Recombinant I-type collagen with low-temperature self-assembly characteristic as well as preparation method and application of recombinant I-type collagen

Abstract

The invention discloses recombinant I-type collagen with a low-temperature self-assembly characteristic as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The recombinant I-type collagen with the low-temperature self-assembly characteristic is characterized in that an amino acid sequence of the recombinant I-type collagen is obtained by taking a human I-type collagen fragment as a basic unit and repeatedly connecting in series for n times, an amino acid sequence of the human I-type collagen fragment is shown as Seq ID NO.1, and n is an integer greater than or equal to 1. According to the invention, nucleotides of repetitive fragments of functional areas of the human-derived I-type collagen are optimized and screened, and co-expressed with hydroxyproline enzyme genes in a pichia pastoris system, so that the recombinant I-type 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 shown in Seq ID NO.4, Seq ID NO.5 or Seq ID NO.6.

[0007] The present application also proposes a method for preparing recombinant type I collagen with low-temperature self-assembly properties, comprising the following steps: 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-COLI; the linearized plasmid pPICZαA-COL I is electrotransformed into Pichia pastoris X33 competent cells, and after culture and positive transformant screening, a recombinant humanized collagen yeast engineering bacterium is obtained; 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 bacterium is used to prepare a recombinant engineering bacteria competent cell, the linearized plasmid pPIC3.5K-P4H is electrotransformed into the recombinant engineering bacteria competent cells, and after culture and co-expression positive transformant screening, a recombinant yeast engineering bacterium X33 / pPICZαA-COL I_P4H is obtained; 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.

[0008] Optionally, the step of constructing a 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-COLI.

[0009] Optionally, the step of constructing a 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.

[0010] Optionally, the step of linearizing the recombinant plasmid pPICZαA-COL I by enzyme digestion to obtain a linearized plasmid pPICZαA-COL I 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 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 the linearized plasmid pPICZαA-COL I.

[0011] Optionally, 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 includes: 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.

[0012] Optionally, the step of linearizing the recombinant plasmid pPIC3.5K-P4H by 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 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 the linearized plasmid pPIC3.5K-P4H.

[0013] Optionally, the step of preparing competent cells of recombinant engineering bacteria 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.

[0014] Optionally, the step of electro-transforming the linearized plasmid pPIC3.5K-P4H into the competent cells of the recombinant engineering bacteria, culturing and screening 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.

[0015] Optionally, 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.

[0016] Optionally, 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.

[0017] Optionally, the step of obtaining a recombinant type I collagen stock solution after purifying the fermentation supernatant 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.

[0018] The present application also proposes an application of recombinant type I collagen with low-temperature self-assembly properties, and the recombinant type I collagen is used to prepare biomaterials, tissue materials or drug carriers.

[0019] In summary, the present application optimizes and screens the nucleotides of the repeated fragments of the functional region of human type I collagen, and co-expresses them with the hydroxyproline enzyme gene in the Pichia pastoris system. Hydroxyproline can enhance the cross-linking and stability between collagen molecules, so as to reduce the problems of low expression level, improper protein folding, poor stability, etc. faced in the process of expressing recombinant proteins. In this way, recombinant type I collagen with low-temperature self-assembly characteristics is successfully obtained, and it retains activity that is superior to natural collagen, has lower production costs, more stable product quality, and can be stably industrialized. It has been verified that the recombinant type I collagen prepared in the present application has a distinct triple helix structure, exhibits obvious self-assembly properties under high protein concentration and low temperature conditions, and its film-forming and adhesion properties are superior to natural collagen without self-assembly characteristics, and thus has broad application prospects in the biomedical field, such as 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 biological activity of biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 This is a spectrum of the recombinant plasmid pPICZαA-COL I01 described in the examples of this application; Figure 2 This is a spectrum of the recombinant plasmid pPICZαA-COL I02 described in the examples of this application; Figure 3 This is a spectrum of the recombinant plasmid pPICZαA-COL I03 described in the examples of this application; Figure 4 This is a spectrum of the recombinant plasmid pPIC3.5K-P4H described in the examples of this application; Figure 5 This is a diagram showing the SDS-PAGE electrophoresis result of the supernatant of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H and X33 / pPICZαA-COL I02_P4H induced expression described in the examples of the present application; Figure 6 This is a SDS-PAGE electrophoresis result diagram of the supernatant of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I03_P4H induced expression described in the examples of this application; Figure 7 This is a diagram of the SDS-PAGE electrophoresis result after high-density fermentation of the target protein described in the examples of this application; Figure 8 This is a graph showing the results of the colorimetric determination of the protein content of the recombinant type I collagen described in the examples of the present application; Fig. 9 This is a graph showing the results of the cell proliferation promoting activity test of the recombinant type I collagen described in the examples of this application; Fig.10 This is a graph showing the results of the cell adhesion promoting activity test of the recombinant type I collagen described in the examples of this application; Fig.11 This is the ultraviolet absorption spectrum of the recombinant type I collagen described in the examples of this application; Fig.12 This is a self-assembly kinetic curve of the recombinant type I collagen COL102 described in the examples of this application; Fig.13 This is a photograph of the self-assembly of the recombinant type I collagen COL102 described in the examples of the present application after being placed at 4° C. for 12 hours; Fig.14 This is an infrared spectrum scan of the bovine type I collagen reference substance described in the examples of this application; Fig.15 This is an infrared spectrum scan of the recombinant type I collagen COL102 described in the examples of this application.

[0022] 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

[0023] 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.

[0024] Description of sequence listing (sequence listing content is provided separately): Seq ID NO.1 shows the amino acid sequence of the human type I collagen fragment in the embodiment of the present application, and can also be expressed as the amino acid sequence of the recombinant type I collagen when n=1; Seq ID NO.2 shows the amino acid sequence of recombinant type I collagen when n=2 in the example of the present application; Seq ID NO.3 shows the amino acid sequence of recombinant type I collagen when n=3 in the example of the present application; Seq ID NO.4 shows the gene sequence of recombinant type I collagen when n=1 in the embodiment of the present application; Seq ID NO.5 shows the gene sequence of recombinant type I collagen when n=2 in the embodiment of the present application; Seq ID NO.6 shows the gene sequence of recombinant type I collagen when n=3 in the embodiment of the present application; Seq ID NO.7 shows the amino acid sequence of the pseudovirus P4H in the examples of this application; Seq ID NO.8 shows the gene sequence of the pseudovirus P4H in the examples of this application.

[0025] 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.

[0026] Example 1 Construction and identification of recombinant yeast and preparation of recombinant type I collagen 1. Construction of expression vector The amino acid sequence of the recombinant type I collagen described in this application is obtained by repeating the human type I collagen fragment n times in series as the basic unit. The amino acid sequence of the human type I collagen fragment is shown in Seq ID NO.1, which is derived from human type I collagen. The human type I collagen sequence reference: UniProt P02461 sequence (https: / / www.uniprot.org / uniprot / P02452). This application analyzes the triple helix region of the mature peptide of human type I collagen, screens out the human type I collagen domain fragment (G932-1156N) as shown in Seq ID NO.1, and then repeats the sequence shown in SeqID NO.1 n times in series as the basic unit, and each repeated sequence is directly connected from head to tail. Preferably, n=1, 2 or 3, that is, the amino acid sequence of the recombinant type I collagen is as 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 properties and retains activity superior to that of natural collagen.

[0027] Furthermore, according to the codon preference of the Pichia pastoris host, the gene sequence encoding the recombinant type I collagen was optimized and designed, and the yeast self-cleavage site "KR" was designed and introduced at the N-terminus, and a terminator was added at the C-terminus. The base sequence AAAAGA was added to the 5' end of the optimized gene sequence of the recombinant type I collagen, that is, between XhoⅠ (Takara, Catalog No.: 1635) and the target gene sequence, and GeneScript Biotech Co., Ltd. was commissioned to synthesize the gene fragment to obtain the gene sequence of the recombinant type I collagen as shown in Seq ID NO.4, SeqID NO.5 or Seq ID NO.6. The synthesized gene fragment was inserted into the Pichia pastoris expression plasmid pPICZαA through the restriction sites of XhoⅠ and NotⅠ (Takara, Catalog No.: 1623), and the recombinant plasmids pPICZαA-COL I01 (such as Figure 1 As shown), pPICZαA-COL I02 (as shown Figure 2 ) and pPICZαA-COL I03 (as Figure 3 as shown).

[0028] Furthermore, in order to co-express recombinant type I collagen with hydroxyproline enzyme gene in Pichia pastoris system, the present application selects proline hydroxylase. Since the complex structure and low activity of animal P4H (prolyl 4-hydroxylase) limit its application, most of the P4H of plants and viruses are monomeric polypeptides and can catalyze proline-rich polypeptide chains. Studies have found that when the pseudo-viral P4H (L593) is co-expressed with a 38kDa collagen chain in Escherichia coli, hydroxylated collagen is obtained, and its hydroxylation rate is relatively high. Therefore, the present application uses pseudo-viral P4H as the amino acid sequence of proline hydroxylase, and the specific sequence is shown in Seq ID NO.7, and the predicted molecular weight is about 28kDa.

[0029] 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 make it more suitable for expression in Pichia pastoris, thereby obtaining the proline hydroxylase gene sequence shown in Seq ID NO.8.

[0030] According to the gene sequence of proline hydroxylase shown in Seq ID NO.8, GenScript Biotech Co., Ltd. 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 sites of EcoRⅠ (Takara, Catalog No.: 1611) and NotⅠ to obtain the recombinant plasmid pPIC3.5K-P4H. Figure 4 shown.

[0031] 2 Construction and screening of recombinant engineered bacteria 2.1 Obtaining recombinant plasmid The above-mentioned recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02, pPICZαA-COL I03 and pPIC3.5K-P4H were expanded and cultured, and high-concentration plasmids were extracted for use (Omega, catalog number: D6904).

[0032] 2.2 Linearization of recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02, and pPICZαA-COL I03 Use QuickCut SacⅠ (Takara, catalog number: 1606) to linearize 15 μg of recombinant plasmids pPICZαA-COL I01, pPICZαA-COL I02 and pPICZαA-COL I03 extracted in 2.1. The enzyme digestion conditions are 37°C, 5h, and the enzyme digestion system is shown in Table 1.

[0033] Table 1 Enzyme digestion system

[0034] Then add 0.1 times the volume of 3M NaAc (pH 5.2) and 2.5 times the volume of anhydrous ethanol, place at -20°C overnight, centrifuge at 4°C, 13000rpm for 20min, discard the supernatant, collect the precipitate and rinse with 700μL 75% ethanol, centrifuge at 4°C, 13000rpm for 20min and discard the supernatant, invert the EP tube on the clean bench absorbent paper for about 10 min to remove water and residual ethanol as much as possible, dissolve with 20μL ddH2O, take 1μL and dilute 10 times, and use one-drop to detect the nucleic acid concentration. Linearized plasmids pPICZαA-COL I01, pPICZαA-COL I02 and pPICZαA-COL I03 were obtained.

[0035] 2.3 Preparation of X33 competent cells After streaking the X33 strain plate, pick a single colony and inoculate it into 20 mL YPD liquid medium, and culture it at 30°C and 225 rpm for 24 h; then transfer it to 50 mL YPD liquid medium at an inoculation ratio of 1:1000, and culture it 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 3000rpm 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 3000rpm 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 3000rpm 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 3000rpm 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 X33 competent cells.

[0036] 2.4 Electrotransformation into X33 competent cells Take 100 μL of X33 competent cells and mix with 10 μL of linearized plasmid pPICZαA-COL I01, pPICZαA-COL I02 or pPICZαA-COL I03, transfer to the pre-cooled electroporation cup, immediately place in an ice bath for 5 minutes, turn 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 min, and then invert and culture in a 30°C incubator for 2d-5d until a single colony grows.

[0037] 2.5 Screening of positive transformants Pick a single colony from the YPD plate and transfer it to the first 96-well culture plate containing 200 μL YPD liquid culture 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 0.1 mg / mL, 0.2 mg / mL and 0.3 mg / mL Zeocin; if the positive transformant can grow on a plate containing a high concentration of 0.3 mg / mL Zeocin, it means that the positive transformant contains more copies of the target gene, and after this step of screening, a recombinant humanized collagen yeast engineered bacteria that can be efficiently expressed can be obtained.

[0038] 2.6 Secondary electroporation of recombinant humanized collagen yeast After streaking the recombinant humanized collagen yeast engineering bacteria plate, a single colony was picked and inoculated into 20 mL YPD liquid medium, and cultured at 30°C and 225 rpm for 24 h; then transferred to 50 mL YPD liquid medium at an inoculation ratio of 1:1000, and cultured at 30°C and 225 rpm until OD 600nmThe absorbance value is 1.3-1.5; the bacterial solution is then transferred to a sterile 50mL centrifuge tube, centrifuged at 4°C and 3000rpm 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 3000rpm 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 3000rpm 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 3000rpm 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 recombinant engineered bacteria competent cells.

[0039] 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 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 the linearized plasmid pPIC3.5K-P4H.

[0040] 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 in a 30°C incubator for 2d-5d until a single colony grew; 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 a 50 mL centrifuge tube and diluted to 10 with sterile water. 5 cells / ml concentration of cell suspension (counted by spectrophotometer, 1OD 600 ≈5×10 7cells / ml), and spread on a YPD plate containing 0.5 mg / mL G418, and culture at 30°C 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 liquid culture medium, and continue to culture at 30°C; 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 more copies of the target gene. After this step of screening, the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H, X33 / pPICZαA-COLI02_P4H and X33 / pPICZαA-COL I03_P4H with high expression efficiency can be obtained.

[0041] 3. Identification of induced expression Pick a single colony grown on the plate containing 4 mg / mL G418 in step 2.6 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 The absorbance value was 2, and the culture was continued at 30°C and 220rpm. 1%, 1.5% and 2% methanol were added every 24h. The bacterial solution samples were taken at 24h, 48h, 72h and 96h after induction, and the expression supernatant was collected by centrifugation. The expression of the target protein was analyzed 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 after 24h induction was as follows Figure 5 As shown; the protein expression of recombinant yeast engineering bacteria X33 / pPICZαA-COL I03_P4H induced for 24 hours is shown Figure 6It can be seen that the recombinant yeast engineering bacteria X33 / pPICZαA-COL I01_P4H can produce about 20kDa recombinant type I collagen after induction, the recombinant yeast engineering bacteria X33 / pPICZαA-COL I02_P4H can produce about 40kDa recombinant type I collagen after induction, and the recombinant yeast engineering bacteria X33 / pPICZαA-COL I03_P4H can produce about 60kDa recombinant type I collagen after induction.

[0042] 4 High-density fermentation Add 2L fermentation medium into a 5L fermenter, place the fermenter in a steam sterilizer and sterilize at 121℃ 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 activate the bacteria and obtain a primary seed solution; The primary seed solution was inoculated into 200 mL YPD liquid medium at a 1% (v / v) inoculation rate and cultured until OD 600nm When the absorbance value is 4-6, the secondary seed solution is obtained; The secondary seed liquid was inoculated into the fermentation tank at an inoculation rate of 10% (v / v), and 8.7 mL PTM4 trace elements were added to perform fermentation culture; the culture temperature was 30° C., the stirring speed was 400 rpm-500 rpm, the ventilation volume was 2 L / min-6 L / min, and 50% (w / w) ammonia water was added to maintain the pH in the fermentation tank at 5.0; When the dissolved oxygen DO value in the fermentation tank continues to rise, it means that the glycerol in the culture medium is exhausted by the cells. At this time, feed medium is added, and the feed rate is controlled to make the dissolved oxygen DO value fluctuate periodically within the range of 20%-50%. Samples of bacterial liquid are taken 24h, 48h, and 72h after fermentation, and the fermentation supernatant is separated by a solid-liquid separation system. The molecular weight of the target protein is analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 7 shown.

[0043] Figure 7 In the experiment, the recombinant yeast engineering strain X33 / pPICZαA-COL I01_P4H showed slow methanol metabolism after 24 hours of fermentation induction, and was unable to effectively metabolize methanol. The fermentation was terminated at 48 hours. This phenomenon occurred in three batches of fermentation, so the strain was subsequently abandoned; the recombinant yeast engineering strains X33 / pPICZαA-COL I02_P4H and X33 / pPICZαA-COL I03_P4H could both ferment normally up to 96 hours, but X33 / pPICZαA-COL I03_P4H showed degradation in the later stage of fermentation.

[0044] Specifically, the PTM4 trace elements are Pichia trace minerals 4 trace element solution, including: CuSO4·5H2O 2.0g / L, NaI 0.08g / L, MnSO4·H2O 3.0g / L, Na2MoO4·2H2O 0.2g / L, H3BO30.02g / L, CaSO4·2H2O 0.5g / L, CoCl20.5g / L, ZnCl27.0g / L, FeSO4·7H2O 22.0g / L, biotin 0.2g / L, H2SO41.0 mL / L.

[0045] 5 Purification 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 a phosphate buffer (20 mM NaH2PO4, pH 4.7) was used to balance the chromatography column until the conductivity value and the A280 absorbance value remained unchanged, the fermentation supernatant was loaded, 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 phosphate buffer until the conductivity and ultraviolet A280 absorbance values ​​were the lowest and no longer changed, and the loading of samples was stopped; then, the corresponding protein was eluted with a NaH2PO4 buffer containing NaCl (1 M) and collected, and after ultrafiltration, liquid replacement and concentration, a recombinant type I collagen stock solution was obtained, wherein the recombinant type I collagen obtained by fermentation of the recombinant yeast engineering bacteria X33 / pPICZαA-COL I02_P4H was recorded as COL102, and the recombinant type I collagen obtained by fermentation of the recombinant yeast engineering bacteria X33 / pPICZαA-COL The recombinant type I collagen obtained by I03_P4H fermentation was denoted as COL103, and this abbreviation will be used in subsequent descriptions.

[0046] Specifically, the culture medium formula used in this embodiment is as follows: YPD solid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 2% agar; YPD liquid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L; MD solid medium: YNB without amino acid nitrogen source 13.4g / L; biotin 0.4mg / L; glucose 20g / L, and contains 2% agar; 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; 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; Fermentation medium: H3PO4 (85%) 26.7 mL / L, CaSO4·2H2O 1.175 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40.0 g / L, PTM4 4.35 mL / L, pH adjusted to 5.0 with aqueous ammonia; Feed medium: 50% glycerol, 0.8% PTM4 trace elements.

[0047] Example 2 Protein content detection The protein content of the recombinant type I collagen COL102 and COL103 prepared in Example 1 of the present application was tested. Refer to Appendix 0731 Method 4 (BCA Method) of the 2020 edition (Part III) of the Chinese Pharmacopoeia. This method is based on the conversion of protein molecules to Cu in an alkaline solution. 2+ Reduction to Cu + , 2,2'-biquinoline-4,4'-dicarboxylic acid (BCA) and Cu + The purple complex is formed by combining. The color depth is proportional to the protein concentration within a certain range. The protein reference solution is used as the standard curve, and the protein content in the test sample is determined by colorimetry.

[0048] In this experiment, the “BCA protein concentration assay kit” (purchased from Bio-Tech, catalog number P0010) was used to detect protein content.

[0049] Test samples: recombinant type Ⅰ collagen COL102 and COL103.

[0050] Specific operation steps (see the kit manual for details): dilute the standard BSA to prepare 0mg / mL, 0.05mg / mL, 0.1mg / mL, 0.15mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL and 0.5mg / mL solutions, add 20μL to a 96-well plate, 2 parallels per group. Add 200μL BCA working solution (BCA reagent A: BCA reagent B = 50:1) to each well, mix well, and place at 37℃ for 25min; measure the absorbance at a wavelength of 562nm to calculate the linear regression equation, R 2 If the value is greater than 0.99, the test is established. The protein concentration of the test solution after treatment is calculated according to the linear regression equation.

[0051] The test results of the kit are as follows Figure 8 As shown, it can be seen that the standard curve R 2 =0.9993, the test was established. After calculation, the protein concentration of the purified recombinant type Ⅰ collagen COL102 was 11.1 mg / mL, and the protein concentration of the recombinant type Ⅰ collagen COL103 was 8.9 mg / mL.

[0052] Example 3 In vitro cell proliferation activity detection The recombinant type I collagen COL102 and COL103 of the present application were tested for promoting cell proliferation. Refer to Appendix "3528" of the 2020 edition (Part 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 / c3T3 cells). The growth of BALB / c 3T3 cells is different due to the difference in biological activity caused by different proteins, so as to detect the biological activity of promoting cell proliferation in vitro.

[0053] Test Materials: Complete cell culture medium: add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C; Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C; Digestion solution: 0.25% trypsin; 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 to 1000 mL, and sterilize by high pressure at 121°C for 15 min; Thiazole blue (MTT) solution: weigh 0.10 g of MTT powder, add 20 mL of PBS to dissolve, filter through a 0.22 μm filter membrane to sterilize, and store at 4°C away from light; BALB / c 3T3 cells (purchased from Wuhan Prosperity Biotechnology Co., Ltd.).

[0054] Samples: recombinant type I collagen (COL102 and COL103), bovine type I collagen reference (purchased from China Food and Drug Inspection Institutes, catalog number 380008-202001).

[0055] Specific implementation method: The test samples COL102, COL103 and bovine type I collagen reference were diluted to 1 mg / mL with PBS buffer. The BALB / c 3T3 cell line was cultured with complete culture medium at 37°C and 5% CO2, and the cell concentration was controlled to be 1.0×10 5 cells / mL-5.0×10 5 cells / mL, and used for biological activity determination 24h-36h after subculture. Discard the culture medium in the culture flask, digest and collect the cells, and use complete culture medium to make 5.0×10 5 cells / mL-8.0×10 5 Cell suspension of 100 μL cells / mL was inoculated into a 96-well cell culture plate, 100 μL per well, and cultured at 37°C, 5% CO2. After 24 hours, the medium was replaced with maintenance medium and cultured at 37°C, 5% CO2 for 24 hours. The maintenance medium was discarded from the prepared cell culture plate, and the standard solution and test solution were added to each well at 100 μL, and cultured at 37°C, 5% CO2 for 64-72 hours; 20 μL of MTT solution was added to each well, and cultured at 37°C, 5% CO2 for 5 hours. The above operations were performed under sterile conditions. After discarding the liquid in the culture plate, 100 μL of DMSO was added to each well, mixed well, and the absorbance was measured at a wavelength of 570 nm on a microplate reader, and the measurement results were recorded. The results are as follows. Fig. 9 shown.

[0056] Depend on Fig. 9 It can be seen that the cell proliferation activities of COL102 and COL103 are basically the same, 1721U / mg and 1687U / mg respectively, and the cell proliferation activity of the bovine type I collagen reference substance is 532U / mg. The results show that the in vitro cell proliferation activity of the recombinant type I collagen prepared in the present application is significantly better than that of the reference substance.

[0057] Example 4 In vitro cell adhesion promoting activity detection Refer to the industry standard "YY / T-1849-2022" Appendix B "Cell Adhesion Assay".

[0058] Test Materials: Complete cell culture medium: add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C; Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C; Digestion solution: 0.25% trypsin; 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 to 1000 mL, and sterilize by high pressure at 121°C for 15 min; Thiazole blue (MTT) solution: weigh 0.10 g of MTT powder, add 20 mL of PBS to dissolve, filter through a 0.22 μm filter membrane to sterilize, and store at 4°C away from light; BALB / c 3T3 cells (purchased from Wuhan Prosperity Biotechnology Co., Ltd.).

[0059] Samples: recombinant type I collagen (COL102 and COL103), bovine type I collagen reference (purchased from China Food and Drug Inspection Institutes, catalog number 380008-202001).

[0060] Specific implementation method: The protein sample was pre-diluted to 0.5 μg / ml with PBS. After the pre-dilution, it was diluted 2 times in a 96-well plate, with a total of 8 dilutions. Each well was set with 50 μL of protein samples of different dilutions, and a negative control (without protein) was set up. 50 μL PBS was added as a control and incubated at 4°C overnight. After the incubation, the liquid in the plate was discarded, and 100 μL PBS was added to each well for washing, and washed 3 times in total. After the washing, 100 μL 30 μg / μL BSA was added to each well for blocking, and the plate was incubated at 37°C for 1 hour. After the incubation, the liquid in the plate was discarded, and 100 μL PBS was added to each well for washing 3 times, and then the fibroblast suspension was added. The cell seeding density was 1.0×10 5 cells / mL, 100uL was inoculated into each well and incubated in an incubator for 5h; the incubated cell plate was washed with PBS 3 times, the cell adhesion was observed under a microscope, and the number of adherent cells was counted at five points except the edge under a 200x microscope, and the titer was obtained based on the fitting curve of the counting results. Fig.10 shown.

[0061] Depend on Fig.10 It can be seen that the cell adhesion promoting activity of COL103 is 7230U / mg, and the cell adhesion promoting activity of COL102 is 6398U / 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 the bovine type I collagen reference substance is about 600U / mg. The results show that the in vitro cell adhesion promoting activity of the recombinant type I collagen prepared in the present application is significantly better than that of the reference substance.

[0062] Example 5 Analysis of the self-assembly phenomenon of recombinant type I collagen Ultraviolet absorption spectra can be used to characterize the structure of proteins. The far ultraviolet region (250nm-170nm) is mainly related to the secondary and tertiary structures of proteins, and the near ultraviolet region (320nm-250nm) is closely related to the aromatic amino acids contained in proteins, among which the 290nm-280nm band is the ultraviolet absorption peak of tyrosine and tryptophan. Collagen is mainly composed of glycine, proline and glutamic acid, and contains only a small amount of aromatic amino acids.

[0063] Samples: recombinant type Ⅰ collagen (COL102 and COL103), fish skin collagen reference.

[0064] Specific implementation steps: COL102, COL103 and fish skin collagen reference were diluted to 1 mg / ml with PBS, and the samples were tested using a multifunctional microplate reader (SpectraMax i3x, model 100-240VAC1). The scanning range was set to 230nm-500nm, and the PBS result was used as the baseline. The test was started and the following results were obtained: Fig.11 The UV absorption spectrum is shown.

[0065] Fig.11 In the results, COL102 and COL103 have only weak absorption peaks at 320nm-250nm, indicating that the content of aromatic amino acids in recombinant type I collagen is very low, and also indicating that the collagen purity is relatively high; and the ultraviolet absorption spectra of COL102 and COL103 are basically consistent, and are close to the characteristic peaks of fish skin collagen reference.

[0066] The self-assembly kinetics of recombinant type I collagen were further monitored by ultraviolet spectroscopy (UV).

[0067] The specific steps include: using PBS buffer solution (NaCl concentration 150mM, pH 7.4) to dilute and mix the recombinant type I collagen COL102 sample evenly to make its concentration 10mg / mL, 8mg / mL, 6mg / mL, 4mg / mL, 2mg / mL, 1mg / mL, and then putting it in a 4°C refrigerator for self-assembly for a certain period of time, wherein the kinetics are sampled at 0h, 2h, 4h, 6h, 8h..... and its absorbance value at 313nm is tested to obtain the self-assembly kinetics curve of recombinant type I collagen COL102, as shown in Fig.12 As shown in the figure, and the self-assembly photos of recombinant type I collagen COL102 with different concentrations after being placed at 4°C for 12 hours, as shown in the figure Fig.13 shown.

[0068] Depend on Fig.12It can be seen that high concentration (above 8 mg / mL) of recombinant type I collagen COL102 will gradually change from liquid to gel state when placed at 4°C for a long time. This is because the storage modulus exceeds the loss modulus after collagen self-assembly, 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 higher the collagen concentration, the more molecules per unit space, and the easier it is for the molecules to aggregate and self-assemble. In other words, the more molecules per unit space, the faster the rate at which collagen molecules aggregate together, and the self-assembly rate also accelerates accordingly.

[0069] from Fig.13 From the photos after self-assembly, it can be seen that the recombinant type I collagen COL102 began to self-assemble after being placed at 4°C for 12 hours, and gradually transformed from a liquid state to a gel state, which is consistent with the kinetic data. The gel state is proportional to the protein concentration. This is because the storage modulus of the collagen exceeds the loss modulus after self-assembly, thus presenting a gel state.

[0070] Example 6 Infrared spectroscopy (FTIR) analysis Infrared spectroscopy is an effective means to study the structure of biological polymers and the configuration of polypeptide chains. The characteristic absorption peaks of collagen groups can be detected by infrared spectroscopy analysis.

[0071] Samples: recombinant type Ⅰ collagen COL102, bovine type Ⅰ collagen reference.

[0072] Specific implementation steps: The samples were freeze-dried and ground into powder with KBr and then pressed into tablets. They were scanned and detected by infrared spectrometer (NICOLET 6700, ThermoFisher) at room temperature and low humidity. The parameters were set as follows: scanning wave number range 4000cm -1 -400cm -1 , scanning resolution 4cm -1 , scanned 16 times. The infrared spectrum scanning diagram of bovine type I collagen reference substance is as follows: Fig.14 As shown in the figure, and the infrared spectrum scanning diagram of recombinant type I collagen COL102 is shown in the figure Fig.15 shown.

[0073] Depend on Fig.14 and Fig.15 It can be seen that the characteristic peaks of the recombinant type I collagen COL102 and the bovine type I collagen reference substance are basically similar, and both have characteristic absorption peaks of the collagen group, indicating that the recombinant type I collagen COL102 prepared in the present application also maintains the triple helix structure of natural collagen.

[0074] In summary, this application optimizes and screens the nucleotides of the repeating fragments of the functional region of human type I collagen, and co-expresses them with the hydroxyproline enzyme gene in the Pichia system to reduce the problems of low expression, improper protein folding, poor stability, etc. faced in the process of expressing recombinant proteins, thereby successfully obtaining recombinant type I collagen with low-temperature self-assembly characteristics, and it retains activity that is superior to natural collagen, with lower production costs and more stable product quality, and can be stably industrialized. It has been verified that the recombinant type I collagen prepared by this application has a clear triple helix structure, exhibits obvious self-assembly characteristics under high protein concentration and low temperature conditions, and its film-forming and adhesion properties are better than natural collagen without self-assembly characteristics, so it has broad application prospects in the biomedical field.

[0075] 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 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. 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.

2. The recombinant type I collagen with low-temperature self-assembly properties according to claim 1, characterized in that: 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.

3. The recombinant type I collagen with low-temperature self-assembly properties according to claim 2, 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.

4. A method for preparing recombinant type I collagen with low-temperature self-assembly properties as claimed in any one of claims 1 to 3, 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.

5. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

6. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

7. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

8. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

9. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

10. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

11. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

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 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.

13. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 12, 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.

14. The method for preparing recombinant type I collagen with low-temperature self-assembly properties according to claim 4, 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.

15. A use of the recombinant type I collagen with low-temperature self-assembly properties as claimed in any one of claims 1 to 3, characterized in that: The recombinant type I collagen is used to prepare biological materials, tissue materials or drug carriers.

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