A recombinant type I collagen with low-temperature self-assembly characteristics, its preparation method and application

Abstract

The present application discloses a recombinant type I collagen with low-temperature self-assembly characteristics, its preparation method and application, which relates to the technical field of genetic engineering. A recombinant type I collagen with low-temperature self-assembly characteristics, the amino acid sequence of the recombinant type I collagen is obtained by repeating and concatenating the human-derived type I collagen fragment as a basic unit for n times, and the amino acid sequence of the human-derived type I collagen fragment is shown in Seq ID NO.1, and n is an integer greater than or equal to 1. In the present application, the nucleotides of the functional region repeat fragment of human-derived type I collagen are optimized and screened, and co-expressed with the hydroxyproline enzyme gene in the Pichia pastoris system, and a recombinant type I collagen with low-temperature self-assembly characteristics is successfully obtained.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a recombinant type I collagen with low-temperature self-assembly characteristics, and a preparation method and application thereof. Background Art

[0002] Type I collagen is a fibrous protein widely found in the human body. It is not only one of the most abundant and important proteins in the human body, but also found in almost all connective tissues, such as bones, skin, tendons, ligaments, sclera, cornea, and blood vessels. The structural characteristics of type I collagen are that it is composed of three α chains tightly wound in the form of a triple helix, forming a stable and tough fiber structure. This collagen plays a vital role in maintaining the integrity and function of the human body structure. The film-forming and adhesion properties of type I collagen with self-assembly properties are better than those of type I collagen without self-assembly properties. The membranes, scaffolds, gels and other materials prepared by type I collagen with self-assembly properties have the advantage of high mechanical strength and are ideal tissue replacement materials. They can be used in artificial bones, artificial joints, artificial teeth, artificial valves, artificial blood vessels, and artificial skin. However, the self-assembly process of type I collagen is complex and is affected by many factors, such as solution conditions, temperature, pH value, etc. The large-scale production of recombinant type I collagen requires efficient production processes and quality control measures. The current expression system may have problems of low yield and high cost, and there are challenges in maintaining the natural structure and function of type I collagen. Therefore, the precise control and regulation of the self-assembly process by synthetic biology recombinant proteins is still limited, and it is difficult to achieve the self-assembly of recombinant type I collagen. Based on this, the present application proposes a recombinant type I collagen with low-temperature self-assembly characteristics and its preparation method and application. Summary of the invention

[0003] The main purpose of the present application is to provide a recombinant type I collagen with low-temperature self-assembly characteristics and a preparation method and application thereof, aiming to solve the technical problem that the prior art is difficult to achieve self-assembly of recombinant type I collagen.

[0004] To achieve the above-mentioned purpose, the present application proposes a recombinant type I collagen with low-temperature self-assembly properties, wherein the amino acid sequence of the recombinant type I collagen is obtained by repeating a human type I collagen fragment as a basic unit n times in series, and the amino acid sequence of the human type I collagen fragment is shown in Seq ID NO.1, where n is an integer greater than or equal to 1.

[0005] Optionally, n is 1, 2 or 3, and the amino acid sequence of the recombinant type I collagen is shown in Seq ID NO.1, Seq ID NO.2 or Seq ID NO.3.

[0006] Optionally, the gene sequence of the recombinant type I collagen is as shown in Seq ID NO.4, Seq ID NO.5 or Seq ID NO.6.

[0007] The present application also provides a method for preparing recombinant type I collagen with low-temperature self-assembly properties, comprising the following steps:

[0008] According to the codon preference of the Pichia pastoris host, the gene sequence encoding the recombinant type I collagen is optimized and designed to obtain the gene sequence of the recombinant type I collagen;

[0009] According to the gene sequence of the recombinant type I collagen and the Pichia pastoris expression plasmid pPICZαA, a recombinant plasmid pPICZαA-COL I is constructed;

[0010] Using virusoid P4H as the amino acid sequence of prolyl hydroxylase, after optimization and design according to the codon preference of the Pichia pastoris host, the gene sequence of prolyl hydroxylase as shown in Seq ID NO.8 is obtained;

[0011] According to the gene sequence of the prolyl hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K, a recombinant plasmid pPIC3.5K-P4H is constructed;

[0012] The recombinant plasmid pPICZαA-COL I is digested and linearized to obtain a linearized plasmid pPICZαA-COLI; the linearized plasmid pPICZαA-COL I is electrotransformed into Pichia pastoris X33 competent cells, and after culturing and screening for positive transformants, a recombinant humanized collagen yeast engineering bacterium is obtained; the recombinant plasmid pPIC3.5K-P4H is digested and linearized to obtain a linearized plasmid pPIC3.5K-P4H; using the recombinant humanized collagen yeast engineering bacterium to prepare recombinant engineering bacterium competent cells, and the linearized plasmid pPIC3.5K-P4H is electrotransformed into the recombinant engineering bacterium competent cells, and after culturing and screening for co-expression positive transformants, a recombinant yeast engineering bacterium X33 / pPICZαA-COL I_P4H is obtained;

[0013] The recombinant yeast engineering bacterium X33 / pPICZαA-COL I_P4H is induced to express to obtain an engineering bacterium with the highest protein expression level, and then high-density fermentation is carried out, and the fermentation supernatant is collected by centrifugation;

[0014] After purifying the fermentation supernatant, a recombinant type I collagen stock solution is obtained.

[0015] Optionally, the step of constructing the recombinant plasmid pPICZαA-COL I according to the gene sequence of the recombinant type I collagen and the Pichia pastoris expression plasmid pPICZαA includes:

[0016] Synthesize a gene fragment according to the gene sequence of the recombinant type I collagen, and insert the synthesized gene fragment into the Pichia pastoris expression plasmid pPICZαA through the restriction enzyme sites Xho I and Not I to obtain the recombinant plasmid pPICZαA-COLI.

[0017] Optionally, the step of constructing the recombinant plasmid pPIC3.5K-P4H according to the gene sequence of the prolyl hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K includes:

[0018] Synthesize a gene fragment according to the gene sequence of the prolyl hydroxylase, and insert the synthesized gene fragment into the Pichia pastoris expression plasmid pPIC3.5K through the restriction enzyme sites EcoR I and Not I to obtain the recombinant plasmid pPIC3.5K-P4H.

[0019] Optionally, the step of digesting the recombinant plasmid pPICZαA-COL I to obtain the linearized plasmid pPICZαA-COL I includes:

[0020] Perform restriction enzyme linearization on the recombinant plasmid pPICZαA-COL I using QuickCut Sac I, with a restriction enzyme digestion temperature of 37°C and a digestion time of 5 h. Then add 3M NaAc and absolute ethanol, place it at -20°C overnight, centrifuge at 4°C and 13,000 rpm for 20 min, discard the supernatant, collect the precipitate and wash it with 75% ethanol, centrifuge again and discard the supernatant. After removing the moisture and residual ethanol in the collected precipitate, dissolve it with ddH2O to obtain the linearized plasmid pPICZαA-COL I.

[0021] Optionally, the step of electrotransforming the linearized plasmid pPICZαA-COL I into Pichia pastoris X33 competent cells, culturing and screening for positive transformants to obtain the recombinant humanized collagen yeast engineering bacteria includes:

[0022] Mix the linearized plasmid pPICZαA-COL I with Pichia pastoris X33 competent cells, after ice bath, perform electroporation, then add sorbitol solution, mix well, transfer it to a sterile EP tube, incubate at 30°C for 1 h - 2 h, spread it on a YPD plate containing Zeocin, let it stand at room temperature for 10 min, and then incubate it inverted at 30°C for 2 d - 5 d until single colonies grow;

[0023] Pick the single colonies on the YPD plate and transfer them to a 96-well culture plate containing YPD liquid medium with Zeocin, and continue to culture at 30 °C to screen and obtain the recombinant humanized collagen yeast engineering bacteria.

[0024] Optionally, the step of digesting and linearizing the recombinant plasmid pPIC3.5K-P4H to obtain the linearized plasmid pPIC3.5K-P4H includes:

[0025] Perform digestion and linearization treatment on the recombinant plasmid pPIC3.5K-P4H using QuickCut SacⅠ, with the digestion temperature at 37 °C and the digestion time at 5 h. Then add 3M NaAc and absolute ethanol, place it at -20 °C overnight, centrifuge at 4 °C and 13,000 rpm for 20 min, discard the supernatant, collect the precipitate and rinse it with 75% ethanol. Centrifuge again and discard the supernatant. After removing the water and residual ethanol in the collected precipitate, dissolve it with ddH2O to obtain the linearized plasmid pPIC3.5K-P4H.

[0026] Optionally, the step of preparing the recombinant engineering bacteria competent cells with the recombinant humanized collagen yeast engineering bacteria includes:

[0027] After streaking the recombinant humanized collagen yeast engineering bacteria on the plate, pick single colonies and inoculate them into YPD liquid medium, and culture at 30 °C and 225 rpm for 24 h; then transfer them to YPD liquid medium at an inoculation ratio of 1:1000 and culture at 30 °C and 225 rpm until the OD 600nm The absorbance value is 1.3 - 1.5; then transfer it to a sterile centrifuge tube, centrifuge at 4 °C and 3000 rpm for 5 min, discard the supernatant and collect the bacterial cells;

[0028] Resuspend the bacterial cells with 50 mL of sterile ultrapure water and centrifuge at 4 °C and 3000 rpm for 5 min. After discarding the supernatant, resuspend the bacterial cells with 50 mL of sterile ultrapure water again and centrifuge at 4 °C and 3000 rpm for 5 min. After discarding the supernatant, resuspend the cell precipitate with 40 mL of sterile 1M sorbitol; then centrifuge at 4 °C and 3000 rpm for 5 min. After discarding the supernatant, resuspend the cell precipitate with 100 μL - 150 μL of sterile 1M sorbitol, rotate and mix well, and place it on ice to obtain the recombinant engineering bacteria competent cells.

[0029] Optionally, the step of electrotransforming the linearized plasmid pPIC3.5K-P4H into the recombinant engineering bacteria competent cells, through culture and screening of co-expressed positive transformants to obtain the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H includes:

[0030] Mix the linearized plasmid pPIC3.5K-P4H with the recombinant engineered bacterial competent cells. After ice-bathing, perform electroporation, then add sorbitol solution, mix well, transfer to a sterile EP tube, incubate at 30°C for 1 h - 2 h, spread on an MD solid plate, let stand at room temperature for 10 min, and then incubate in an inverted position at 30°C for 2 d - 5 d until single colonies grow.

[0031] Scrape the His + transformants on the MD solid plate, dilute them, and spread on a YPD plate containing G418. Incubate in an inverted position at 30°C until single colonies appear, then pick the single colonies on the YPD plate and transfer them to a 96-well culture plate containing YPD liquid medium, and continue to culture at 30°C to screen for co-expression positive transformants that can grow on a YPD plate containing 4 mg / mL G418, and obtain the recombinant yeast engineered strain X33 / pPICZαA-COL I_P4H.

[0032] Optionally, the step of inducing the expression of the recombinant yeast engineered strain X33 / pPICZαA-COL I_P4H to obtain the engineered strain with the highest protein expression level includes:

[0033] Pick single colonies growing on a YPD plate containing 4 mg / mL G418 and inoculate them into BMGY yeast growth medium, culture at 30°C and 220 rpm for 24 h until the OD 600nm absorbance is 2 - 6, then centrifuge at a rate of 3000 rpm for 10 min, collect the bacterial cells and resuspend them with BMMY yeast induction medium with the same volume as the BMGY yeast growth medium to make the initial OD 600nm absorbance 2, then continue to culture at 30°C and 220 rpm, add 1%, 1.5%, and 2% methanol every 24 h respectively, and take samples at 24 h, 48 h, 72 h, and 96 h after induction to detect the protein expression level by SDS-PAGE electrophoresis to obtain the engineered strain with the highest protein expression level.

[0034] Optionally, the step of performing high-density fermentation and centrifuging to collect the fermentation supernatant includes:

[0035] Add the fermentation medium to the fermenter, place the fermenter in a steam sterilizer and sterilize at 121°C for 15 min, cool down and calibrate the electrode, and wait for inoculation;

[0036] Inoculate the engineered strain with the highest protein expression level into YPD liquid medium and culture overnight at 30°C and 220 rpm to obtain a primary seed solution;

[0037] Inoculate the primary seed liquid into the YPD liquid medium at an inoculation amount of 1% (v / v), and amplify the culture until the OD 600nm The absorbance value is 4 - 6 to obtain the secondary seed liquid;

[0038] Inoculate the secondary seed liquid into the fermenter at an inoculation amount of 10% (v / v), and simultaneously add PTM4 trace elements for fermentation culture; the culture temperature is 30°C, the stirring speed is 400 rpm - 500 rpm, the ventilation volume is 2 L / min - 6 L / min, and 50% (w / w) ammonia water is added to control and maintain the pH in the fermenter at 5.0;

[0039] When the dissolved oxygen DO value in the fermenter continuously rises, add the feeding medium and control the feeding rate to keep the dissolved oxygen DO value between 20% - 50%. After 96 hours, stop the fermentation, and separate the fermentation supernatant through a solid-liquid separation system.

[0040] Optionally, the step of obtaining the recombinant type I collagen stock solution after purifying the fermentation supernatant includes:

[0041] Use a cation exchange medium to balance the chromatography column with a phosphate buffer until the conductivity value and the A280 absorbance value remain unchanged. Load the fermentation supernatant with a loading flow rate of 20 cm / h and detect the ultraviolet A280 absorbance value; after the loading is completed, re-balance the chromatography column with the phosphate buffer until the conductivity and the ultraviolet A280 absorbance value are the lowest and no longer change, and stop sampling; then elute with a phosphate buffer containing NaCl and collect the corresponding protein. After ultrafiltration, buffer exchange, and concentration, the recombinant type I collagen stock solution is obtained.

[0042] This application also proposes an application of the recombinant type I collagen with low-temperature self-assembly characteristics, and the recombinant type I collagen is applied to the preparation of biomaterials, tissue materials, or drug carriers.

[0043] In summary, the present application optimizes and screens the nucleotides of the repetitive fragments of the functional region of human type I collagen, and co-expresses them with the hydroxylase gene in the Pichia pastoris system. Hydroxyproline can enhance the cross-linking and stability between collagen molecules, so as to reduce problems such as low expression level, improper protein folding, and poor stability faced during the process of expressing recombinant proteins. As a result, recombinant type I collagen with low-temperature self-assembly characteristics is successfully obtained, and it retains better activity than natural collagen, has lower production costs and more stable product quality, and can be stably produced industrially. After verification, the recombinant type I collagen prepared in the present application has an obvious triple-helix structure, shows obvious self-assembly characteristics at high protein concentration and low temperature conditions, and its film-forming property and adhesiveness are superior to those of natural collagen without self-assembly characteristics. Therefore, it has broad application prospects in the biomedical field. For example, using recombinant type I collagen nanofibers to prepare biological scaffolds, which have good biocompatibility and biodegradability; using recombinant type I collagen to prepare tissue replacement materials; combining drugs with recombinant type I collagen and using its nanofibers as drug carriers to achieve targeted drug delivery; using recombinant type I collagen nanofibers for surface modification of biomaterials to improve the bioactivity of biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0045] Figure 1 It is the spectrum of the recombinant plasmid pPICZαA-COL I01 described in the embodiment of the present application;

[0046] Figure 2 It is the spectrum of the recombinant plasmid pPICZαA-COL I02 described in the embodiment of the present application;

[0047] Figure 3 It is the spectrum of the recombinant plasmid pPICZαA-COL I03 described in the embodiment of the present application;

[0048] Figure 4 It is the spectrum of the recombinant plasmid pPIC3.5K-P4H described in the embodiment of the present application;

[0049] Figure 5SDS-PAGE electrophoresis result diagram of the supernatant induced and expressed by the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H and X33 / pPICZαA-COL I02_P4H described in the embodiments of the present application;

[0050] Figure 6 SDS-PAGE electrophoresis result diagram of the supernatant induced and expressed by the recombinant yeast engineering bacteria X33 / pPICZαA-COL I03_P4H described in the embodiments of the present application;

[0051] Figure 7 SDS-PAGE electrophoresis result diagram of the target protein after high-density fermentation described in the embodiments of the present application;

[0052] Figure 8 Colorimetric method determination result diagram of the protein content of the recombinant type I collagen described in the embodiments of the present application;

[0053] Figure 9 Promoting cell proliferation activity detection result diagram of the recombinant type I collagen described in the embodiments of the present application;

[0054] Figure 10 Promoting cell adhesion activity detection result diagram of the recombinant type I collagen described in the embodiments of the present application;

[0055] Figure 11 Ultraviolet absorption spectrum diagram of the recombinant type I collagen described in the embodiments of the present application;

[0056] Figure 12 Self-assembly kinetic curve diagram of the recombinant type I collagen COL102 described in the embodiments of the present application;

[0057] Figure 13 Self-assembly photo diagram of the recombinant type I collagen COL102 after being placed at 4°C for 12 hours described in the embodiments of the present application;

[0058] Figure 14 Infrared spectrum scanning diagram of the bovine type I collagen reference substance described in the embodiments of the present application;

[0059] Figure 15 Infrared spectrum scanning diagram of the recombinant type I collagen COL102 described in the embodiments of the present application.

[0060] The realization, functional features and advantages of the present application will be further described in combination with the embodiments with reference to the accompanying drawings. Detailed implementation manners

[0061] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts belong to the scope of protection of the present application.

[0062] Sequence Listing Description (the sequence listing content is provided separately):

[0063] The amino acid sequence of the human type I collagen fragment in the embodiment of the present application is shown in Seq ID NO.1, and can also be expressed as the amino acid sequence of recombinant type I collagen when n = 1;

[0064] The amino acid sequence of recombinant type I collagen when n = 2 in the embodiment of the present application is shown in Seq ID NO.2;

[0065] The amino acid sequence of recombinant type I collagen when n = 3 in the embodiment of the present application is shown in Seq ID NO.3;

[0066] The gene sequence of recombinant type I collagen when n = 1 in the embodiment of the present application is shown in Seq ID NO.4;

[0067] The gene sequence of recombinant type I collagen when n = 2 in the embodiment of the present application is shown in Seq ID NO.5;

[0068] The gene sequence of recombinant type I collagen when n = 3 in the embodiment of the present application is shown in Seq ID NO.6;

[0069] The amino acid sequence of the virus-like P4H in the embodiment of the present application is shown in Seq ID NO.7;

[0070] The gene sequence of the virus-like P4H in the embodiment of the present application is shown in Seq ID NO.8.

[0071] For the convenience of those skilled in the art to understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

[0072] Example 1 Construction, Identification of Recombinant Yeast Engineering Bacteria and Preparation of Recombinant Type I Collagen

[0073] 1 Construction of Expression Vector

[0074] The amino acid sequence of the recombinant type I collagen described in this application is obtained by repeating and concatenating the human-derived type I collagen fragment as the basic unit n times. The amino acid sequence of the human-derived type I collagen fragment is shown in Seq ID NO.1, which is derived from human-derived type I collagen. The reference sequence of human-derived type I collagen is: UniProt P02461 sequence (https: / / www.uniprot.org / uniprot / P02452). In this application, by analyzing the triple helix region of the mature peptide of human-derived type I collagen, a human-derived type I collagen domain fragment (G932-1156N) shown in Seq ID NO.1 was screened out, and then repeated and concatenated n times based on the sequence shown in Seq ID NO.1, with the head and tail of each repeated sequence directly connected. Preferably, n = 1, 2 or 3, that is: the amino acid sequence of the recombinant type I collagen is shown in Seq ID NO.1, Seq ID NO.2 or Seq ID NO.3. At this time, the recombinant type I collagen has good low-temperature self-assembly characteristics and retains better activity than natural collagen.

[0075] Furthermore, according to the codon preference of the Pichia pastoris host, the gene sequence encoding recombinant type I collagen was optimized and designed. A yeast self-cleavage site "KR" was designed and introduced at the N-terminus, and a terminator was added at the C-terminus. At the 5' end of the optimized gene sequence of recombinant type I collagen, that is, between XhoⅠ (Takara, catalog number: 1635) and the target gene sequence, the base sequence AAAAAGA was added, and GenScript Biotech Corporation was commissioned to synthesize the gene fragment to obtain the gene sequence of recombinant type I collagen shown in Seq ID NO.4, Seq ID NO.5 or Seq ID NO.6. The synthesized gene fragment was inserted into the Pichia pastoris expression plasmid pPICZαA through the restriction enzyme sites of XhoⅠ and NotⅠ (Takara, catalog number: 1623) to obtain recombinant plasmids pPICZαA-COL I01 (as Figure 1 shown), pPICZαA-COL I02 (as Figure 2 shown) and pPICZαA-COL I03 (as Figure 3 shown).

[0076] Furthermore, to co-express recombinant type I collagen and the hydroxyproline enzyme gene in the Pichia pastoris system, this application selects prolyl hydroxylase. Since the complex structure and low activity of animal P4H (prolyl 4-hydroxylase) limit its application, most P4Hs from plants and viruses are monomeric polypeptides and can catalyze proline-rich polypeptide chains. It has been found that co-expressing the P4H (L593) of virusoid with a 38 kDa collagen chain in Escherichia coli yields hydroxylated collagen with a relatively high hydroxylation rate. Therefore, this application uses the amino acid sequence of virusoid P4H as the prolyl hydroxylase, and the specific sequence is shown in Seq ID NO.7, with a predicted molecular weight of approximately 28 kDa.

[0077] The gene sequence of prolyl hydroxylase was optimized and designed according to the codon preference of the Pichia pastoris host, and a 6×His tag sequence and a terminator were added at the C-terminus to make it more suitable for expression in Pichia pastoris, obtaining the gene sequence of prolyl hydroxylase shown in Seq ID NO.8.

[0078] According to the gene sequence of prolyl hydroxylase shown in Seq ID NO.8, GenScript Biotech Corporation was commissioned to synthesize the gene fragment, and the synthesized gene fragment was inserted into the Pichia pastoris expression plasmid pPIC3.5K through the restriction enzyme sites of EcoRⅠ (Takara, catalog number: 1611) and NotⅠ, obtaining the recombinant plasmid pPIC3.5K-P4H, as Figure 4 shown.

[0079] 2 Construction and screening of recombinant engineering bacteria

[0080] 2.1 Obtaining recombinant plasmids

[0081] The above-mentioned recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02, pPICZαA-COL I03 and pPIC3.5K-P4H were amplified and cultured, and then high-concentration plasmids were extracted for standby (Omega, catalog number: D6904).

[0082] 2.2 Linearization of recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02 and pPICZαA-COL I03

[0083] Use QuickCut SacⅠ (Takara, catalog number: 1606) to digest and linearize the 15 μg recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02 and pPICZαA-COL I03 extracted in 2.1. The digestion conditions are 37 °C for 5 h, and the digestion system is shown in Table 1 below.

[0084] Table 1 Digestion system

[0085]

[0086] Add 0.1 volume of 3M NaAc (pH 5.2) and 2.5 volumes of absolute ethanol. After standing overnight at -20°C, centrifuge at 4°C and 13,000 rpm for 20 min. Discard the supernatant, collect the precipitate, and wash it with 700 μL of 75% ethanol. Then centrifuge at 4°C and 13,000 rpm for 20 min again and discard the supernatant. Invert the EP tube on the absorbent paper in the laminar flow hood for about 10 min to remove moisture and residual ethanol as much as possible. Dissolve it with 20 μL of ddH2O, take 1 μL and dilute it 10 times, and detect the nucleic acid concentration with one-drop. Obtain the linearized plasmids pPICZαA-COL I01, pPICZαA-COL I02, and pPICZαA-COL I03.

[0087] 2.3 Preparation of X33 competent cells

[0088] After streaking the X33 strain on a plate, pick a single colony and inoculate it into 20 mL of YPD liquid medium. Culture it at 30°C and 225 rpm for 24 h; then transfer it to 50 mL of YPD liquid medium at an inoculation ratio of 1:1000 and culture it at 30°C and 225 rpm until the OD 600nm absorbance value is 1.3 - 1.5; then transfer the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and collect the bacterial cells; then resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water and centrifuge at 4°C and 3000 rpm for 5 min. After discarding the supernatant, resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water again; centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 40 mL of pre-cooled sterile 1M sorbitol; then centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 100 μL - 150 μL of pre-cooled sterile 1M sorbitol. Gently rotate and mix well, place it on ice for use, and obtain X33 competent cells.

[0089] 2.4 Electroporation into X33 competent cells

[0090] Take 100 μL of X33 competent cells and mix them with 10 μL of linearized plasmid pPICZαA-COL I01, pPICZαA-COL I02, or pPICZαA-COL I03. Transfer the mixture into a pre-chilled electroporation cuvette, immediately ice-bath for 5 min, then place it on the electroporator. Select the yeast mode of the electroporator for electroporation. Immediately after that, add 1 mL of pre-chilled 1 M sorbitol solution to the electroporation cuvette, mix well, and transfer the mixture to a sterile EP tube. After incubating statically in a 30 °C incubator for 1 h - 2 h, take 100 μL - 200 μL of the incubated bacterial liquid and spread it on a YPD plate containing 0.1 mg / mL Zeocin (bleomycin). Let it stand at room temperature for 10 min, and then incubate it upside down in a 30 °C incubator for 2 d - 5 d until single colonies grow.

[0091] 2.5 Screening of positive transformants

[0092] Pick the single colonies from the YPD plate and transfer them to the first 96-well culture plate containing 200 μL of YPD liquid medium (containing 0.1 mg / mL Zeocin), and continue to culture at 30 °C. After 48 h, blow the bacterial liquid evenly, and transfer 10 μL of the bacterial liquid from each well to the second 96-well culture plate (containing 190 μL of YPD). After continuing to culture for 24 h, blow the bacterial liquid evenly, and transfer 10 μL of the bacterial liquid from each well to the third 96-well culture plate. After 24 h, blow the bacterial liquid in the third 96-well culture plate evenly, and take 1 μL and spot it on YPD plates containing 0.1 mg / mL, 0.2 mg / mL, and 0.3 mg / mL Zeocin respectively for continued culture. If the positive transformants can grow on the plate containing 0.3 mg / mL high-concentration Zeocin, it indicates that the positive transformants contain more copies of the target gene. Through this step of screening, a recombinant humanized collagen yeast engineering bacterium with high expression efficiency can be obtained.

[0093] 2.6 Secondary electroporation of recombinant humanized collagen yeast engineering bacteria

[0094] After streaking the recombinant humanized collagen yeast engineering bacteria on a plate, pick single colonies and inoculate them into 20 mL of YPD liquid medium, and culture them at 30 °C and 225 rpm for 24 h. Then transfer them to 50 mL of YPD liquid medium at an inoculation ratio of 1:1000, and culture them at 30 °C and 225 rpm until OD 600nmThe absorbance value is 1.3 - 1.5; then transfer the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant and collect the bacterial cells; then resuspend the bacterial cell precipitate with 50 mL of pre-cooled sterile ultrapure water, and centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and then resuspend the bacterial cell precipitate with 50 mL of pre-cooled sterile ultrapure water; centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and then resuspend the cell precipitate with 40 mL of pre-cooled sterile 1 M sorbitol; then centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and resuspend the cell precipitate with 100 μL - 150 μL of pre-cooled sterile 1 M sorbitol, gently rotate and mix evenly, place on ice for use, and obtain the competent cells of the recombinant engineering bacteria.

[0095] Use QuickCut SacⅠ to perform enzymatic digestion and linearization treatment on the recombinant plasmid pPIC3.5K - P4H. The enzymatic digestion temperature is 37°C and the enzymatic digestion time is 5 h. Then add 3 M NaAc and absolute ethanol, place at -20°C overnight, centrifuge at 4°C and 13000 rpm for 20 min, discard the supernatant, collect the precipitate and wash it with 75% ethanol, centrifuge again and discard the supernatant. After removing the moisture and residual ethanol in the collected precipitate, dissolve it with ddH2O to obtain the linearized plasmid pPIC3.5K - P4H.

[0096] Mix the linearized plasmid pPIC3.5K - P4H with the competent cells of the recombinant engineering bacteria, after ice bath, perform electroporation, then add the sorbitol solution, mix evenly, transfer to a sterile EP tube, incubate at 30°C for 1 h - 2 h, then coat on the MD solid plate, let it stand at room temperature for 10 min, and then incubate in an inverted position in a 30°C incubator for 2 d - 5 d until single colonies grow;

[0097] Add 2 mL of sterile double-distilled water to the surface of the MD solid plate, and then gently scrape the His + transformants on the plate surface with a sterile triangular spreader and transfer them to a 50 mL centrifuge tube, dilute them with sterile water to a cell suspension with a concentration of 10 5 cells / ml (count with a spectrophotometer, 1 OD 600 ≈5×10 7cells / ml), and spread them on YPD plates containing 0.5 mg / mL G418. After inverted incubation at 30 °C for 3 - 4 days until single colonies appear; then pick the single colonies from the YPD plates and transfer them to the first 96-well culture plate containing 200 μL of YPD liquid medium, and continue to culture at 30 °C; after 48 h, blow the bacterial liquid evenly, and transfer 10 μL of the bacterial liquid from each well to the second 96-well culture plate (containing 190 μL of YPD); continue to culture for 24 h, then blow the bacterial liquid evenly, and transfer 10 μL of the bacterial liquid from each well to the third 96-well culture plate; after 24 h, blow the bacterial liquid in the third 96-well culture plate evenly, and take 1 μL and spot it on YPD plates containing 1 mg / mL, 2 mg / mL, and 4 mg / mL G418 respectively for continued culture; if the co-expression positive transformants can grow on the plates containing 4 mg / mL high-concentration G418, it indicates that the co-expression positive transformants contain more copies of the target gene. Through this step of screening, the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H, X33 / pPICZαA-COLI02_P4H, and X33 / pPICZαA-COL I03_P4H that can be highly expressed can be obtained.

[0098] 3 Identification of Induced Expression

[0099] Pick the single colonies that grew on the plates containing 4 mg / mL G418 in step 2.6 and inoculate them into BMGY yeast growth medium, and culture them at 30 °C and 220 rpm until the OD 600nm The absorbance value is 2 - 6, then centrifuge at a rate of 3000 rpm for 10 min, collect the bacterial cells and resuspend them with BMMY yeast induction medium with the same volume as the BMGY yeast growth medium to make the initial OD 600nm The absorbance value is 2, and then continue to culture at 30 °C and 220 rpm. Add 1%, 1.5%, and 2% methanol respectively every 24 h. Take bacterial liquid samples at 24 h, 48 h, 72 h, and 96 h after induction, centrifuge to collect the expression supernatant, and analyze the expression of the target protein by SDS-PAGE electrophoresis. Among them, the protein expression of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H and X33 / pPICZαA-COLI02_P4H at 24 h after induction is as shown in Figure 5 shown; the protein expression of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I03_P4H at 24 h after induction is as shown in Figure 6As shown, the recombinant yeast engineering strain X33 / pPICZαA-COL I01_P4H can produce recombinant type I collagen of about 20 kDa after induction, the recombinant yeast engineering strain X33 / pPICZαA-COL I02_P4H can produce recombinant type I collagen of about 40 kDa after induction, and the recombinant yeast engineering strain X33 / pPICZαA-COL I03_P4H can produce recombinant type I collagen of about 60 kDa after induction.

[0100] 4 High-density fermentation

[0101] Add 2 L of fermentation medium to a 5 L fermenter, place the fermenter in a steam sterilizer and sterilize at 121 °C for 15 min. After cooling, calibrate the electrode and wait for inoculation;

[0102] Inoculate the engineering strain with the highest protein expression level into YPD liquid medium and culture overnight at 30 °C and 220 rpm to activate the cells and obtain a primary seed solution;

[0103] Inoculate the primary seed solution into 200 mL of YPD liquid medium at an inoculation amount of 1% (v / v), and amplify the culture until the OD 600nm Absorbance value is 4 - 6 to obtain a secondary seed solution;

[0104] Inoculate the secondary seed solution into the fermenter at an inoculation amount of 10% (v / v), and simultaneously add 8.7 mL of PTM4 trace elements for fermentation culture; the culture temperature is 30 °C, the stirring speed is 400 rpm - 500 rpm, the ventilation volume is 2 L / min - 6 L / min, and 50% (w / w) ammonia water is added to control the pH in the fermenter to 5.0;

[0105] When the dissolved oxygen DO value in the fermenter continuously rises, it indicates that the glycerol in the medium has been exhausted by the cells. At this time, add the feeding medium and control the feeding rate to make the dissolved oxygen DO value show a periodic fluctuation within the range of 20% - 50%. Take bacterial liquid samples at 24 h, 48 h, and 72 h after fermentation, separate the fermentation supernatant through a solid-liquid separation system, and analyze the molecular weight of the target protein by SDS-PAGE electrophoresis. The results are as Figure 7 shown.

[0106] Figure 7In the process, after 24 hours of fermentation induction of the recombinant yeast engineering strain X33 / pPICZαA-COL I01_P4H, a phenomenon of slow methanol metabolism occurred, and it was unable to effectively metabolize methanol. The fermentation terminated at 48 hours. This phenomenon occurred in all three batches of fermentation of this strain, so this strain was discarded subsequently; both the recombinant yeast engineering strains X33 / pPICZαA-COL I02_P4H and X33 / pPICZαA-COL I03_P4H could be normally fermented to 96 hours, but X33 / pPICZαA-COL I03_P4H showed a degradation phenomenon in the later stage of fermentation.

[0107] Specifically, the PTM4 trace elements are the Pichia trace minerals 4 trace element solution, including: 2.0 g / L of CuSO4·5H2O, 0.08 g / L of NaI, 3.0 g / L of MnSO4·H2O, 0.2 g / L of Na2MoO4·2H2O, 0.02 g / L of H3BO3, 0.5 g / L of CaSO4·2H2O, 0.5 g / L of CoCl2, 7.0 g / L of ZnCl2, 22.0 g / L of FeSO4·7H2O, 0.2 g / L of biotin, and 1.0 mL / L of H2SO4.

[0108] 5 Purification

[0109] Using a cation exchange medium (the chromatography packing material is SP Purose 6 High Performance produced by Qianchun, loaded on the GE Akta chromatography system), equilibrate the chromatography column with a phosphate buffer solution (20 mM NaH2PO4, pH 4.7) until the conductivity value and the A280 absorbance value remain unchanged. Load the fermentation supernatant, set the loading flow rate at 20 cm / h, and detect the ultraviolet A280 absorbance value; after the loading is completed, re-equilibrate the chromatography column with the phosphate buffer solution until the conductivity and the ultraviolet A280 absorbance value are the lowest and no longer change, and stop sample collection; then elute with a NaH2PO4 buffer solution containing NaCl (1 M) and collect the corresponding protein. After ultrafiltration, buffer exchange, and concentration, the recombinant type I collagen stock solution is obtained. Among them, the recombinant type I collagen obtained by fermenting the recombinant yeast engineering strain X33 / pPICZαA-COL I02_P4H is denoted as COL102, and the recombinant type I collagen obtained by fermenting the recombinant yeast engineering strain X33 / pPICZαA-COL I03_P4H is denoted as COL103. The following descriptions will all use this abbreviation.

[0110] Specifically, the medium formula used in this example is as follows:

[0111] YPD solid medium: 10 g / L of yeast extract, 20 g / L of peptone, 20 g / L of glucose, and contains 2% agar;

[0112] YPD liquid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose;

[0113] MD solid medium: 13.4 g / L YNB without amino acid nitrogen source; 0.4 mg / L biotin; 20 g / L glucose, and contains 2% agar;

[0114] BMGY yeast growth medium: 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L K2HPO4, add water to 890 mL, sterilize at 121 °C for 20 minutes, then after the temperature drops to 60 °C, add 100 mL of 10×YNB (13.4 g / L), 1 mL of 500×biotin (4×10 -4 g / L), 10 mL of glycerol on the laminar flow bench;

[0115] BMMY yeast induction medium: 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L K2HPO4, add water to 895 mL, sterilize at 121 °C for 20 minutes, then after the temperature drops to 60 °C, add 100 mL of 100×YNB (13.4 g / L), 1 mL of 500×biotin (4×10 -4 g / L), 5 mL of methanol;

[0116] Fermentation medium: 26.7 mL / L H3PO4 (85%), 1.175 g / L CaSO4·2H2O, 18.2 g / L K2SO4, 14.9 g / L MgSO4·7H2O, 4.13 g / L KOH, 40.0 g / L glycerol, 4.35 mL / L PTM4, adjust the pH value to 5.0 with ammonia water;

[0117] Feeding medium: 50% glycerol, 0.8% PTM4 trace elements.

[0118] Example 2 Protein content detection

[0119] The protein contents of the recombinant type I collagen COL102 and COL103 prepared in Example 1 of this application were detected. Refer to the "Fourth Method (BCA Method)" in Appendix "0731" of the Chinese Pharmacopoeia (2020 Edition, Part III). This method is based on the reduction of Cu 2+ to Cu + by protein molecules in an alkaline solution, and the combination of 2,2'-biquinoline-4,4'-dicarboxylic acid (BCA) with Cu + to form a purple complex. Within a certain range, the color depth is proportional to the protein concentration. A standard curve is made with the protein reference solution, and the protein content in the test sample is determined by colorimetry.

[0120] In this experiment, a "BCA Protein Concentration Assay Kit" (purchased from Beyotime Biotechnology, catalog number P0010) was used to detect protein content.

[0121] Test samples: Recombinant type I collagen COL102 and COL103.

[0122] Specific operation steps (see the kit instruction manual for details): Dilute the standard BSA to prepare solutions of 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL. Add 20 μL of each solution to a 96-well plate, with 2 parallels in each group. Add 200 μL of BCA working solution (BCA reagent A: BCA reagent B = 50:1) to each well, mix well, and incubate at 37 °C for 25 min; measure the absorbance at a wavelength of 562 nm to calculate the linear regression equation, with R 2 greater than 0.99, and the detection is valid. Calculate the protein concentration of the treated test sample solution according to the linear regression equation.

[0123] The detection results of the kit are as Figure 8 shown. It can be seen that the standard curve R 2 = 0.9993, and the detection is valid. After calculation, the protein concentration of the purified recombinant type I collagen COL102 is 11.1 mg / mL, and the protein concentration of recombinant type I collagen COL103 is 8.9 mg / mL.

[0124] Example 3 Detection of in vitro cell proliferation-promoting activity

[0125] Perform a cell proliferation-promoting test on the recombinant type I collagen COL102 and COL103 of this application. Refer to Appendix "3528" of the 2020 Edition (Volume III) of the Chinese Pharmacopoeia. This method is based on the fact that recombinant collagen has a stimulating effect on the growth of mouse embryonic fibroblasts (BALB / c 3T3 cells), and the growth status of BALB / c 3T3 cells varies due to the biological activity differences caused by different proteins, so as to detect the biological activity of promoting cell proliferation in vitro.

[0126] Test materials:

[0127] Complete cell culture medium: 1640 culture medium (containing double antibodies) supplemented with 10% fetal bovine serum, stored at 4 °C;

[0128] Serum-free medium: 1640 culture medium (containing double antibodies), stored at 4 °C;

[0129] Digestive solution: 0.25% trypsin;

[0130] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate. Dissolve in water and make up the volume to 1000 mL. Autoclave at 121 °C for 15 min;

[0131] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder, dissolve it in 20 mL of PBS, filter and sterilize through a 0.22 μm filter membrane, and store in the dark at 4 °C;

[0132] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0133] Samples: Recombinant type I collagen (COL102 and COL103), bovine type I collagen reference standard (purchased from the National Institutes for Food and Drug Control, product number 380008-202001).

[0134] Specific implementation method:

[0135] Dilute the test articles COL102, COL103, and bovine type I collagen reference standard to 1 mg / mL with PBS buffer respectively; Culture the BALB / c 3T3 cell line in complete culture medium at 37 °C and 5% CO2, and control the cell concentration to be 1.0×10 5 cells / mL - 5.0×10 5 cells / mL. Use the cells for biological activity determination 24 h - 36 h after passage. Discard the culture medium in the culture flask, digest and collect the cells, and prepare a cell suspension with a concentration of 5.0×10 5 cells / mL - 8.0×10 5 cells / mL. Inoculate the cell suspension into a 96-well cell culture plate, 100 μL per well, and culture at 37 °C and 5% CO2. After 24 h, change to maintenance culture medium and culture at 37 °C and 5% CO2 for 24 h. Discard the maintenance medium from the prepared cell culture plate, add the standard solution and the test article solution, 100 μL per well, and culture at 37 °C and 5% CO2 for 64 h - 72 h; Add 20 μL of MTT solution to each well and culture at 37 °C and 5% CO2 for 5 h. The above operations are carried out under sterile conditions. After discarding the liquid in the culture plate, add 100 μL of DMSO to each well, mix well, and measure the absorbance at a wavelength of 570 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and record the measurement results. The results are as Figure 9 shown.

[0136] From Figure 9It can be seen that the cell proliferation-promoting activities of COL102 and COL103 are basically the same, which are 1721 U / mg and 1687 U / mg respectively, and the cell activity-promoting activity of bovine type I collagen reference substance is 532 U / mg. The results show that the in vitro cell proliferation-promoting activity of the recombinant type I collagen prepared in this application is significantly better than that of the reference substance.

[0137] Example 4 Detection of in vitro cell adhesion activity

[0138] Refer to Appendix B "Determination of Cell Adhesion" of the industry standard "YY / T - 1849 - 2022".

[0139] Test materials:

[0140] Complete cell culture medium: 1640 medium (containing double antibodies) added with 10% fetal bovine serum, stored at 4°C;

[0141] Serum-free medium: 1640 medium (containing double antibodies), stored at 4°C;

[0142] Digestive solution: 0.25% trypsin;

[0143] PBS buffer solution: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up the volume to 1000 mL, sterilize at 121°C for 15 min;

[0144] Thiazolyl blue (MTT) solution: Weigh 0.10 g of MTT powder, add 20 mL of PBS to dissolve it, filter and sterilize through a 0.22 μm filter membrane, and store it in the dark at 4°C;

[0145] BALB / c 3T3 cells (purchased from Wuhan Punosai).

[0146] Samples: Recombinant type I collagen (COL102 and COL103), bovine type I collagen reference substance (purchased from the National Institutes for Food and Drug Control, product number 380008 - 202001).

[0147] Specific implementation method:

[0148] Pre-dilute the protein sample to 0.5 μg / ml with PBS. After pre-dilution, perform a two-fold serial dilution in a 96-well plate, with a total of 8 dilution steps. Set 50 μL of protein samples at different dilutions in each well, and establish a negative control (without protein), adding 50 μL of PBS as a control. Incubate overnight at 4°C; after incubation, discard the liquid in the plate, add 100 μL of PBS to each well for washing, and wash 3 times in total; after washing, add 100 μL of 30 μg / μL BSA to each well for blocking, and incubate in a 37°C incubator for 1 h; after incubation, discard the liquid in the plate, add 100 μL of PBS to each well for washing 3 times, and then add fibroblast cell suspension. The cell seeding density is 1.0×10 5 cells / mL, seed 100 μL in each well, and incubate in an incubator for 5 h; wash the cell plate after incubation 3 times with PBS, observe the cell adhesion under a microscope, select five points except at the edge under a 200-fold microscope to count the number of adherent cells, and fit a curve based on the counting results to obtain the titer. The test results are as Figure 10 shown.

[0149] As Figure 10 can be seen, the cell adhesion promoting activity of COL103 is 7230 U / mg, the cell adhesion promoting activity of COL102 is 6398 U / mg, the cell adhesion promoting activity of COL103 is slightly higher than that of COL102, but the difference is not significant. The cell adhesion promoting activity of bovine type I collagen reference is about 600 U / mg. The results show that the recombinant type I collagen prepared in this application has significantly better in vitro cell adhesion promoting activity than the reference.

[0150] Example 5 Analysis of the self-assembly phenomenon of recombinant type I collagen

[0151] Ultraviolet absorption spectroscopy can be used to characterize the structure of proteins. The far ultraviolet region (250 nm - 170 nm) is mainly related to the secondary and tertiary structures of proteins, and the near ultraviolet region (320 nm - 250 nm) is closely related to the aromatic amino acids contained in proteins. Among them, the 290 nm - 280 nm band is the ultraviolet absorption peak of tyrosine and tryptophan. Collagen is mainly composed of glycine, proline and glutamic acid, and only contains a small amount of aromatic amino acids.

[0152] Samples: Recombinant type I collagen (COL102 and COL103), fish skin collagen reference.

[0153] Specific implementation steps: Dilute COL102, COL103 and fish skin collagen reference to 1 mg / ml with PBS respectively, and use a multifunctional microplate reader (SpectraMax i3x, model 100 - 240VAC1) to test the samples. Set the scanning range from 230 nm to 500 nm, use the PBS result as the baseline, start the test, and obtain asFigure 11 The ultraviolet absorption spectrogram shown

[0154] Figure 11 In it, COL102 and COL103 only have very weak absorption peaks at 320nm - 250nm, indicating that the content of aromatic amino acids in recombinant type I collagen is very low, and at the same time indicating that the purity of collagen is relatively high; and the ultraviolet absorption spectra of COL102 and COL103 are basically the same, and are similar to the characteristic peaks of the fish skin collagen reference substance.

[0155] Furthermore, the self-assembly kinetic process of recombinant type I collagen was monitored by ultraviolet spectroscopy (UV).

[0156] The specific steps include: diluting and mixing the recombinant type I collagen COL102 sample evenly with PBS buffer solution (NaCl concentration 150mM, pH 7.4) to make its concentrations 10mg / mL, 8mg / mL, 6mg / mL, 4mg / mL, 2mg / mL, 1mg / mL respectively, and then putting it into a 4°C refrigerator for self-assembly for a certain period of time. Among them, for the kinetics, samples are taken at 0h, 2h, 4h, 6h, 8h..... etc. to measure the absorbance value at 313nm, and the self-assembly kinetic curve diagram of recombinant type I collagen COL102 is obtained, as Figure 12 shown, as well as the self-assembly photo diagram of recombinant type I collagen COL102 with different concentrations after being placed at 4°C for 12h, as Figure 13 shown.

[0157] As Figure 12 can be seen, high-concentration (above 8mg / mL) recombinant type I collagen COL102 will gradually change from a liquid state to a gel state after being placed at 4°C for a long time. This is because after collagen self-assembly, the storage modulus exceeds the loss modulus, presenting a gel state, and the gel time and state are proportional to the protein concentration; at the same time, as the protein concentration gradually increases, the self-assembly rate gradually accelerates. This may be because the greater the collagen concentration, the more molecules in a unit space, and the molecules are more likely to aggregate and undergo self-assembly. That is to say, the more molecules in a unit space, the faster the collagen molecules will aggregate together, and the self-assembly rate will also accelerate accordingly.

[0158] From Figure 13 the self-assembly photos, it can be seen that recombinant type I collagen COL102 starts to self-assemble after being placed at 4°C for 12h, gradually changing from a liquid state to a gel state, which is consistent with the kinetic data, and the gel state is proportional to the protein concentration. This is because after collagen self-assembly, the storage modulus exceeds the loss modulus, thus presenting a gel state.

[0159] Example 6 Infrared spectroscopy detection (FTIR) analysis

[0160] Infrared spectroscopy is an effective means to study the structure of biological macromolecules and the configuration of polypeptide chains. The characteristic absorption peaks of collagen groups can be detected by infrared spectroscopy analysis.

[0161] Samples: Recombinant type I collagen COL102, bovine type I collagen reference substance.

[0162] Specific implementation steps: After freeze-drying the samples, they are ground into powder with KBr and then pressed into tablets. Under room temperature and low humidity conditions, they are scanned and detected by an infrared spectrometer (NICOLET 6700, ThermoFisher). The parameter settings are as follows: the scanning wavenumber range is 4000 cm -1 -400 cm -1 The scanning resolution is 4 cm -1 , and it is scanned 16 times. The infrared spectrum scanning diagram of the bovine type I collagen reference substance is as shown in Figure 14 , and the infrared spectrum scanning diagram of the recombinant type I collagen COL102 is as shown in Figure 15 .

[0163] It can be seen from Figure 14 and Figure 15 that the characteristic peaks of the recombinant type I collagen COL102 and the bovine type I collagen reference substance are basically similar, and the characteristic absorption peaks of the collagen groups all appear, indicating that the recombinant type I collagen COL102 prepared in this application also maintains the triple helix structure of natural collagen.

[0164] In summary, in this application, the nucleotides of the functional region repeat fragments of human-derived type I collagen are optimized and screened, and co-expressed with the hydroxyproline enzyme gene in the Pichia pastoris system to reduce problems such as low expression level, improper protein folding, and poor stability faced during the process of expressing recombinant proteins. Thus, a recombinant type I collagen with low-temperature self-assembly characteristics is successfully obtained, and it retains better activity than natural collagen, has lower production costs and more stable product quality, and can be stably produced industrially. And after verification, the recombinant type I collagen prepared in this application has an obvious triple helix structure, shows obvious self-assembly characteristics under high protein concentration and low temperature conditions, and its film-forming property and adhesion property are superior to those of natural collagen without self-assembly characteristics. Therefore, it has broad application prospects in the biomedical field.

[0165] The above are only optional embodiments of this application, and do not limit the patent scope of this application. Any equivalent structural transformation made under the inventive concept of this application, or direct / indirect application in other related technical fields, is included in the patent protection scope of this application.

Claims

1. A recombinant type I collagen with low-temperature self-assembly properties, characterized in that: The amino acid sequence of the recombinant type I collagen is obtained by repeating the human type I collagen fragment as a basic unit n times in series, where n is 1, 2 or 3. The amino acid sequence of the recombinant type I collagen is shown in Seq ID NO.1, Seq ID NO.2 or Seq ID NO.

3.

2. The recombinant type I collagen with low-temperature self-assembly properties according to claim 1, characterized in that: The gene sequence of the recombinant type I collagen is shown in Seq ID NO.4, Seq ID NO.5 or Seq ID NO.

6.

3. A method for preparing recombinant type I collagen with low-temperature self-assembly properties as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: According to the codon preference of the Pichia pastoris host, the gene sequence encoding the recombinant type I collagen is optimized and designed to obtain the gene sequence of the recombinant type I collagen; According to the gene sequence of the recombinant type I collagen and the Pichia pastoris expression plasmid pPICZαA, a recombinant plasmid pPICZαA-COL I is constructed; The pseudovirus P4H was used as the amino acid sequence of proline hydroxylase, and after optimization design according to the codon preference of the Pichia pastoris host, the gene sequence of proline hydroxylase as shown in Seq ID NO.8 was obtained; According to the gene sequence of the proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K, a recombinant plasmid pPIC3.5K-P4H was constructed; The recombinant plasmid pPICZαA-COL I is linearized by enzyme digestion to obtain a linearized plasmid pPICZαA-COL I; the linearized plasmid pPICZαA-COL I is electrotransformed into Pichia pastoris X33 competent cells, and recombinant humanized collagen yeast engineering bacteria are obtained after culturing and positive transformant screening; The recombinant plasmid pPIC3.5K-P4H is linearized by enzyme digestion to obtain a linearized plasmid pPIC3.5K-P4H; the recombinant humanized collagen yeast engineering bacteria are used to prepare recombinant engineering bacteria competent cells, the linearized plasmid pPIC3.5K-P4H is electrotransformed into the recombinant engineering bacteria competent cells, and the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H is obtained after culturing and screening of co-expression positive transformants; Inducing the expression of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H to obtain the engineering bacteria with the highest protein expression, then performing high-density fermentation, and collecting the fermentation supernatant by centrifugation; After the fermentation supernatant is purified, a recombinant type I collagen stock solution is obtained.

4. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of constructing the recombinant plasmid pPICZαA-COL I according to the gene sequence of the recombinant type I collagen and the Pichia pastoris expression plasmid pPICZαA comprises: Gene fragments were synthesized according to the gene sequence of the recombinant type I collagen, and the synthesized gene fragments were inserted into the Pichia pastoris expression plasmid pPICZαA through restriction sites Xho Ⅰ and Not Ⅰ to obtain the recombinant plasmid pPICZαA-COL I.

5. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of constructing the recombinant plasmid pPIC3.5K-P4H according to the gene sequence of the proline hydroxylase and the Pichia pastoris expression plasmid pPIC3.5K comprises: Gene fragments were synthesized according to the gene sequence of the proline hydroxylase, and the synthesized gene fragments were inserted into the Pichia pastoris expression plasmid pPIC3.5K through restriction sites EcoR Ⅰ and Not Ⅰ to obtain the recombinant plasmid pPIC3.5K-P4H.

6. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of linearizing the recombinant plasmid pPICZαA-COL I by enzyme digestion to obtain the linearized plasmid pPICZαA-COLI comprises: The recombinant plasmid pPICZαA-COL I was linearized by enzyme digestion using QuickCut 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, it was dissolved with ddH2O to obtain the linearized plasmid pPICZαA-COL I.

7. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of electro-transforming the linearized plasmid pPICZαA-COL I into Pichia pastoris X33 competent cells, culturing and screening positive transformants to obtain recombinant humanized collagen yeast engineering bacteria comprises: The linearized plasmid pPICZαA-COL I was mixed with Pichia pastoris X33 competent cells, and after ice bath, the cells were electroporated, and then sorbitol solution was added, mixed, and then transferred to a sterile EP tube. After incubation at 30°C for 1h-2h, the cells 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 was grown; A single colony on the YPD plate was picked up, transferred to a 96-well culture plate containing a YPD liquid culture medium containing Zeocin, and cultured at 30° C. to screen and obtain recombinant humanized collagen yeast engineering bacteria.

8. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of linearizing the recombinant plasmid pPIC3.5K-P4H with enzyme digestion to obtain the linearized plasmid pPIC3.5K-P4H comprises: The recombinant plasmid pPIC3.5K-P4H was linearized by enzyme digestion using QuickCut 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, it was dissolved with ddH2O to obtain the linearized plasmid pPIC3.5K-P4H.

9. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of preparing recombinant engineering bacteria competent cells using the recombinant humanized collagen yeast engineering bacteria comprises: After streaking the recombinant humanized collagen yeast engineering bacteria plate, a single colony was picked and inoculated into YPD liquid medium, and cultured at 30°C and 225 rpm for 24 h; then the inoculation ratio was transferred to YPD liquid medium at 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, 3000rpm for 5 minutes, discard the supernatant and collect the bacteria; Resuspend the bacteria with 50 mL of sterile ultrapure water, centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, resuspend the bacteria with 50 mL of sterile ultrapure water, centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 40 mL of sterile 1 M sorbitol; centrifuge at 4°C, 3000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with 100 μL-150 μL of sterile 1 M sorbitol, rotate to mix, and place on ice to obtain recombinant engineered bacteria competent cells.

10. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of electro-transforming the linearized plasmid pPIC3.5K-P4H into the competent cells of the recombinant engineering bacteria, culturing and screening the co-expression positive transformants to obtain the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H comprises: The linearized plasmid pPIC3.5K-P4H was mixed with the competent cells of the recombinant engineering bacteria, 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 inverted culture is carried out at 30°C 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 liquid culture medium, and culture is continued at 30°C. The co-expression positive transformants that can grow on the YPD plate containing 4 mg / mL G418 are screened to obtain the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H.

11. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 10, characterized in that: The step of inducing expression of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I_P4H to obtain the engineering bacteria with the highest protein expression level comprises: A single colony growing on a YPD plate containing 4 mg / mL G418 was picked and inoculated into BMGY yeast growth medium and cultured at 30°C and 220 rpm for 24 h 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 The absorbance value was 2, and the culture was continued at 30°C and 220rpm, with 1%, 1.5% and 2% methanol added every 24h. Samples were taken 24h, 48h, 72h and 96h after induction, and the protein expression was detected by SDS-PAGE electrophoresis to obtain the engineered bacteria with the highest protein expression.

12. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 11, characterized in that: The step of performing high-density fermentation and collecting the fermentation supernatant by centrifugation comprises: Add the fermentation medium into the fermenter, place the fermenter in a steam sterilizer and sterilize at 121° C. for 15 min, calibrate the electrode after cooling, and wait for inoculation; The engineered bacteria with the highest protein expression amount are inoculated into a YPD liquid culture medium, and cultured overnight at 30° C. and 220 rpm to obtain a primary seed solution; The primary seed solution was inoculated into YPD liquid medium at a rate of 1% (v / v) and cultured until OD 600nm When the absorbance value is 4-6, the secondary seed solution is obtained; The secondary seed liquid is inoculated into the fermentation tank at an inoculation rate of 10% (v / v), and PTM4 trace elements are added to perform fermentation culture; the culture temperature is 30°C, the stirring speed is 400rpm-500rpm, the ventilation volume is 2L / min-6L / min, and 50% (w / w) ammonia water is added to maintain the pH in the fermentation tank at 5.0; When the dissolved oxygen DO value in the fermenter continues to rise, feed culture medium is added, and the feed rate is controlled to maintain the dissolved oxygen DO value between 20% and 50%. After 96 hours, the fermentation is stopped and the fermentation supernatant is separated by a solid-liquid separation system.

13. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 3, characterized in that: The step of purifying the fermentation supernatant to obtain a recombinant type I 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 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. Then, the corresponding protein was eluted with a phosphate buffer containing NaCl and collected. After ultrafiltration, liquid replacement, and concentration, a recombinant type I collagen stock solution was obtained.

14. A use of the recombinant type I collagen with low-temperature self-assembly properties as claimed in any one of claims 1 to 2, characterized in that: The recombinant type I collagen is used for preparing biomaterials.

15. The use of the recombinant type I collagen with low-temperature self-assembly property according to claim 14, characterized in that: The biological material is a tissue material or a drug carrier.

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