanine at position 78 in the wild-type collagen peptide molecule is replaced by tyrosine;

[0108] Collagen peptide mutant 4 is a combined mutant in which alanine at position 66 is replaced by arginine and lysine at position 75 is replaced by arginine in the wild-type collagen peptide molecule;

[0109] Collagen peptide mutant 5 is a combined mutant in which alanine at position 66 is replaced by arginine and alanine at position 78 is replaced by tyrosine in the wild-type collagen peptide molecule;

[0110] Collagen peptide mutant 6 is a combined mutant in which the lysine at position 75 in the wild-type collagen peptide molecule is replaced by arginine and the alanine at position 78 is replaced by tyrosine;

[0111] Collagen peptide mutant 7 is a combined mutant in which alanine at position 66 is replaced by arginine, lysine at position 75 is replaced by arginine, and alanine at position 78 is replaced by tyrosine in the wild-type collagen peptide molecule.

[0112] Specifically, quantitative analysis was performed on the difference in intermolecular binding free energy and non-covalent forces before and after the mutation of different collagen peptide mutants. The results are shown in Table 1 below. The difference in non-covalent forces between different collagen peptide mutants and wild-type collagen peptides is shown in Figure 1. Figure 1-Figure 7 shown.

[0113] Table 1 Analysis of the difference in intermolecular binding free energy and the number of non-covalent forces of different collagen peptide mutants before and after mutation

[0114]

[0115] From Table 1 and Figure 1-Figure 7 It can be seen that compared with the wild-type collagen peptide, the intermolecular binding free energy of collagen peptide mutants 1-7 is lower, and more non-covalent forces such as hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waals forces can be formed.

[0116] Furthermore, the wild-type collagen peptide and 7 collagen peptide mutants were expressed in series 4 times as basic units, and 8 recombinant humanized collagen amino acid sequences were obtained, as shown in Seq ID NO.11 (wild-type recombinant human collagen), Seq ID NO.12 (recombinant human collagen mutant 1), Seq ID NO.13 (recombinant human collagen mutant 2), Seq ID NO.14 (recombinant human collagen mutant 3), Seq ID NO.15 (recombinant human collagen mutant 4), Seq ID NO.16 (recombinant human collagen mutant 5), Seq ID NO.17 (recombinant human collagen mutant 6), and Seq ID NO.18 (recombinant human collagen mutant 7), which have a larger molecular weight, are more conducive to the long-term effect of recombinant human collagen in vivo, and are also conducive to the cross-linking and stability between protein molecules.

[0117] According to the recombinant humanized collagen amino acid sequences shown in Seq ID NO.11, Seq ID NO.12, Seq ID NO.13, Seq ID NO.14, Seq ID NO.15, Seq ID NO.16, Seq ID NO.17 or Seq ID NO.18, terminators were added to the C-terminus, and the Pichia pastoris codon preference was optimized to improve the expression efficiency of the recombinant humanized collagen in Pichia pastoris, and 8 recombinant humanized collagen gene sequences were obtained, namely Seq ID NO.19 (wild-type recombinant human collagen), Seq ID NO.20 (recombinant human collagen mutant 1), Seq ID NO.21 (recombinant human collagen mutant 2), Seq ID NO.22 (recombinant human collagen mutant 3), Seq ID NO.23 (recombinant human collagen mutant 4), Seq ID NO.24 (recombinant human collagen mutant 5), Seq ID NO. NO.25 (recombinant human collagen mutant 6) or Seq ID NO.26 (recombinant human collagen mutant 7).

[0118] The base sequence AAAAGA was added to the 5' end of the optimized recombinant humanized collagen gene sequence, i.e., between Xho Ⅰ and the target gene sequence. Gene fragment synthesis was entrusted to GenScript Biotech Co., Ltd., and the synthesized gene fragment was inserted into the pGAPZαA plasmid through the restriction sites of Xho Ⅰ and Not Ⅰ to obtain 8 recombinant humanized collagen plasmids containing the gene sequences of wild-type recombinant human collagen and recombinant human collagen mutants 1-7, respectively.

[0119] Furthermore, in order to co-express recombinant humanized collagen and hydroxyproline enzyme gene in the Pichia pastoris system, the present application selects proline hydroxylase. Since the hydroxylation rate of human P4H is relatively high and the risk of xenobiotic allergic reaction can be avoided, the present application uses the human gene P4H (sequence reference NCBI: XP_047304323.1) as the amino acid sequence of proline hydroxylase, as shown in SEQ ID No. 27, and the predicted molecular weight is approximately 28 kDa.

[0120] The proline hydroxylase gene sequence was optimized according to the codon preference of the Pichia pastoris host, and a 6×His tag sequence and a terminator were added to the C-terminus to improve the expression efficiency of the recombinant proline hydroxylase in Pichia pastoris. The gene sequence of the recombinant proline hydroxylase was obtained, as shown in Seq ID NO.28.

[0121] The optimized gene sequence of recombinant proline hydroxylase was entrusted to GenScript Biotech Co., Ltd. for gene fragment synthesis, and the synthesized gene fragment was inserted into the pPIC3.5K plasmid through the restriction sites of EcoR Ⅰ and Not Ⅰ to obtain the recombinant proline hydroxylase plasmid.

[0122] 2 Construction and screening of yeast engineering strains co-expressing proline hydroxylase with recombinant human collagen

[0123] 2.1 Obtaining recombinant plasmid

[0124] The above-mentioned recombinant humanized collagen plasmid and recombinant proline hydroxylase plasmid are expanded and cultured, and high-concentration plasmids are extracted for later use.

[0125] 2.2 Linearization of recombinant humanized collagen particles

[0126] QuickCut Sac Ⅰ was used to linearize the 20 μg recombinant humanized collagen protein extracted in 2.1. The enzyme digestion conditions were 37 °C for 5 h. The enzyme digestion system is shown in Table 2 below.

[0127] Table 2 Enzyme digestion system

[0128]

[0129] After electrophoresis verification, 0.1 times the volume of 3M NaAc (pH 5.2) and 2.5 times the volume of anhydrous ethanol were added, and after being placed at -20°C overnight, the mixture was centrifuged at 4°C and 13,000 rpm for 20 min, the supernatant was discarded, the precipitate was collected and rinsed with 700 μL 75% ethanol, and then centrifuged at 4°C and 13,000 rpm for 20 min and the supernatant was discarded. The EP tube was inverted on the absorbent paper on the clean bench for about 10 min to remove water and residual ethanol as much as possible, and then 20 μL ddH2O was used for dissolution, 1 μL was taken for dilution 10 times, and the nucleic acid concentration was detected by one-drop. Linearized recombinant human collagen particles were obtained.

[0130] 2.3 Preparation of GS115 competent cells

[0131] After streaking the GS115 strain plate, a single colony was picked and inoculated into 20 mL YPD solid medium and cultured at 30°C and 225 rpm for 24 h; then the colony was transferred to 50 mL YPD liquid medium at an inoculation ratio of 1:1000 and cultured at 30°C and 225 rpm until OD 600nm The absorbance value is 1.3-1.5; the bacterial solution is then transferred to a sterile 50mL centrifuge tube, centrifuged at 4°C and 1500rpm for 5min, and the supernatant is discarded to collect the bacteria; the bacterial pellet is resuspended with 50mL of pre-cooled sterile ultrapure water, and centrifuged at 4°C and 1500rpm for 5min, and the supernatant is discarded, and the bacterial pellet is resuspended with 50mL of pre-cooled sterile ultrapure water; centrifuged at 4°C and 1500rpm for 5min, and the supernatant is discarded, and the cell pellet is resuspended with 40mL of pre-cooled sterile 1M sorbitol; centrifuged at 4°C and 1500rpm for 5min, and the supernatant is discarded, and the cell pellet is resuspended with 100μL-150μL of pre-cooled sterile 1M sorbitol, gently rotated to mix, and placed on ice for standby use to obtain GS115 competent cells.

[0132] 2.4 Electrotransformation into GS115 competent cells

[0133] Take 100 μL of GS115 competent cells and mix with 10 μL of linearized recombinant human collagen protein particles, transfer to the pre-cooled electroporation cup, immediately put in an ice bath for 5 minutes, put on the electroporator, select the yeast mode of the electroporator, and perform electroporation. Then immediately add 1 mL of pre-cooled 1 M sorbitol solution to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, incubate in a 30°C incubator for 1h-2h, take 100 μL-200 μL of the incubated bacterial solution and spread it on a YPD plate containing 0.1 mg / mL Zeocin (bleomycin), let it stand at room temperature for 10 minutes, and then invert and culture in a 30°C incubator for 2d-5d until a single colony grows.

[0134] 2.5 Screening of positive transformants

[0135] Pick a single colony from the YPD plate and transfer it to the first 96-well culture plate containing 200 μL YPD medium (containing 0.1 mg / mL Zeocin), and continue to culture at 30°C; after 48 hours, blow the bacterial solution evenly, and transfer 10 μL of the bacterial solution from each well to the second 96-well culture plate (containing 190 μL YPD); after continuing to culture for 24 hours, blow the bacterial solution evenly, and transfer 10 μL of the bacterial solution from each well to the third 96-well culture plate; after 24 hours, blow the bacterial solution in the third 96-well culture plate evenly, and draw 1 μL and continue to culture on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL Zeocin; if the positive transformant can grow on a plate containing 3 mg / mL high concentration Zeocin, it means that the positive transformant contains multiple copies of the target gene, and after this step of screening, a recombinant human collagen yeast engineered bacteria that can be efficiently expressed can be obtained.

[0136] 2.6 Identification of positive transformants

[0137] Take 50 μL of the remaining bacterial solution in the first 96-well culture plate in step 2.5 above, boil it in a boiling water bath for 10 min, freeze it in liquid nitrogen for 30 min, boil it in a boiling water bath for 10 min, and after repeated freeze-thaw cycles, centrifuge it at 12,000 rpm for 5 min. Take the supernatant as a PCR template and identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0138] Upstream primer: 5'AOX1 (5'-GACTGGTTCCAATTGACAAGC-3')

[0139] Downstream primer: 3'AOX1 (5'-GCAAATGGCATTCTGACATCC-3')

[0140] PCR conditions: pre-denaturation at 98°C for 5 min, thermal denaturation at 98°C for 50 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 35 cycles; annealing at 72°C for 10 min.

[0141] The amplified product was subjected to 1.0% agarose gel electrophoresis to determine whether the expected size of the gene fragment was amplified. Figure 8 shown. Figure 8 In the middle, lane M: protein marker, the PCR results of the transformants of 8 recombinant human collagens were all positive, about 1790 bp.

[0142] 2.7 Expression of recombinant human collagen

[0143] Pick a single colony grown on the plate containing 3 mg / mL high concentration of Zeocin in step 2.5 and inoculate it into BMGY yeast growth medium. Cultivate it at 30°C and 220 rpm until OD 600nm The absorbance value was 2-6, and then centrifuged at 3000 rpm for 10 min, the cells were collected and resuspended in BMMY yeast induction medium with an equal volume of BMGY yeast growth medium to make the initial OD 600nm The absorbance value was 2, and the culture was continued at 30°C and 220rpm. 0.5% methanol was added every 24h. The supernatant samples were taken at 72h after induction. The molecular weight and expression of the target protein were analyzed by SDS-PAGE electrophoresis. The results are shown in Fig. 9 shown.

[0144] Depend on Fig. 9 It can be seen that the eight recombinant human collagens were successfully expressed in GS115 competent cells, and the expression levels did not decrease significantly before and after mutation, with a molecular weight of approximately 39 kDa.

[0145] 2.8 Secondary electroporation of recombinant human collagen yeast

[0146] The recombinant proline hydroxylase plasmid was subjected to linearization treatment by enzyme digestion with SacⅠ, the enzyme digestion temperature was 37°C, the enzyme digestion time was 5h, 3M NaAc and anhydrous ethanol were added, and after being placed at -20°C overnight, the mixture was centrifuged at 4°C and 13000 rpm for 20min, the supernatant was discarded, the precipitate was collected and rinsed with 75% ethanol, the mixture was centrifuged again and the supernatant was discarded, the water and residual ethanol in the collected precipitate were removed, and the mixture was dissolved with ddH2O to obtain a linearized recombinant proline hydroxylase plasmid;

[0147] The yeast engineered bacteria with high expression of recombinant human collagen screened in step 2.5 were streaked and single colonies were picked and inoculated into 20 mL YPD solid medium, and cultured at 30°C and 225 rpm for 24 h; then the colonies were transferred to 50 mL YPD liquid medium at an inoculation ratio of 1:1000 and cultured at 30°C and 225 rpm until OD 600nm The absorbance value is 1.3-1.5; then transfer to a sterile 50mL centrifuge tube, centrifuge at 4°C and 1500 rpm for 5 minutes, discard the supernatant and collect the engineered bacteria;

[0148] Resuspend the engineered bacteria with 50 mL of sterile ultrapure water, centrifuge at 4 ° C., 1500 rpm for 5 min, discard the supernatant, resuspend the engineered bacteria with 50 mL of sterile ultrapure water, centrifuge at 4 ° C., 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet with 40 mL of sterile 1 M sorbitol, centrifuge at 4 ° C., 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet with 40 mL of sterile 1 M sorbitol, rotate to mix, and place on ice to obtain recombinant engineered bacteria competent cells;

[0149] The linearized recombinant proline hydroxylase plasmid was mixed with the competent cells of the recombinant engineering bacteria, and after an ice bath, electric shock was performed, and then sorbitol solution was added, mixed, and transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, it was spread on the MD solid plate, allowed to stand at room temperature for 10min, and then inverted and cultured at 30°C for 2d-5d until a single colony grew.

[0150] 2.9 Screening of co-expression positive transformants

[0151] Add 2 mL of sterile double distilled water to the surface of the MD solid plate, and then use a sterile triangular applicator to gently scrape off the His on the surface of the plate. + The transformants were transferred to 50 mL centrifuge tubes and diluted to 10 with sterile water. 5 cells / ml concentration of cell suspension (counted by spectrophotometer, 1OD 600 ≈5×10 7 cells / ml), and spread it on a YPD plate containing 0.5mg / mL G418 (Geneticin), and culture it upside down at 30℃ for 3d-4d until a single colony appears; then pick up the single colony on the YPD plate and transfer it to the first 96-well culture plate containing 200μL YPD culture medium, and continue to culture at 30℃; after 48h, blow the bacterial solution evenly, and draw 10μL of the bacterial solution from each well and transfer it to the second 96-well culture plate (containing 190μL YPD); after continuing to culture for 24h, blow the bacterial solution evenly, and draw 10μL of the bacterial solution from each well and transfer it to the third 96-well culture plate; after 24h, blow the bacterial solution in the third 96-well culture plate evenly, and draw 1μL and spot it on the plates containing 1mg / mL, 2mg / mL and 4mg / mL Continue to culture on the YPD plate containing G418; if the co-expression positive transformant can grow on the plate containing 4 mg / mL high concentration G418, it means that the co-expression positive transformant contains multiple copies of the target gene. After this step of screening, a recombinant human collagen yeast engineering strain that efficiently co-expresses proline hydroxylase can be obtained.

[0152] 2.10 Identification of co-expression positive transformants

[0153] Pick a small amount of the single colony in step 2.7 above, dissolve it in 10μL-20μL sterile water, boil it in a boiling water bath for 10 min, freeze it in liquid nitrogen for 30 min, boil it in a boiling water bath for 10 min, and after repeated freeze-thaw cycles, centrifuge it at 12000rpm for 5min. Take the supernatant as a PCR template and identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0154] P4H-F: 5'-TTCGAAGGATCCTACGTAGAAT- 3'

[0155] P4H-R: 5'-CCGCTCAATGATGATGATGATG- 3'

[0156] PCR conditions: pre-denaturation at 98°C for 5 min, thermal denaturation at 98°C for 50 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 30 cycles; annealing at 72°C for 10 min.

[0157] The amplified product was subjected to 1.0% agarose gel electrophoresis to identify whether the gene fragment of the expected size was amplified. Fig.10 As shown, it can be seen that the P4H transformant identification result is positive, about 780bp.

[0158] 3 Inducible expression of recombinant human collagen co-expressing proline hydroxylase in yeast

[0159] Pick a single colony grown on the plate containing 4 mg / mL G418 in step 2.9 and inoculate it into BMGY yeast growth medium. Cultivate at 30°C and 220 rpm until OD 600nm The absorbance value was 2-6, and then centrifuged at 3000 rpm for 10 min, the cells were collected and resuspended in BMMY yeast induction medium with an equal volume of BMGY yeast growth medium to make the initial OD 600nm When the absorbance value was 2, the culture was continued at 30°C and 220 rpm, and 0.5% methanol, 1% trace elements (PTM1 trace elements) and ascorbic acid (final concentration 10 mM) were added every 24 h. At 72 h after induction, the expression bacterial precipitate, the supernatant after the bacterial precipitate was broken, and the fermentation liquid supernatant were taken respectively.

[0160] The bacterial precipitate and the supernatant after the bacterial precipitate was broken were identified by Western-bolt (the primary antibody was rabbit anti-P4HA1 polyclonal antibody, purchased from abcam, catalog number: ab127564). The results are as follows Fig.11 As shown, there is an obvious positive band at 28 kDa, which is consistent with the theoretical molecular weight value of P4H, indicating that P4H is successfully expressed in the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria, which is intracellular expression.

[0161] The fermentation supernatant was subjected to SDS-PAGE electrophoresis to analyze the molecular weight and expression level of the target protein. Fig.12 As shown, it can be seen that the 8 recombinant human collagen engineering bacteria were successfully expressed in the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria, which was secretory expression.

[0162] Specifically, the trace elements of PTM1 include: CuSO4·5H2O 6.0g / L, NaI 0.088g / L, MnSO4·H2O3.0g / L, Na2MoO4·2H2O 0.2g / L, H3BO30.02g / L, CoCl20.5g / L, ZnCl220.0g / L, FeSO4·7H2O65.0g / L, and biotin 0.2g / L.

[0163] 4. Purification by cation exchange chromatography

[0164] A cation exchange medium (chromatographic filler is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system) was used, and the chromatography column was balanced with phosphate buffer (20mM NaH2PO4, pH 5.0) until the conductivity value and A280 absorbance value remained unchanged, the fermentation broth supernatant was loaded, the loading flow rate was set to 20cm / h, and the ultraviolet A280 absorbance value was detected; after the loading was completed, the chromatography column was re-equilibrated with phosphate buffer until the conductivity and ultraviolet A280 absorbance values ​​were the lowest and no longer changed, and the sampling was stopped; then the corresponding protein was eluted with a buffer containing NaH2PO4-NaCl (0.5M) and the recombinant humanized collagen stock solution was obtained by ultrafiltration. The purity of the recombinant humanized collagen stock solution sample was detected by SDS-PAGE electrophoresis, and the results were as follows: Fig.13 As shown, the purity of the eight recombinant humanized collagens is very high.

[0165] Specifically, the culture medium formula used in this embodiment is as follows:

[0166] YPD solid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 2% agar;

[0167] YPD liquid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L;

[0168] MD solid medium: YNB without amino acid nitrogen source 13.4g / L; biotin 0.4mg / L; glucose 20g / L, and contains 2% agar;

[0169] BMGY yeast growth medium: yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 890mL, sterilize at 121℃ for 20 minutes, then add 10×YNB 100mL (13.4 g / L), 500×biotin 1mL (4×10 -4 g / L), glycerol 10mL;

[0170] BMMY yeast induction medium: yeast extract 10g / L, peptone 20g / L, K2HPO4 3g / L, K2HPO4 11.8g / L, add water to 895mL, sterilize at 121℃ for 20 minutes, then add 100×YNB 100mL (13.4 g / L), 500×biotin 1mL (4×10 -4 g / L), methanol 5mL.

[0171] Example 2 Analysis of Hydroxyproline Content

[0172] The oxidation product produced by hydroxyproline under the action of an oxidant reacts with dimethylaminobenzaldehyde to present a purple-red color, and its content can be inferred according to the depth of its color. In this embodiment, the hydroxyproline content in the recombinant humanized collagen sample was detected using a hydroxyproline test kit (Nanjing Jiancheng Bioengineering Institute, item number: A030-1-1). The detection method refers to the operating instructions of the test kit. At the same time, a commercially available collagen peptide (Naibo, item number: NB-650) sample was set as a positive control, and the wild-type recombinant human collagen that did not co-express hydroxyproline enzyme in Example 1 was taken as a negative control. The test results are shown in Table 3 below.

[0173] Table 3 Detection results of hydroxyproline content in recombinant human collagen

[0174]

[0175] As can be seen from Table 3, the hydroxyproline content in the wild-type recombinant human collagen and the recombinant human collagen mutants 1-7 is above 2 μg / mg, the hydroxyproline content of the commercially available natural collagen peptide is 0.97 μg / mg, and the hydroxyproline content of the wild-type recombinant human collagen without co-expressing hydroxyprolinease is 0.70 μg / mg, indicating that the wild-type recombinant human collagen and the 7 recombinant human collagen mutants of the present application have a higher hydroxyproline content than the commercially available natural collagen.

[0176] Example 3 Micro-differential scanning calorimetry (DSC) test of thermal stability

[0177] Differential scanning calorimetry (DSC) is an effective technique for evaluating the thermal stability of proteins. It works by measuring the difference in heat flow between a sample and a reference during heating. This experiment was commissioned to the Public Technology Center of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences for testing. First, the sample protein was weighed, the instrument was calibrated using standard substances (such as indium), the experimental parameters were set, the DSC was started, and the melting point detection experiment was performed. The experimental results of 8 recombinant human collagen Tm values ​​were obtained through software analysis. The test results are shown in Table 4 below.

[0178] Table 4 Differential scanning calorimetry (DSC) test results of Tm values ​​of 8 recombinant human collagens

[0179]

[0180] As can be seen from Table 4, the recombinant human collagen mutant 7 has the highest Tm value, which can reach 85.67°C, which is 30.44°C higher than the Tm value of the wild-type recombinant human collagen. Since the recombinant human collagen mutant 7 has the highest Tm value and the best thermal stability, it will be further analyzed later.

[0181] Example 4 High temperature denaturation analysis of recombinant human collagen mutant 7

[0182] 1 Effects of high temperature on the appearance and low-temperature self-assembly properties of recombinant human collagen mutant 7

[0183] Low-temperature self-assembly is one of the most important physical and chemical properties of natural collagen, and is also the basis for determining whether the physical properties of recombinant collagen are similar to those of natural collagen.

[0184] Specific test method: Heat the water bath to 100°C, take out the recombinant human collagen mutant 7 sample to be tested (the sample is in a sealed vial) from the 4°C refrigerator, put it into boiling water and heat it for 30 minutes, then take out the sample and observe the appearance change. The results are as follows: Fig.14 As shown; then sample was taken for SDS-PAGE electrophoresis detection, the results are as follows Fig.15 As shown; then put the heated sample back into the 4℃ refrigerator for 1 hour and observe the state. The result is as follows Fig.16 shown.

[0185] Depend on Fig.14 It can be seen that the recombinant human collagen mutant 7 stock solution sample changed from a gel state to a clear transparent liquid without any protein precipitation. Fig.15 It can be seen that the molecular weight of the recombinant human collagen mutant 7 stock solution sample did not change significantly after high-temperature boiling, indicating that it was not degraded; Fig.16It can be seen that after the recombinant human collagen mutant 7 stock solution sample was heated and then refrigerated, its state changed from liquid to gel, indicating that the recombinant human collagen mutant 7 has low-temperature self-assembly properties.

[0186] 2 Effect of high temperature on the cell proliferation activity of recombinant human collagen mutant 7

[0187] The detection method is based on the fact that recombinant human collagen has a stimulating effect on the growth of mouse embryonic fibroblasts (BALB / c 3T3 cells). The growth conditions of BALB / c 3T3 cells vary depending on the biological activity of recombinant human collagen, so the biological activity of recombinant human collagen is detected.

[0188] The recombinant human collagen mutant 7 sample to be tested (the sample was placed in a vial and sealed) was taken out from the 4°C refrigerator, and placed in a 100°C water bath for 30 minutes. The samples before and after the heating treatment of the recombinant human collagen mutant 7 were taken to detect the cell proliferation activity. The detection method was based on the 2020 edition of the "Pharmacopoeia of the People's Republic of China" (Part III) General Rules and Guidelines 3527 "Cell Proliferation Method / MTT Colorimetric Method". At the same time, naturally extracted rat tail collagen (purchased from Sigma, item number C7661) was set as a positive control, and the wild-type recombinant human collagen prepared in Example 1 was set as a negative control. The test results are shown in Table 5 below.

[0189] Table 5 Effect of high temperature on the cell proliferation activity of recombinant human collagen mutant 7

[0190]

[0191] As can be seen from Table 5, after being treated in a 100°C water bath for 30 minutes, the recovery rate of the cell proliferation activity of the naturally extracted rat tail collagen was only 14.0%, the recovery rate of the cell proliferation activity of the wild-type recombinant human collagen prepared in Example 1 was 25.2%, and the recovery rate of the cell proliferation activity of the recombinant human collagen mutant 7 reached 93.5%. This indicates that the recombinant human collagen mutant 7 has better heat resistance than the naturally extracted rat tail collagen and the wild-type recombinant human collagen prepared in Example 1, and the treatment in a 100°C water bath for 30 minutes has no significant effect on the cell proliferation activity of the recombinant human collagen mutant 7.

[0192] Example 5 In vitro enzymatic resistance test of recombinant human collagen mutant 7

[0193] Matrix metalloproteinase-1 (MMP-1) is a collagenase that mainly cleaves type I collagen and type III collagen. The enzymatic hydrolysis resistance test of recombinant human collagen tests its ability to resist enzymatic hydrolysis by adding MMP-1 to the sample to simulate the conditions and methods of enzymatic hydrolysis in vivo.

[0194] 1. Test materials

[0195] Sample to be tested: the recombinant human collagen mutant 7 sample prepared in Example 1;

[0196] Positive control: commercially available collagen, purchased from Sigma, catalog number C7661;

[0197] Blank control: PBS;

[0198] MMP-1 collagenase: human fibroblast collagenase, purchased from Sigma, catalog number SRP3117.

[0199] 2. The method steps are as follows

[0200] Recombinant human collagen mutant 7 samples and commercial collagen were taken separately, and the final protein concentration was 1 mg / mL with PBS, and then MMP-1 with a working concentration of 2.5 μg / mL was added, and the reaction was allowed to stand at 37°C for 1 hour. The enzyme digestion products were subjected to SDS-PAGE electrophoresis to analyze the enzymatic hydrolysis and the resistance of recombinant human collagen mutant 7 to enzymatic hydrolysis. The results are shown in Fig.17 shown.

[0201] 3. Results Analysis

[0202] After commercially available collagen was hydrolyzed by MMP-1, a large number of degradation bands appeared, and the protein bands before hydrolysis disappeared, indicating that it was completely hydrolyzed; however, the recombinant human collagen mutant 7 sample did not show obvious degradation bands, which were consistent with the protein bands of the sample before hydrolysis, indicating that recombinant human collagen mutant 7 has good resistance to collagenase hydrolysis.

[0203] Example 6 Transdermal absorption test and long-term effect analysis of recombinant human collagen mutant 7

[0204] The recombinant human collagen transdermal absorption test is mainly based on in vitro models to evaluate the penetration, absorption and long-term effect of proteins in the skin.

[0205] 1. Test materials

[0206] Test sample: recombinant human collagen mutant 7 sample prepared in Example 1, 1 mg / mL;

[0207] Positive control: commercially available collagen (Naibo, catalog number: NB-650), 1 mg / mL;

[0208] Negative control: sterile saline;

[0209] Antibody: Anti-Collagen III polyclonal antibody (abcam, catalog number: ab7778).

[0210] 2. The method steps are as follows

[0211] Anesthetize SD rats, scrape the hair off the abdominal skin, wash the skin with clean water, take 100 μL of the sample to be tested and apply it on the skin, apply once every 10 minutes, for a total of 3 times, 100 μL each time. Set up positive and negative controls at the same time; take rat skin tissue samples from two groups of different time points at 4h and 24h after application, gently wipe off the skin surface samples, kill the rats by drawing blood from the heart, and then use a blade to cut off the skin in contact with collagen; prepare sections, dewax and hydrate, block endogenous peroxidase, antigen repair to expose antigenic determinants, block nonspecific proteins, incubate antibodies, color develop, counterstain, dehydrate, make transparent, and seal for observation. After the sample was applied to the abdominal skin of SD rats for 4h, samples were taken and sections were prepared. After immunohistochemistry, the test results are as follows: Fig.18 24 hours after the sample was applied, the rat skin was sampled for immunohistochemical detection. The test results were as follows: Fig.19 shown.

[0212] 3. Results Analysis

[0213] Depend on Fig.18 It can be seen that 4 hours after applying the sample, the test results of the recombinant human collagen mutant 7 test group and the commercially available collagen peptide control group were both positive, and a large number of collagen-positive reaction areas were detected in the basal layer of the skin. The test result of the normal saline control group was negative, and no collagen was detected. Fig.19 It can be seen that 24 hours after applying the sample, the commercially available collagen peptide was hydrolyzed by protease in the basal layer of the skin, and only a small number of positive reaction areas were detected, while the recombinant human collagen mutant 7 test group still detected a large number of positive reaction areas in the basal layer of the skin, while the normal saline control group tested negative. The above results show that recombinant human collagen mutant 7 can penetrate the epidermis to reach the basal layer, has a good transdermal absorption effect, and is more long-lasting in the body than commercially available collagen peptides.

[0214] In summary, the recombinant human collagen prepared in the present application has low-temperature self-assembly properties similar to those of natural collagen, and can form a stable collagen gel without the addition of cross-linking agents or gel excipients. It has high stability, heat resistance and is not easily degraded. It is not easily enzymatically hydrolyzed in vitro and has longer effectiveness in vivo. It has been verified that recombinant human collagen mutant 7 has the best heat resistance stability, and still has good stability and biological activity after being heated at 100°C for 30 minutes. It can reform the gel when placed at low temperature and is not easily denatured or degraded by high temperature. The results of in vitro enzymatic hydrolysis tests showed that it has good in vitro resistance to collagenase enzymatic hydrolysis; the results of in vivo transdermal absorption tests showed that the recombinant human collagen mutant 7 has good transdermal absorption effect and is long-lasting.

[0215] The above description is only an optional embodiment of the present application, and does not limit the patent scope of the present application. All equivalent structural transformations made by using the contents of the present application specification and drawings under the inventive concept of the present application, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability, characterized in that: The gene sequence of the recombinant humanized collagen is shown as Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.

26.

2. A method for preparing a recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability as claimed in claim 1, characterized in that: The following steps are involved: Connecting human type I collagen peptide fragment and human type III collagen peptide fragment end to end to form wild-type collagen peptide, wherein the wild-type collagen peptide does not contain the main MMPs cleavage site; the main MMPs cleavage site includes -Gly-|-Ile-, -Pro-Gln-Gly-|-Ile-Ala-Gly-Gln-, -Asn / Asp-|-Leu-Gly-|-Ile-Asn-|-Phe-Gln-|-Thr, -Ala-|-Gln-, -Ala-|-Leu- and -Tyr-|-Leu-; Based on the wild-type collagen peptide, computer-aided protein design was used to perform structural simulation and energy calculation to obtain 7 collagen peptide mutants; The wild-type collagen peptide and the seven collagen peptide mutants are respectively used as basic units and are repeatedly connected in series four times to obtain a recombinant humanized collagen amino acid sequence as shown in Seq ID NO.11, Seq ID NO.12, Seq ID NO.13, Seq ID NO.14, Seq ID NO.15, Seq ID NO.16, Seq ID NO.17 or Seq ID NO.18; Based on the recombinant humanized collagen amino acid sequence, after optimizing the codon preference of Pichia pastoris, a recombinant humanized collagen gene sequence is obtained; Based on the recombinant humanized collagen gene sequence and the Pichia pastoris expression plasmid pGAPZαA, a recombinant humanized collagen plasmid is constructed; The human gene P4H was used as the amino acid sequence of proline hydroxylase, and the gene sequence of recombinant proline hydroxylase was obtained after optimizing the design according to the codon preference of the Pichia pastoris host. Based on the gene sequence of the recombinant proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K, a recombinant proline hydroxylase plasmid is constructed; The recombinant humanized collagen protein plasmid is subjected to enzyme cutting and linearization to obtain a linearized recombinant human collagen protein plasmid; the linearized recombinant human collagen protein plasmid is electro-transformed into Pichia pastoris GS115 competent cells, and after culture and positive transformant screening, a recombinant human collagen yeast engineering bacterium is obtained; the recombinant proline hydroxylase plasmid is subjected to enzyme cutting and linearization to obtain a linearized recombinant proline hydroxylase plasmid; the recombinant human collagen yeast engineering bacterium is used to prepare a recombinant engineering bacterium competent cell, the linearized recombinant proline hydroxylase plasmid is electro-transformed into the recombinant engineering bacterium competent cells, and after culture and co-expression positive transformant screening, a recombinant human collagen co-expressing proline hydroxylase yeast engineering bacterium is obtained; Inducing the expression of the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria, and collecting the fermentation broth supernatant by centrifugation; After the fermentation broth supernatant is purified, a recombinant humanized collagen stock solution is obtained.

3. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of obtaining the recombinant humanized collagen gene sequence after optimizing the codon preference of Pichia pastoris based on the recombinant humanized collagen amino acid sequence comprises: According to the recombinant humanized collagen amino acid sequence shown in Seq ID NO.11, Seq ID NO.12, Seq ID NO.13, Seq ID NO.14, Seq ID NO.15, Seq ID NO.16, Seq ID NO.17 or Seq ID NO.18, a terminator was added to the C-terminus, and the Pichia pastoris codon preference was optimized to obtain the recombinant humanized collagen gene sequence shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.

26.

4. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 3, characterized in that: The step of constructing a recombinant humanized collagen plasmid based on the recombinant humanized collagen gene sequence and the Pichia pastoris expression plasmid pGAPZαA comprises: Gene fragments were synthesized according to the recombinant humanized collagen gene sequences shown in Seq ID NO.19, Seq ID NO.20, Seq ID NO.21, Seq ID NO.22, Seq ID NO.23, Seq ID NO.24, Seq ID NO.25 or Seq ID NO.26, and the synthesized gene fragments were inserted between the restriction sites Xho Ⅰ and Not Ⅰ on the Pichia pastoris expression plasmid pGAPZαA to obtain recombinant humanized collagen plasmids.

5. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: In the step of obtaining the gene sequence of recombinant proline hydroxylase by using the human gene P4H as the amino acid sequence of proline hydroxylase and optimizing the design according to the codon preference of the Pichia pastoris host, the gene sequence of the recombinant proline hydroxylase is shown in Seq ID NO.

28.

6. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 5, characterized in that: The step of constructing a recombinant proline hydroxylase plasmid based on the gene sequence of the recombinant proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K comprises: According to the gene sequence of the recombinant proline hydroxylase as shown in Seq ID NO.28, the gene fragment was synthesized, and the synthesized gene fragment was inserted between the restriction sites EcoR Ⅰ and Not Ⅰ on the Pichia pastoris expression plasmid pPIC3.5K to obtain the recombinant proline hydroxylase plasmid.

7. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of linearizing the recombinant humanized collagen protein by enzyme digestion to obtain linearized recombinant human collagen protein protein comprises: The recombinant humanized collagen protein particle was subjected to enzymatic linearization treatment using QuickCut SacⅠ, the enzymatic cutting temperature was 37°C, the enzymatic cutting time was 5h, and then 3M NaAc and anhydrous ethanol were added. After being placed at -20°C overnight, the particle was centrifuged at 4°C and 13000rpm for 20min, the supernatant was discarded, the precipitate was collected and rinsed with 75% ethanol, centrifuged again and the supernatant was discarded. After removing the water and residual ethanol in the collected precipitate, it was dissolved with ddH2O to obtain a linearized recombinant human collagen protein particle.

8. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of electro-transforming the linearized recombinant human collagen protein plasmid into Pichia pastoris GS115 competent cells, culturing and screening positive transformants to obtain recombinant human collagen yeast engineering bacteria comprises: The linearized recombinant human collagen protein particles were mixed with Pichia pastoris GS115 competent cells, and after ice bath, they were electroporated, and then sorbitol solution was added, mixed, and transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, they were spread on a YPD plate containing Zeocin, and allowed to stand at room temperature for 10min, and then inverted and cultured at 30°C for 2d-5d until a single colony grew; A single colony on the YPD plate was picked up, transferred to a 96-well culture plate containing a YPD medium containing Zeocin, and cultured at 30° C. to screen and obtain recombinant human collagen yeast engineering bacteria.

9. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of linearizing the recombinant proline hydroxylase plasmid by enzyme digestion to obtain a linearized recombinant proline hydroxylase plasmid comprises: The recombinant proline hydroxylase plasmid was linearized by enzyme digestion with SacⅠ, the enzyme digestion temperature was 37°C, the enzyme digestion time was 5h, and then 3M NaAc and anhydrous ethanol were added. After being placed at -20°C overnight, the mixture was centrifuged at 4°C and 13,000 rpm for 20min, the supernatant was discarded, the precipitate was collected and rinsed with 75% ethanol, centrifuged again and the supernatant was discarded. After removing the water and residual ethanol in the collected precipitate, the mixture was dissolved with ddH2O to obtain a linearized recombinant proline hydroxylase plasmid.

10. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of preparing competent cells of recombinant engineering bacteria using the recombinant human collagen yeast engineering bacteria comprises: After streaking the recombinant human collagen yeast engineering bacteria plate, a single colony was picked and inoculated into YPD solid medium, and cultured at 30°C and 225 rpm for 24 h; then transferred to YPD liquid medium at an inoculation ratio of 1:1000, and cultured at 30°C and 225 rpm until OD 600nm The absorbance value is 1.3-1.5; then transfer to a sterile centrifuge tube, centrifuge at 4°C and 1500 rpm for 5 minutes, discard the supernatant and collect the engineered bacteria; The engineered bacteria were resuspended with sterile ultrapure water, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the engineered bacteria were resuspended with sterile ultrapure water, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the cell pellet was resuspended with sterile 1 M sorbitol, and centrifuged at 4°C, 1500 rpm for 5 min. After discarding the supernatant, the cell pellet was resuspended with sterile 1 M sorbitol, and the mixture was rotated to mix, and then placed on ice to obtain recombinant engineered bacteria competent cells.

11. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of electro-transforming the linearized recombinant proline hydroxylase plasmid into the competent cells of the recombinant engineering bacteria, culturing and screening co-expression positive transformants to obtain the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria comprises: The linearized recombinant proline hydroxylase plasmid and the competent cells of the recombinant engineering bacteria were mixed, and after ice bath, electric shock was performed, and then sorbitol solution was added, mixed, and transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, it was spread on an MD solid plate, and allowed to stand at room temperature for 10min, and then inverted and cultured at 30°C for 2d-5d, until a single colony grew; Scrape off the His on the MD solid plate + After the transformant is diluted, it is spread on a YPD plate containing G418, and cultured at 30°C upside down until a single colony appears. Then, the single colony on the YPD plate is picked and transferred to a 96-well culture plate containing YPD medium, and cultured at 30°C. The co-expression positive transformants that can grow on a plate containing 4 mg / mL of G418 are screened to obtain the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria.

12. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 11, characterized in that: The step of inducing expression of the recombinant human collagen co-expressing proline hydroxylase yeast engineering bacteria and collecting the fermentation broth supernatant by centrifugation comprises: A single colony growing on a plate containing 4 mg / mL G418 was picked and inoculated into BMGY yeast growth medium and cultured at 30°C and 220 rpm until OD 600nm The absorbance value was 2-6, and then centrifuged at 3000 rpm for 10 min, the cells were collected and resuspended in BMMY yeast induction medium with an equal volume of the BMGY yeast growth medium to make the initial OD 600nm When the absorbance value was 2, the culture was continued at 30°C and 220 rpm, and 0.5% methanol, 1% trace elements and ascorbic acid were added every 24 hours. At 72 hours after induction, the expression bacterial precipitate, the supernatant after the bacterial precipitate was broken, and the supernatant of the fermentation liquid were taken respectively.

13. The method for preparing the recombinant humanized collagen capable of low-temperature self-assembly and long-term heat-resistant stability according to claim 2, characterized in that: The step of purifying the fermentation broth supernatant to obtain a recombinant humanized collagen stock solution comprises: A cation exchange medium was used, and the chromatography column was equilibrated with a phosphate buffer until the conductivity value and the A280 absorbance value remained unchanged. The fermentation broth supernatant was loaded, and the loading flow rate was set to 20 cm / h, and the ultraviolet A280 absorbance value was detected. After the loading was completed, the chromatography column was re-equilibrated with a phosphate buffer until the conductivity and the ultraviolet A280 absorbance values ​​were the lowest and no longer changed, and the sampling was stopped; and then the corresponding protein was eluted with a phosphate buffer containing NaCl and collected to obtain a recombinant humanized collagen stock solution.

14. An application of the low-temperature self-assembly and long-lasting heat-stable recombinant humanized collagen as claimed in claim 1, characterized in that: The recombinant humanized collagen that can self-assemble at low temperature and has long-term heat resistance and stability is applied to cream-type cosmetics.

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