High-molecular-weight recombinant humanized collagen as well as synthesis method and application thereof

Abstract

The invention discloses a high molecular weight recombinant humanized collagen as well as a synthesis method and application thereof, the sequence is 100% homologous with the sequence of a humanized III type collagen, the full length is 2059aa, the molecular weight is 183.15 kDa, the protein has better biocompatibility, and the activity of promoting cell adhesion, fibroblast proliferation and keratinocyte migration, and can be used for preparing a high molecular weight recombinant humanized collagen. Meanwhile, the capabilities of promoting skin injury repair and stimulating collagen regeneration are realized. Furthermore, the high-molecular-weight collagen also shows better thermal stability and supporting property, and is suitable for being used as a tissue filling material to be applied to the field of medical instruments.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a high molecular weight recombinant humanized collagen protein and a synthesis method and application thereof. Background Art

[0002] There are three types of biomaterials used in tissue engineering: natural polymers (such as collagen and alginate), decellularized tissue matrices (such as bladder submucosa and small intestinal submucosa) and synthetic polymers, such as polyglycolic acid (PGA), polylactic acid (PLA) and polylactic-coglycolic acid (PLGA). These biomaterials have good biocompatibility, while natural polymers and decellularized tissue matrices have potential biorecognition advantages over synthetic polymers.

[0003] As a natural polymer material, collagen not only has good biocompatibility, degradability, and low immunogenicity, but also has the advantages of high tensile strength and hemostasis. It is precisely because of these advantages that collagen has become the new favorite in the medical beauty industry. In 1981, the first collagen implant, Zyderm I dermal injection, was approved by the US FDA for marketing. Its composition is 95% type I and 5% type III bovine collagen suspended in 3% lidocaine saline. However, research data shows that when Zyderm bovine collagen injection is used to correct shallow and moderate wrinkles, although it can play a good filling effect, its duration is short, and there is also the problem of delayed allergic reaction (1.5-3.0%). In 2001, Shuangmei applied collagen technology to SPF pigs. Although the structure of porcine collagen is more similar to that of human skin collagen and collagen is more stable after injection, there is still a great risk of immune rejection.

[0004] Recombinant humanized collagen is based on the original sequence of human type III collagen. The parts with strong water solubility and high biological activity are selected through splicing and recombination or tandem duplication, and the recombinant humanized type III collagen sequence is obtained after codon optimization. Large-scale production can be achieved by selecting appropriate hosts and fermentation technology. More and more studies have confirmed that recombinant humanized collagen has good water solubility and high biological activity. Its performance is better than that of natural human collagen, and it has broad application prospects in the fields of biomedical materials, beauty cosmetics, food and health care.

[0005] At present, the patents of recombinant type III collagen and the recombinant collagen raw materials sold in the market are all focused on the preparation of low molecular weight collagen. For example, the core sequence of type III collagen of Jiangsu Jiangshan Juyuan is 599aa and the molecular weight is 55.0kDa; the core sequence of type III collagen of Jiangsu Chuangjian is 474aa and the molecular weight is 42.5kDa; the molecular weight of type III collagen of Shanxi Jinbo is even lower, which is a short peptide of GERGAPGFRGPAGPNGIPGEKGPAGERGAP (2.8kDa) repeated 16 to 32 times in series, with a molecular weight of 44.8 to 89.6kDa. When designing short low molecular weight collagen, the protease degradation sites or unstable amino acid sequences in the expression host are usually removed, so it has better stability during fermentation and purification. However, the low molecular weight collagen has a simple structure and is difficult to be used as an injection filling material. At the same time, the low molecular weight has a fast degradation rate in the body, making it more difficult to maintain a long-term filling or anti-wrinkle effect. Therefore, more and more researchers have focused on improving the molecular weight and structural stability of collagen by cross-linking, by adding chemical cross-linking agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), silane coupling agents, glutaraldehyde, succinaldehyde, genipin, glutaraldehyde or epichlorohydrin to carry out cross-linking reactions, and then reduce the residual cross-linking agent concentration through multiple washings. Although this method can obtain high molecular weight and high support collagen, the residual chemical cross-linking agents still have certain safety risks, which have blocked the steps of some beauty seekers.

[0006] It should be noted that the above information disclosed in this background technology section is only used to understand the background technology of the present application concept, and therefore, the above description is not considered to constitute information of the prior art. Summary of the invention

[0007] In view of the above problems, the present invention aims to provide a high molecular weight recombinant humanized collagen and its synthesis method and application. The sequence is 100% homologous to the sequence of human type III collagen, with a total length of 2059aa and a molecular weight of 183.15kDa. The protein has better biocompatibility, better activity in promoting cell adhesion, promoting fibroblast proliferation and keratinocyte migration, and under in vitro simulated skin keratin damage conditions, the high molecular weight collagen has better ability to promote skin damage repair and stimulate collagen regeneration; at the same time, the high molecular weight collagen can be assembled into a hydrogel without the introduction of any harmful reagents, showing better support, thermal stability and structural stability, and is more suitable as a tissue filling material in the field of medical devices.

[0008] In an optional embodiment, the collagen sequence involved in the present invention can also be selected from any one or more full-length triple helix structure regions of human collagen. The obtained collagen sequence can refer to the above-mentioned construction method and be repeated in series multiple times to obtain the corresponding high molecular weight collagen. The human type III collagen disclosed in this embodiment is only used as an embodiment.

[0009] Another aspect of the present invention provides a method for synthesizing high molecular weight recombinant humanized collagen, comprising the following steps:

[0010] (1) The amino acids 168 to 1196 of human type III collagen were selected as the initial sequence C3A1, Bgl II was added to the N-terminus of the sequence, and BamH I and Xho I restriction sites were added to the C-terminus of the sequence, and the codons were optimized according to the codon preference of the expression host before synthesis;

[0011] (2) The pPIC9KM plasmid was double-digested with BamH I and Xho I;

[0012] (3) The fragment C3A1 in step (1) was double-digested with Bgl II and Xho I, and ligated with the vector in step (2) to obtain the recombinant plasmid pPIC9KM-C3A1;

[0013] (4) Design primers to amplify the C3A1 fragment again and use Bgl II and Xho I for double digestion;

[0014] (5) The pPIC9KM-C3A1 plasmid was double-digested with BamH I and Xho I, and ligated with the fragment in step (4) to obtain the recombinant plasmid pPIC9KM-C3A1-II;

[0015] (6) linearizing the recombinant plasmid pPIC9KM-C3A1-II obtained in step (5) with Sal I or Sac I, and then electrotransforming it into Pichia pastoris competent cells to obtain positive transformants by screening;

[0016] (7) Placing the positive transformants obtained in step (6) on a high-concentration antibiotic plate for high-copy screening to obtain high-copy transformants;

[0017] (8) The obtained high-copy transformants were inoculated into 96-well deep-well plates for culture, induced expression, and high-expression strains were screened by SDS-PAGE;

[0018] (9) fermenting the obtained high-expression strain on a large scale to obtain a large amount of fermentation liquid, and centrifuging and filtering the fermentation liquid to obtain a crude protein liquid;

[0019] (10) Purifying the crude protein solution using a phenyl hydrophobic column to obtain high molecular weight recombinant humanized collagen with a purity of 95% to 99%;

[0020] (11) By repeating the above steps (4) and (5), a higher molecular weight recombinant collagen sequence can be obtained, and the corresponding plasmids are named pPIC9KM-C3A1-III, pPIC9KM-C3A1-IV and pPIC9KM-C3A1-V respectively;

[0021] (12) Based on step (11), steps (6) to (10) are repeated to obtain corresponding high molecular weight recombinant collagens, which are named C3A1-III, C3A1-IV and C3A1-V, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 C3A1 gene retrieval map

[0023] Figure 2 E.coli Top10 / pPIC9KM-C3A1 transformation and colony PCR verification

[0024] Figure 3 pPIC9KM-C3A1 Plasmid Map

[0025] Figure 4 Transformation and colony PCR verification of Pichia pastoris KM71H / pPIC9KM-C3A1

[0026] Figure 5 Pichia pastoris KM71H / pPIC9KM-C3A1 high copy screening map

[0027] Figure 6 Pichia pastoris KM71H / pPIC9KM-C3A1 high expression screening diagram

[0028] Figure 7 SDS-PAGE analysis of protein expression in 5L fermenter of Pichia pastoris KM71H / pPIC9KM-C3A1

[0029] Figure 8 Purification results of Pichia pastoris KM71H-C3A1 analyzed by SDS-PAGE

[0030] Fig. 9 C3A1 gene retrieval map

[0031] Fig.10E.coli Top10 / pPIC9KM-C3A1-II transformation and colony PCR verification

[0032] Fig.11 pPIC9KM-C3A1-II Plasmid Map

[0033] Fig.12 Transformation and colony PCR verification of Pichia pastoris KM71H / pPIC9KM-C3A1-II

[0034] Fig.13 Pichia pastoris KM71H / pPIC9KM-C3A1-II high copy screening diagram

[0035] Fig.14 Pichia pastoris KM71H / pPIC9KM-C3A1-II high expression screening diagram

[0036] Fig.15 SDS-PAGE analysis of protein expression in Pichia pastoris KM71H / pPIC9KM-C3A1-II 5L fermenter

[0037] Fig.16 Purification results of Pichia pastoris KM71H-C3A1-II analyzed by SDS-PAGE

[0038] Fig.17 Comparison of cytotoxicity between C3A1 and C3A1-II on L-929 fibroblasts

[0039] Fig.18 Comparison of the proliferation effects of C3A1 and C3A1-II on L-929 fibroblasts

[0040] Fig.19 Comparison of cell scratch assay between C3A1 and C3A1-II on HaCAT keratinocytes

[0041] Fig. 20 Comparison of cell adhesion effects of C3A1 and C3A1-II on L-929 fibroblasts

[0042] Fig.21 Comparison of the effects of C3A1 and C3A1-II in stimulating collagen regeneration

[0043] Fig. 22 Comparison of the efficacy of C3A1 and C3A1-II in repairing zebrafish embryos

[0044] Fig.23Comparison of self-assembled hydrogel experiments between C3A1 and C3A1-II

[0045] Fig.24 Scanning electron microscopy images of freeze-dried samples of C3A1 and C3A1-II

[0046] Fig.25 Determination of Tm values ​​of C3A1 and C3A1-II by DSC

[0047] Fig.26 Protein structure display of C3A1 and C3A1-II

[0048] Fig. 27 Comparison of RMSD between C3A1 and C3A1-II DETAILED DESCRIPTION

[0049] Definition of terms

[0050] Herein, the term "bioengineering bacteria" refers to those cells used for transformation, i.e., cells used to express a target gene. Bioengineering bacteria can be isolated cells or cell lines cultured in culture, or cells present in living tissues or organisms. In the context of the present invention, host cells are preferably cells that can grow in culture. The cells described in the present invention are bacteria or fungi, and the present invention is shown and described in examples with Escherichia coli and Pichia pastoris.

[0051] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or devices not specifically noted in the following embodiments all adopt conventional equipment or devices in the art. In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combined connection relationship between one or more devices / devices mentioned in the present invention does not exclude the presence of other devices / devices before and after the combination device / device or the insertion of other devices / devices between these explicitly mentioned two devices / devices, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the order of arrangement of each method step or limiting the scope of the present invention. The change or adjustment of its relative relationship, without substantial change in the technical content, should also be regarded as the scope of the present invention.

[0052] Example 1 Construction of recombinant engineered bacteria E. coli Top10 / pPIC9KM-C3A1

[0053] In order to construct a high-molecular-weight collagen sequence, the patent of this invention selected the triple-helix region of human type III collagen (GenBanK accession number: NM_000090.4) as the initial sequence, corresponding to the 168th to 1196th amino acids of the full length, named C3A1, and codon optimization was performed according to the codon preference of Pichia pastoris, and the removal of BamH I, Xho I, Bgl II, Sac I and Sal I restriction sites was avoided. Bgl II and Xho I restriction sites were added at both ends of the optimized sequence and handed over to Nanjing GenScript Biotechnology Co., Ltd. for synthesis.

[0054] The synthesized C3A1 sequence was amplified by designing primers. The sequence is shown in SEQ ID NO.1. The PCR reaction system (50 μL) is as follows: 2×Phanta Master Mix 25.0 μL, template 1.0 μL, upstream and downstream primers 1.0 μL each, and ddH2O is added to 50 μL. SEQ ID NO.1:

[0055] GSGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAG

[0056] KDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGL

[0057] PGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGA

[0058] KGEVGPAGSPSGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGL

[0059] MGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDG

[0060] SPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGV

[0061] PGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGA

[0062] PGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGP

[0063] PGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPLAGAPGLRGGAGPPGPEGGKG

[0064] AAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIG

[0065] PPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGA

[0066] PGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGGPGAAGFPGARGLPGPPGSNGNP

[0067] GPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGI

[0068] TGARGLAGPPGMGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGR

[0069] DGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGP

[0070] AGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSP

[0071] The PCR amplification program for GPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGS was as follows: (1) denaturation at 95°C for 10 min, (2) denaturation at 94°C for 10 s, (3) annealing at 55°C for 30 s, (4) extension at 72°C for 3 min 30 s, and steps (2) to (4) were repeated for 35 cycles. Finally, extension was performed at 72°C for 10 min, and the PCR amplification product was stored at 4°C.

[0072] PCR results are as follows Figure 1 As shown in the figure, a C3A1 band of 3087 bp was successfully obtained by PCR. After purification and recovery using a gel recovery purification kit, it was ligated with the pPIC9KM plasmid double-digested with BamH I and Xho I using T4 DNA ligase for 2 h. The ligation solution was then transformed into E. coli Top10 competent cells and spread on a plate containing 200 μg mL -1 After the transformants grow on the ampicillin-resistant plate ( Figure 2 ) was used for colony PCR verification. The results of colony PCR are shown in Figure 2 As shown in the figure, PCR successfully amplified a band of about 3087 bp, proving that the recombinant plasmid pPIC9KM-C3A1 was successfully constructed. The plasmid map is shown in Figure 3 After the plasmids were extracted from the corresponding bacteria, they were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing, and the strains were preserved after the sequencing was correct.

[0073] Example 2 Construction of recombinant engineering bacteria Pichia pastoris KM71H / pPIC9KM-C3A1

[0074] The recombinant plasmid pPIC9KM-C3A1 successfully sequenced above was extracted using the Tiangen medium extraction kit, and the extracted plasmid was linearized by single enzyme digestion using Sac I or Sal I. The enzyme digestion system (50 μL) was as follows: 10×Q-cut buffer 5 μL, Sac I or Sal 1 μL, plasmid 1-2.0 μg, ddH2O was added to 50 μL, and the enzyme digestion reaction was carried out at 37°C for 2-4 hours. After the enzyme digestion was completed, the Tiangen large-scale PCR product recovery kit was used to purify and recover the enzyme digestion product for later use.

[0075] Pick a single colony from the Pichia pastoris KM71H streak plate and inoculate it into a sterile shaking tube containing 3 mL of YPD medium. Incubate it at 30°C, 250 rpm overnight. The next day, transfer 1 mL of the seed solution to a 500 mL conical flask containing 50 mL of YPD medium and incubate it at 30°C, 250 rpm until the OD 600The pH value is 1.0, then collect the bacterial solution in a 50mL round-bottom inner spiral centrifuge tube, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Resuspend the bacteria with 40mL pre-cooled sterile water, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Repeat the above sterile water washing step once. Resuspend the bacteria with 10mL pre-cooled 1M sorbitol, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Then add 300μL pre-cooled 1M sorbitol, gently pipette and mix with a pipette tip, and dispense the competent cells into 1.5mL centrifuge tubes at 100μL per tube for later use.

[0076] Take 10 μL of the purified product and add it to the Pichia pastoris KM71H competent cells prepared above, and place it on ice for 5 min. Then transfer the mixture to a 0.2 cm electric shock cup, and adjust the electroporator to the SC2 program for electric shock. After the electric shock, add 1 mL of pre-cooled 1 M sorbitol, and then place it in a shaker, and culture it at 30 ° C, 150 rpm for 1 h, and then spread it to contain 0.3 mg·mL -1 G418 YPD plate, after the transformants grow ( Figure 4 ), and then colony PCR was performed to verify the result. Figure 4 As shown in the figure, it can be seen that PCR successfully amplified a C3A1 band of about 3087 bp, indicating that the linearized product was successfully integrated into the yeast genome.

[0077] Since homologous recombination between yeast genome and linearized plasmid is a random integration event, recombinant bacteria with multiple copies of target gene integration will appear. Therefore, in order to screen recombinant bacteria with high copy integration, the positive transformants obtained above were inoculated in 1.0 mg mL -1 and 4.0 mg mL -1 G418 was screened on a high-resistance YPD plate. The screening results of the high-resistance plate are shown in Figure 5 As shown in the figure, all bacteria can be -1 G418 grew on YPD plates, but only 50% of the bacteria could grow at 4.0 mg mL -1 The bacteria that grow on the YPD plates with G418 resistance may be high-copy strains.

[0078] In order to screen and obtain high expression strains, the high copy strains ( Figure 5 ) were sequentially plated in 96-well plates for culture and induction of expression, and the specific steps were as follows:

[0079] (1) The colonies that can grow on the high-concentration G418 plate were picked up and placed in a 96-well deep-well plate containing 300 μL YPD medium and cultured at 30° C. and 250 rpm for 12 to 24 h.

[0080] (2) 10-100 μL of bacterial solution was transferred to two 48-well deep-well plates containing 500 μL of BMGY medium and cultured at 30°C and 250 rpm for 12-18 h.

[0081] (3) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 500 μL BMMY medium, and induce at 28°C, 250 rpm for 24 to 48 h. Add 1% methanol at 24 h.

[0082] (4) After induction, centrifuge at 4000 rpm for 10 min, use a pistol to pipette 20 μL of supernatant into a 96-well PCR tube, add 5 μL of 5×Sample Loading Buffer and denature at room temperature for 5 min, centrifuge at 4000 rpm for 10 min, and pipette 10 μL of supernatant for SDS-PAGE analysis. After Coomassie Brilliant Blue staining and decolorization, you can take pictures.

[0083] The results of SDS-PAGE screening are as follows Figure 6 As shown in the figure, target bands were observed between 95 and 140 kDa in lanes 5, 7 and 9, indicating that three high-expression strains were obtained through screening, and then these three high-copy strains were preserved.

[0084] Example 3 Fermentation of recombinant engineered bacteria Pichia pastoris KM71H / pPIC9KM-C3A1 in a 5 L fermenter

[0085] Pichia pastoris KM71H / pPIC9KM-C3A1 was streaked in a flask containing 0.3 mg mL -1 G418 was cultured on YPD plates at 30°C for 72 h, and a single colony was picked and inoculated into 50 mL of BMG medium and continued to be cultured until OD 600 The inoculum was transferred to 100 mL BMG medium at a rate of 10% and cultured until the OD reached 8.0-12.0. The secondary seed solution was transferred to a 5 L fermentation tank and 4.35 mL L -1 PTM 1 trace elements and 4.35mL·L -1 0.02% biotin, start fermentation tank culture, when DO quickly rises to 50-70%, start glycerol feeding, glycerol feeding rate is 10-20mL·h -1 ·L -1 , the DO was maintained at 25-30% by fine-tuning the glycerol feeding rate, and the feeding was stopped after 5-6 hours of feeding. After "starvation" for 30-60 minutes, methanol induction was started. The methanol flow rate was 0.5-7.2 mL·h -1 ·L -1The induction temperature was 28°C, and samples were taken during the period to detect the wet weight and protein concentration of the bacteria.

[0086] Seed culture medium BMG (1L): 10 g glycerol, 2.3 g K2HPO4, 11.81 g KH2PO4, dilute to 900 mL, sterilize at 121 °C for 15 min, add 100 mL 13.4% YNB and 2 mL 0.02% biotin after cooling;

[0087] Fermentation medium BSM (1L): 80g glycerol, 26.7mL H3PO4, 1.175g CaSO4, 18.2g K2SO4, 7.28g MgSO4, 4.13g KOH, add water to dissolve and make up to 2000mL. After sterilization, add 4.35mL PTM1;

[0088] Feed growth medium: 50% (w / v) glycerol (containing 12 mL·L -1 PTM1)

[0089] Fermentation induction medium: 100% methanol (containing 12 mL·L -1 PTM1).

[0090] PTM1 ingredients (1L): CuSO4·5H2O 6.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O0.2g, H3BO3 0.02g, CoCl2 0.5g, ZnCl2 20.0g, FeSO4·7H2O 65.0g, H2SO4 5mL·L -1 0.2 g of biotin was sterilized by filtration and stored at 4°C for later use.

[0091] Protein expression during the fermentation of Pichia pastoris KM71H / pPIC9KM-C3A1 Figure 7 As shown in the figure, with the extension of fermentation time, the expression level of C3A1 protein also showed a significant increase, reaching 10 g·L at 72 h. -1 .

[0092] Example 4 C3A1 protein purification

[0093] The fermented liquid after the fermentation is centrifuged to remove the bacterial cells, and the supernatant is obtained. The supernatant is clarified by a 50-200 nm ceramic membrane, and the supernatant is collected. Further, the supernatant is concentrated by a 3-50 kDa hollow fiber membrane (Shandong Bona Biotechnology Group Co., Ltd.), and after the concentration is completed, the pH is adjusted to 4.5 and the conductivity is 100-150 mS / cm for standby use.

[0094] The prepared samples were purified using buffer A (20 mM KH2PO4, 2 M NaCl, pH 4.5) and buffer B (20 mM KH2PO4, pH 4.5) in combination with a protein purifier (SCG-P100 series from Suzhou Saipu Instrument Co., Ltd., loaded with Phenyl Purose 6Fast Flow chromatographic column, and phenyl hydrophobic filler purchased from Jiaxing Qianchun Biotechnology Co., Ltd.). The specific steps were as follows: first, the chromatographic column was equilibrated with 5 CV buffer A, and the sample was loaded after the equilibration (the sample amount was 200 mg). After the loading, the column was rinsed with buffer A until A 210 The protein purity of C3A1 was detected by SDS-PAGE. Figure 8 As shown in the figure, it can be seen that after purification, the protein purity of C3A1 is significantly improved, and the band is single. After the purified proteins are concentrated and grayscale analysis is performed, the purity can reach 95%.

[0095] Example 5 Construction of recombinant engineered bacteria E. coli Top10 / pPIC9KM-C3A1-II

[0096] The plasmid pPIC9KM-C3A1 obtained above was double-digested with BamH I and Xho I. After the reaction was completed, it was purified and recovered using a gel recovery purification kit and set aside.

[0097] Primers were designed to amplify the C3A1 sequence. The PCR reaction system (50 μL) included 25.0 μL of 2×Phanta Master Mix, 1.0 μL of template, 1.0 μL of upstream and downstream primers, and ddH2O was added to make up to 50 μL.

[0098] The PCR amplification program was as follows: (1) denaturation at 95°C for 10 min, (2) denaturation at 94°C for 10 s, (3) annealing at 55°C for 30 s, and (4) extension at 72°C for 3 min 30 s. Steps (2) to (4) were repeated for 35 cycles, and finally extension was performed at 72°C for 10 min. The PCR amplification product was stored at 4°C.

[0099] PCR results are as follows Fig. 9 As shown in the figure, a C3A1 band of 3087 bp was successfully obtained by PCR and purified and recovered using a gel recovery purification kit. It was further ligated with the double-digested pPIC9KM-C3A1 plasmid using T4 DNA ligase for 2 h, and then the ligation solution was transformed into E. coli Top10 competent cells and spread on a plate containing 200 μg mL -1After the transformants grow on the ampicillin-resistant plate ( Fig.10 ) was used for colony PCR verification. The results of colony PCR are shown in Fig.10 As shown in the figure, PCR successfully amplified a band of about 6174 bp, and the sequence is shown in SEQ ID NO.2, proving that the recombinant plasmid pPIC9KM-C3A1-II was successfully constructed. The plasmid map is shown in Fig.11 After the plasmids were extracted from the corresponding bacteria, they were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing, and the strains were preserved after the sequencing was correct.

[0100] SEQ ID NO.2:

[0101] GSGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAG

[0102] KDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGL

[0103] PGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGA

[0104] KGEVGPAGSPSGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGL

[0105] MGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDG

[0106] SPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGV

[0107] PGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGA

[0108] PGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGP

[0109] PGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPLAGAPGLRGGAGPPGPEGGKG

[0110] AAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIG

[0111] PPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGA

[0112] PGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGGPGAAGFPGARGLPGPPGSNGNP

[0113] GPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGI

[0114] TGARGLAGPPGMGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGR

[0115] DGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGP

[0116] AGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSP

[0117] GPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGS

[0118] GLAGYPGPAGPPGPPPGTGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPPGAIGPSGPAGK

[0119] DGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLP

[0120] GENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAK

[0121] GEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLM

[0122] GARGPPGPAGANGAPGLRGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSP

[0123] GEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVP

[0124] GGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAP

[0125] GKNGERGGPGGPGPQGPPGKNGETGPQGPPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPP

[0126] GENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPLAGAPGLRGGAGPPGPEGGKGA

[0127] AGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGP

[0128] PGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAP

[0129] GEKGEGGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGGPGAAGFPGARGLPGPPGSNGNPG

[0130] PPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGIT

[0131] GARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRD

[0132] GNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPA

[0133] GAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPG

[0134] PAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGS

[0135] Example 6 Construction of recombinant engineering bacteria Pichia pastoris KM71H / pPIC9KM-C3A1-II

[0136] The recombinant plasmid pPIC9KM-C3A1 successfully sequenced above was extracted using the Tiangen medium extraction kit, and the extracted plasmid was linearized by single enzyme digestion using Sac I or Sal I. The enzyme digestion system (50 μL) was as follows: 10×Q-cut buffer 5 μL, Sac I or Sal 1 μL, plasmid 1-2.0 μg, ddH2O was added to 50 μL, and the enzyme digestion reaction was carried out at 37°C for 2-4 hours. After the enzyme digestion was completed, the Tiangen large-scale PCR product recovery kit was used to purify and recover the enzyme digestion product for later use.

[0137] Pick a single colony from the Pichia pastoris KM71H streak plate and inoculate it into a sterile shaking tube containing 3 mL of YPD medium. Incubate it at 30°C, 250 rpm overnight. The next day, transfer 1 mL of the seed solution to a 500 mL conical flask containing 50 mL of YPD medium and incubate it at 30°C, 250 rpm until the OD 600 The pH value is 1.0, then collect the bacterial solution in a 50mL centrifuge tube, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Resuspend the bacteria with 40mL pre-cooled sterile water, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Repeat the above sterile water washing step once. Resuspend the bacteria with 10mL pre-cooled 1M sorbitol, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Then add 300μL pre-cooled 1M sorbitol, gently pipette and mix with a pipette tip, and dispense the competent cells into 1.5mL centrifuge tubes at 100μL per tube for later use.

[0138] Take 10 μL of the purified product and add it to the Pichia pastoris KM71H competent cells prepared above, and place it on ice for 5 min. Then transfer the mixture to a 0.2 cm electric shock cup, and adjust the electroporator to the SC2 program for electric shock. After the electric shock, add 1 mL of pre-cooled 1 M sorbitol, and then place it in a shaker, and culture it at 30 ° C, 150 rpm for 1 h, and then spread it to contain 0.3 mg·mL -1 G418 YPD plate, after the transformants grow ( Fig.12 ), and then colony PCR was performed to verify the result. Fig.12 As shown in the figure, it can be seen that PCR successfully amplified a C3A1 band of about 3087 bp, indicating that the linearized product was successfully integrated into the yeast genome.

[0139] Since homologous recombination between yeast genome and linearized plasmid is a random integration event, recombinant bacteria with multiple copies of target gene integration will appear. Therefore, in order to screen recombinant bacteria with high copy integration, the positive transformants obtained above were inoculated in 1.0 mg mL -1 and 4.0 mg mL -1 Screening was performed on the high-resistance YPD plate of G418, and the bacteria growing on the high-resistance plate may be high-copy strains.

[0140] The growth of Pichia pastoris KM71H / pPIC9KM-C3A1-II on high resistance plates is as follows Fig.13 As shown, most bacteria can grow in the presence of 1.0 mg mL -1 G418 grew on YPD plates, but only about 30% of the bacteria could grow on YPD plates containing 4.0 mg mL -1 These bacteria grew on G418 plates and may be high-copy strains.

[0141] In order to screen and obtain high-expression strains, the high-copy strains obtained above were sequentially inoculated into 96-well plates for culture and induced expression. The specific steps are as follows:

[0142] (1) The colonies that can grow on the high-concentration G418 plate were picked with a toothpick and placed in a 96-well deep-well plate containing 300 μL YPD medium. The plates were cultured at 30°C and 250 rpm for 12 to 24 h.

[0143] (2) 10-100 μL of bacterial solution was transferred to a 48-well deep-well plate containing 500 μL of BMGY medium and cultured at 30°C and 250 rpm for 12-18 h.

[0144] (3) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 500 μL BMMY medium, and induce at 28°C, 250 rpm for 24 to 48 h. Add 1% methanol after 24 h.

[0145] (4) After induction, centrifuge at 4000 rpm for 10 min, use a pistol to pipette 20 μL of supernatant into a 96-well PCR tube, add 5 μL of 5×Sample Loading Buffer and denature at room temperature for 5 min, centrifuge at 4000 rpm for 10 min, and pipette 10 μL of supernatant for SDS-PAGE analysis. After Coomassie Brilliant Blue staining and decolorization, you can take pictures.

[0146] The results of SDS-PAGE screening are as follows Fig.14 As shown in the figure, the target bands were observed above 190 kDa in lanes 2 and 5, indicating that two high-expression strains were obtained through screening, and then these two high-copy strains were preserved.

[0147] Example 7 Fermentation of recombinant engineered bacteria Pichia pastoris KM71H / pPIC9KM-C3A1-II in a 5 L fermenter

[0148] Pichia pastoris KM71H / pPIC9KM-C3A1-II was streaked in a flask containing 0.3 mg mL -1 G418 was cultured on YPD plates at 30°C for 72 h, and a single colony was picked and inoculated into 50 mL of BMG medium and continued to be cultured until OD 600 The inoculum was transferred to 100 mL BMG medium at a rate of 10% and cultured until the OD reached 8.0-12.0. The secondary seed solution was transferred to a 5 L fermentation tank and 4.35 mL L -1 PTM 1 trace elements and 4.35mL·L -1 0.02% biotin, start fermentation tank culture, when DO quickly rises to 50-70%, start glycerol feeding, glycerol feeding rate is 10-20mL·h -1 ·L -1 , the DO was maintained at 25-30% by fine-tuning the glycerol feeding rate, and the feeding was stopped after 5-6 hours of feeding. After "starvation" for 30-60 minutes, methanol induction was started. The methanol flow rate was 0.5-7.2 mL·h -1 ·L -1 The induction temperature was 28°C, and samples were taken during the period to detect the wet weight and protein concentration of the bacteria.

[0149] Seed culture medium BMG (1L): 10 g glycerol, 2.3 g K2HPO4, 11.81 g KH2PO4, dilute to 900 mL, sterilize at 121 °C for 15 min, add 100 mL 13.4% YNB and 2 mL 0.02% biotin after cooling;

[0150] Fermentation medium BSM (1L): 80g glycerol, 26.7mL H3PO4, 1.175g CaSO4, 18.2g K2SO4, 7.28g MgSO4, 4.13g KOH, add water to dissolve and make up to 2000mL. After sterilization, add 4.35mL PTM1;

[0151] Feed growth medium: 50% (w / v) glycerol (containing 12 mL·L -1 PTM1)

[0152] Fermentation induction medium: 100% methanol (containing 12 mL·L -1 PTM1).

[0153] PTM1 ingredients (1L): CuSO4·5H2O 6.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O0.2g, H3BO3 0.02g, CoCl2 0.5g, ZnCl2 20.0g, FeSO4·7H2O 65.0g, H2SO4 5mL·L -1 0.2 g of biotin was sterilized by filtration and stored at 4°C for later use.

[0154] Protein expression during the fermentation of Pichia pastoris KM71H / pPIC9KM-C3A1-II is shown in Fig.15 As shown in the figure, with the extension of fermentation time, the expression level of C3A1-II protein also showed a significant increase, reaching 10 g·L at 72 h. -1 .

[0155] Example 8 C3A1-II protein purification

[0156] The fermented liquid after the fermentation is centrifuged to remove the bacterial cells, and the supernatant is obtained. The supernatant is clarified by a 50-200 nm ceramic membrane, and the supernatant is collected. Further, the supernatant is concentrated by a 3-50 kDa hollow fiber membrane (Shandong Bona Biotechnology Group Co., Ltd.), and after the concentration is completed, the pH is adjusted to 4.5 and the conductivity is 100-150 mS / cm for standby use.

[0157] The prepared samples were purified using buffer A (20 mM KH2PO4, 2 M NaCl, pH 4.5) and buffer B (20 mM KH2PO4, pH 4.5) in combination with a protein purifier (SCG-P100 series from Suzhou Saipu Instrument Co., Ltd., loaded with Phenyl Purose 6Fast Flow chromatographic column, and phenyl hydrophobic filler purchased from Jiaxing Qianchun Biotechnology Co., Ltd.). The specific steps were as follows: first, the chromatographic column was equilibrated with 5 CV buffer A, and the sample was loaded after the equilibration (the sample amount was 200 mg). After the loading, the column was rinsed with buffer A until A 210 The protein purity of C3A1 was detected by SDS-PAGE. Fig.16 As shown in the figure, it can be seen that after purification, the protein purity of C3A1-II is significantly improved, and the band is single. After the purified proteins are concentrated and grayscale analysis is performed, the purity can reach 99%.

[0158] Example 8 Cytotoxicity Experiment of C3A1 and C3A1-II

[0159] L-929 fibroblasts in good growth state and in the logarithmic growth phase were taken, digested with 0.25% trypsin, neutralized with complete medium (10% FBS, 1% penicillin-streptomycin, RPMI 1640 medium), counted, and gradiently diluted to prepare a final cell density of 8.0×10 4 / mL cell suspension; after mixing the cell suspension, place it in a sterile sample tank, use a spray gun to seed a 96-well plate, the inoculation volume is 100μL / well, the inoculation amount of cells is 8000 / well, every 6 duplicate wells are regarded as a parallel, PBS solution is added to the edge wells of the well plate (to prevent drying), after the well plate is inoculated, it is placed on the desktop and left to stand for 10 minutes, and then placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity, and cultured continuously for 24 hours; aspirate the complete culture medium and divide the 96-well plate into a blank control group, an experimental control group and an experimental treatment group: the experimental treatment group was added with different concentrations of C3A1-II collagen (1, 5, 10, 20, 40, 60, 80 and 100μg·mL -1) RPMI 1640 complete medium; the experimental control group was added with RPMI 1640 complete medium containing different concentrations of C3A1-I collagen (1, 5, 10, 20, 40, 60, 80 and 100 μg / mL); the blank control group was added with the corresponding volume of RPMI 1640 complete medium without any collagen; then, the cells were placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity for 24 hours; the old culture medium was removed, and a CCK-8 solution with a final concentration of 10% in RPMI 1640 complete medium was added to each well; after incubation at 37°C in the dark for 1 to 3 hours, the absorbance value (OD value) of each well was measured at 450nm using a full-band microplate reader. The cell survival rate was calculated according to the mean absorbance of each group according to the following formula:

[0160] Cell survival rate (%) = [(OD of experimental group) - (OD of blank group)] / [(OD of control group) - (OD of blank group)] × 100%

[0161] The experimental results are as follows Fig.17 As shown in the figure, it can be seen that the cell survival rates in the experimental groups with different concentrations of C3A1-I and C3A1-II collagen were close to 100%, indicating that C3A1-I and C3A1-II were non-cytotoxic to L-929 fibroblasts and had good biocompatibility.

[0162] Example 9 Cell proliferation assay (colony formation assay) of C3A1 and C3A1-II

[0163] The cell colony formation assay was used to compare the cell proliferation effects of two types of recombinant humanized type III collagen.

[0164] L-929 fibroblasts in good growth state and in logarithmic growth phase were taken, digested with 0.25% trypsin, neutralized with complete culture medium (10% FBS, 1% penicillin-streptomycin, RPMI1640 culture medium), counted, and gradiently diluted to prepare a cell suspension with a cell density of 100 cells / mL; 200 cells were planted in each well of a 6-well cell culture plate, and every 3 wells were used as a parallel, among which: the final concentration of 50 μg mL was added to each well of the experimental group -1C3A1 collagen and C3A1-II collagen solution; the blank control group did not add any collagen, and continued to be placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity for 7 to 10 days. When clones visible to the naked eye appear, the culture is terminated (do not move the culture dish during the clone culture to avoid affecting the formation of clones). After the culture is completed, the culture medium is discarded, washed once with PBS buffer, fixed with methanol solution for 20 minutes, washed again with PBS, stained with 1% crystal violet for 4 hours, washed with running water to remove the staining solution, and placed in the air to dry and count. The number of clones is counted by naked eye observation or microscopy. In theory, each clone should contain >50 cells. After the statistics are completed, the cell planting rate and cell survival rate are calculated according to the following formula.

[0165] Cell implantation rate (%) = (number of cell clones in the culture dish of the control group / number of cells implanted) × 100%

[0166] Cell survival rate (%) = (cell seeding rate of treatment group (%) / cell seeding rate of control group (%)) × 100%

[0167] The experimental results are as follows Fig.18 As shown in the figure, the cell survival rates of the blank control group, C3A1 and C3A1-II were 55%, 61% and 67%, respectively, which indicates that compared with the control, C3A1 and C3A1-II can significantly promote the colony formation of L-929 fibroblasts, and C3A1-II shows better cell proliferation promotion effect than C3A1.

[0168] Example 10 Cell scratch assay of C3A1 and C3A1-II

[0169] Keratinocytes HaCaT were used to simulate the keratinocyte layer of the skin to explore the skin damage repair effect of collagen solution. Use a ruler and a marker to evenly draw horizontal lines on the back of a 6-well plate, approximately every 0.5 cm (at least 5 lines per well); take keratinocytes HaCaT cells that are in good growth and in the logarithmic growth phase, digest them with 0.25% trypsin, neutralize them with full culture medium (10% FBS, 1% penicillin-streptomycin, high-glucose DMEM cell culture medium), count them, and dilute them in a gradient to prepare a cell density of 1.5×10 5 2 mL of the above cell suspension was inoculated in 6-well culture plates, with an inoculation volume of 3.0 × 10 cells / mL per well. 5After that, the cells were placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity. The cells were observed regularly during the culture process. When the cell confluence rate was ≥90%, the serum-free medium was replaced and starvation culture was continued for 24 hours. After starvation, a 200μL pipette tip was used to gently push down the horizontal line on the back; the cells were rinsed with PBS 2 to 3 times, and after confirming that the cells were clean under a microscope, 100μg·mL of DMEM high-glucose basal medium was added to the experimental groups. -1 Two types of collagen III solutions (three replicate wells were set up), and the blank control group was added with the same volume of DMEM high-glucose basal medium; after culturing in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity for 0h, 24h and 48h, the horizontal scratches on the back of the 6-well plate were wiped off, and photos were taken under a 10x objective lens to ensure that the scratches were centered and vertical, and pay attention to the consistency of the background; samples were taken at 0h and 48h, and after taking photos and recording, the images were opened using Image J software, 6 to 8 horizontal lines were randomly drawn, the mean of the distance between cells or the mean of the scratch area was calculated, and the cell migration rate was calculated according to the following formula.

[0170] Cell migration rate = (t 0h Initial scratch area-t 48h Time scratch area) / t 0h Initial scratch area.

[0171] The experimental results are as follows Fig.19 As shown in the figure, there are no obvious cells in the central scratch area at 0h. After culturing in the culture medium containing two collagen solutions for 48h, HaCaT cells gradually migrate to the central scratch area. In the culture medium containing C3A1-II collagen, the migration rate of HaCaT cells is 38%, which is significantly higher than that of the group containing C3A1 collagen (27%). This indicates that compared with C3A1, C3A1-II collagen has a significant ability to promote keratinocyte migration, that is, the C3A1-II collagen prepared in Example 1 of the present invention has better potential to promote skin damage repair.

[0172] Example 10 Cell Adhesion Experiment of C3A1 and C3A1-II

[0173] Cell adhesion is a bridge between cells and the outside world, and plays an important role in cell proliferation and differentiation. In this example, a cell adhesion experiment was used to explore the effects of two types of collagen III on cell adhesion ability.

[0174] First, 50 μL of 1% gelatin solution was added to each well of a 96-well plate culture dish as a plate coating solution to prepare an artificial basement membrane glue solution, which was placed in a cell culture incubator for 2 h; the coating solution was removed, 200 μL of 1% BSA was added, and the plate was incubated in a 37°C incubator for 1 h. The plate was washed with serum-free culture medium 3 times to remove excess glue for later use; L-929 fibroblasts in good growth state and in the logarithmic growth phase were taken, digested with 0.25% trypsin, neutralized with complete culture medium (10% FBS, 1% penicillin-streptomycin, RPMI 1640 culture medium), and counted at 5×10 4 The cells were inoculated into 96-well plates at a density of 100 μL per well. C3A1 and C3A1-II solutions were added to the experimental groups to reach a final concentration of 50 μg mL -1 , the control group did not add collagen solution, and a blank control well with no cells but only culture medium was set up; 5 replicate wells were set up for each group of cells, and the edge wells were filled with PBS solution; the culture plate was placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity, and cultured continuously for 1 to 2 hours (the culture time can be adjusted appropriately according to the cell state and experimental requirements); the cell culture plate was removed, the culture medium was aspirated, and PBS was gently rinsed 3 times, and a CCK-8 solution with a final concentration of 10% in RPMI 1640 complete culture medium was added to each well; after incubation at 37°C in the dark for 1 to 3 hours, the absorbance value (OD value) of each well was measured at 450nm using a full-band microplate reader. The cell adhesion rate was calculated according to the mean absorbance of each group according to the following formula:

[0175] Cell adhesion rate = [(experimental group cell OD-blank OD) / (control group cell OD-blank OD)] × 100%

[0176] The experimental results are as follows Fig. 20 As shown in the figure, the cell adhesion rates of the control group, C3A1 and C3A1-II were 39%, 45% and 51%, respectively, which indicates that compared with the control, both C3A1 and C3A1-II can promote the adhesion of L-929 fibroblasts, and C3A1-II shows better cell adhesion-promoting effect than C3A1.

[0177] Example 11 Experiment on stimulating collagen regeneration by C3A1 and C3A1-II

[0178] Type I collagen is the only component of collagen fibers in the dermis, accounting for more than 80% of the skin, and is the most abundant component in the dermal connective tissue; the role of collagen in the skin is like the springs in a mattress. Sufficient collagen can help the skin lock in moisture and maintain skin firmness and elasticity. The reason why the skin has wrinkles is due to the aging of collagen in the dermis. The difference in the ability of the two types of recombinant humanized type III collagen to promote collagen regeneration was determined by qualitative and quantitative analysis of collagen production in fibroblast L-929 cell lysate after exogenous addition of the two types of collagen.

[0179] ① The effect of C3A1-II prepared by this method on the regeneration of type I and type III collagen was qualitatively detected by Western Blotting protein gel electrophoresis. First, 100 μg mL -1 Protein was extracted from 3-day-old fibroblast L-929 cells treated with C3A1 and C3A1-II. The extracted cell lysate was quantified using the BCA protein quantification kit of Beyotime, and the quantified protein solution was subjected to SDS-PAGE electrophoresis. The samples were divided into a blank control group without any treatment and an experimental treatment group with two types of recombinant collagen. The gel after electrophoresis was subjected to gel transfer and immunoreaction. The PVDF membrane after immunoreaction was subjected to chemiluminescence and image analysis.

[0180] The experimental results are as follows Fig.21 As shown in the figure, compared with the blank control group and the C3A1-treated group, the exogenous addition of C3A1-II collagen can increase the type I collagen content of the fibroblast L-929 to a certain extent, which indicates that C3A1-II collagen has the potential to resist wrinkles and firm the skin.

[0181] ② The total collagen content in the cells after exogenous addition of the two types of collagen was quantitatively detected by the Sirius Red detection kit; the specific method was as follows: first, protein extraction was performed on the cells treated with the two types of collagen for 3 days; 100 μL of protein lysate obtained in the above steps was aspirated, and 500 μL of Sirius Red solution was added, and the mixture was vortexed continuously at room temperature for 20 minutes; centrifuged at 10,000 rpm and 4°C for 3 minutes, and the supernatant was carefully aspirated and discarded; 500 μL of washing solution was continued to be added to the test tube, and the mixture was centrifuged at 10,000 rpm and 4°C for 3 minutes, and the supernatant was carefully aspirated and discarded; 250 μL of extraction buffer was added to the test tube with the remaining particle precipitation, and the particles were carefully vortexed until they were completely dissolved; 200 μL of the dissolved solution was transferred from the test tube to a 96-well plate, and the absorbance was read at 530 nm using a full-band microplate reader; the collagen concentration (μg / mL) in the sample was calculated using the attached standard curve and regression analysis.

[0182] The experimental results are as follows Fig.21 As shown in the figure, compared with the blank control group, the total collagen in the C3A1 and C3A1-II treatment groups increased to 107% and 114% of the control group, respectively, that is, compared with the C3A1 treatment group, the exogenously added C3A1-II collagen can significantly increase the total collagen content in fibroblast L-929 cells. The above results indicate that C3A1-II has the ability to stimulate skin collagen regeneration.

[0183] In summary, the experimental results of Examples 8 to 11 all show that C3A1 and C3A1-II have high biocompatibility and bioactivity. In comparison, C3A1-II has better ability to promote cell adhesion, promote fibroblast proliferation, promote keratinocyte migration and stimulate skin collagen regeneration, which indicates that C3A1-II has a higher application value in promoting skin damage repair and anti-wrinkle and firming.

[0184] Example 12 Evaluation of the repair efficacy of C3A1 and C3A1-II on zebrafish

[0185] The zebrafish embryo tail amputation inflammation model is a traumatic inflammation model. After the tail of the zebrafish is amputated, local damage to the zebrafish tail fin will be induced, prompting the zebrafish to have an autoimmune response. After the tail fin is cut off, the wound will immediately shrink and be covered with epithelial cells. An inflammatory reaction will occur within a few hours, and the number of neutrophils in the wound will increase. The neutrophils of the transgenic neutrophil fluorescent zebrafish can be clearly observed under a fluorescent microscope, and the AB type wild zebrafish can be seen to regenerate the damaged part of the tail fin under a microscope. Therefore, the repair efficacy of the sample can be evaluated by the regeneration speed of the tail fin or the number of neutrophils near the wound.

[0186] After hatching, AB wild zebrafish embryos were placed under a stereo microscope. Tricaine was used to anesthetize the zebrafish embryos in advance, or the zebrafish embryos were placed on an agar plate and the caudal fin of the embryos was cut. After cutting the tail, a zebrafish tail-cut inflammation model was constructed. Each embryo was photographed and numbered. The zebrafish without tail-cutting were taken as the blank group, and the tail-cut zebrafish were divided into a model group and a sample group. The blank group and the model group were cultured with culture water, and the sample group was cultured with different concentrations of C3A1 and C3A1-Ⅱ collagen (concentrations of 2.0, 4.0 and 10.0 mg·mL -1 ) and placed in a 28.5°C incubator for 2 days. After the incubation, the corresponding numbered zebrafish tail fins of each group were photographed, and ImageJ was used to compare the area of ​​the tail fins just after the tail was cut. The repair rate of the tail fins of each group of zebrafish embryos was calculated according to the following formula, and the repair rates of each group were compared to evaluate the repair efficacy of the tested samples.

[0187]

[0188] The experimental results are as follows Fig. 22 (A) and Fig. 22 As shown in (B), after 48 h of exposure to different concentrations of collagen, the resected tail fin of zebrafish embryos gradually recovered. When the concentration reached 10.0 mg·mL -1 This indicates that both C3A1 and C3A1-Ⅱ collagens can repair the damaged parts of the zebrafish tail fin, thereby promoting the regeneration of the zebrafish tail fin, and the strength of the repair ability is positively correlated with the concentration of collagen.

[0189] At the same time, the repair ability of C3A1 and C3A1-Ⅱ collagen was compared. The results are as follows: Fig. 22 As shown in (C), it can be seen from the figure that under the three concentrations tested, the repair ability of zebrafish embryos exposed to C3A1-Ⅱ collagen is significantly higher than that of C3A1 collagen, indicating that without affecting the normal growth and development of zebrafish embryos, the repair effect of C3A1-Ⅱ collagen is significantly better than that of C3A1 collagen.

[0190] Example 13 Self-assembly hydrogel experiment

[0191] The purified C3A1 and C3A1-II proteins were replaced with pH 7.0, 0.2M Na2HPO4 buffer and then placed at 4°C for self-assembly experiments. The self-assembly effects were observed and photographed. The final concentrations of C3A1 and C3A1-II were 6.8 and 7.3 g·L, respectively. -1 .

[0192] The experimental results after 24 hours of self-assembly are as follows Fig.23 As shown in the figure, C3A1-II is in a hydrogel state, while C3A1 is still in a solution state under the same conditions, which indicates that the structure of C3A1-II may be more complex, making the interaction between chains more obvious and easier to form a hydrogel, thus having better stability and support. The obtained hydrogel samples were freeze-dried and their microstructures were further observed by SEM. The results are shown in Fig.24 As shown in the figure, it can be seen that compared with C3A1, the structure of C3A1-II presents a dense network structure, which increases the specific surface area of ​​the material and significantly improves the mechanical properties of the material; at the same time, the increase in surface area helps to enhance the charge storage capacity, thereby enhancing the interaction within and between chains, thereby making the protein structure more stable; and this porous structure can serve as a scaffold for cell growth, promoting cell attachment and growth, thereby contributing to wound healing and tissue repair.

[0193] Example 14 Determination of collagen melting point by DSC method

[0194] In this example, differential scanning calorimetry (DSC) was used to measure the melting points (T m ), the specific experimental method is: respectively take 10 μL of the protein solution of C3A1 and C3A1-II replaced with pH 7.0, 0.2M Na2HPO4 and add them into the crucible, weigh the mass of the protein solution, input the mass into the determination method, seal the cover and start the determination, the determination procedure is: 25℃ equilibrium for 3min, temperature scanning range 25~90℃, temperature increase rate 10℃ / min, after the scanning, the corresponding protein DSC curve can be obtained, and the corresponding melting point can be calculated by integration.

[0195] The Tm values ​​of C3A1 and C3A1-II were determined as follows: Fig.25 As shown in the figure, it can be seen that the melting point of C3A1 is 54.03±1.73, and the melting point of C3A1-II is 67.73±1.00, which indicates that C3A1-II has better thermal stability.

[0196] Example 15 Comparison of protein structure stability

[0197] To further explain why C3A1-II is more effective than C3A1, we attempted to analyze the protein structures of C3A1 and C3A1-II. We used AlphaFold 2 software to perform structural modeling and obtain accurate protein structure models. The structural models of the two proteins are shown in Figure 2. Fig.26 As shown in the figure, it can be seen that the C3A1-II structure is more complex and overall compact than C3A1, and therefore may be more stable.

[0198] Then, the two proteins were simulated for 10 ns each, and the RMSD value of each frame was recorded to compare the time required for them to reach a stable conformation and calculate the average RMSD value when they finally reached equilibrium. The results of the molecular dynamics simulation are shown in Figure 2. Fig. 27 As shown in the figure, the conformation of C3A1-II has reached equilibrium at 6 ns, while the conformation of C3A1 has not yet been observed to have an obvious equilibrium trend at the end of the simulation. At the same time, during the simulation, the overall RMSD of C3A1-II is significantly lower than that of C3A1. the latter RMSD average difference The fluctuation difference is quite significant for protein structure. These results indicate that the structure of C3A1-II is more stable than that of C3A1 and is more suitable for use as an injectable filling material in the field of medical devices.

[0199] In summary, the present invention provides a high molecular weight recombinant humanized collagen and a method for synthesizing the same. The collagen sequence of the present invention is 100% homologous to the human type III collagen sequence, and can also be selected from any one or more full-length triple helix structure regions of human collagen. No amino acid sequence of non-human collagen is introduced, which can greatly reduce the risk of immunogenicity caused by sequence rejection. In addition, the sequence construction does not contain any purification tags, and there is no need to perform a tag removal step. The purity can reach 95% to 99% after one-step hydrophobic chromatography purification. At the same time, the present invention provides an application of high molecular weight recombinant humanized collagen. The high molecular weight collagen C3A1-II obtained by the present invention has better biocompatibility, better activity in promoting cell adhesion, promoting fibroblast proliferation and keratinocyte migration. In the case of simulated skin keratin damage in vitro, C3A1-II has better ability to promote skin damage repair and stimulate collagen regeneration. At the same time, C3A1-II exhibits better support, thermal stability and structural stability, and is more suitable as a tissue filling material in the field of medical devices.

[0200] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A high molecular weight recombinant humanized collagen, characterized in that: The core monomer sequence of the high molecular weight recombinant humanized collagen is selected from the triple helix structure region of the human type III collagen sequence, and the amino acid sequence is shown in SEQ ID NO:

1. The high molecular weight recombinant humanized collagen is obtained by multiple tandem repetitions of the amino acid sequence shown in SEQ ID NO:

1.

2. The high molecular weight recombinant humanized collagen according to claim 1, characterized in that: The high molecular weight recombinant humanized collagen is obtained by tandemly repeating the amino acid sequence shown in SEQ ID NO: 1 1 to 5 times.

3. The high molecular weight recombinant humanized collagen according to claim 2, characterized in that: The number of tandem repetitions is 2, and the obtained amino acid sequence is shown in SEQ ID NO: 2, which contains 2059 aa and has a molecular weight of 183.15 kDa.

4. The high molecular weight recombinant humanized collagen according to claim 3, characterized in that: A Bgl II restriction site was added to the N-terminus of the sequence shown in SEQ ID NO: 2, and BamH I and Xho I restriction sites were added to the C-terminus.

5. The high molecular weight recombinant humanized collagen according to claim 3, characterized in that: The sequence shown in SEQ ID NO: 2 contains GPP, KGD, RGD, and GER integrin binding site sequences, and has good activity in promoting cell adhesion, fibroblast proliferation, and keratinocyte migration.

6. The high molecular weight recombinant humanized collagen according to claim 1, characterized in that: The core monomer sequence can be selected from any one or more full-length triple helical structure regions of human collagen.

7. A method for synthesizing high molecular weight recombinant humanized collagen according to any one of claims 1 to 6, characterized in that: The steps include: (1) Amino acids 168 to 1196 of human collagen were selected as the core monomer sequence C3A1, Bgl II was added to the N-terminus of the sequence, and BamH I and Xho I restriction sites were added to the C-terminus, and the codons were optimized according to the codon preference of the expression host before synthesis; (2) The pPIC9KM plasmid was double-digested with BamH I and Xho I; (3) The fragment C3A1 in step (1) was double-digested with Bgl II and Xho I, and ligated with the vector in step (2) to obtain the recombinant plasmid pPIC9KM-C3A1; (4) Design primers to amplify the C3A1 fragment again and use Bgl II and Xho I for double digestion; (5) The pPIC9KM-C3A1 plasmid was double-digested with BamH I and Xho I, and ligated with the fragment in step (4) to obtain the recombinant plasmid pPIC9KM-C3A1-II; (6) linearizing the recombinant plasmid pPIC9KM-C3A1-II obtained in step (5) with Sal I or Sac I, and then electrotransforming it into Pichia pastoris competent cells to obtain positive transformants by screening; (7) Placing the positive transformants obtained in step (6) on a high-concentration antibiotic plate for high-copy screening to obtain high-copy transformants; (8) The obtained high-copy transformants were inoculated into deep-well plates for culture, induced to express, and high-expressing strains were screened by SDS-PAGE; (9) fermenting the obtained high-expression strain on a large scale to obtain a large amount of fermentation liquid, and centrifuging and filtering the fermentation liquid to obtain a crude protein liquid; (10) The crude protein solution is purified using a phenyl hydrophobic column, an MMC column, or an SP column to obtain a high molecular weight recombinant humanized collagen with a purity of 95% to 99%.

8. The synthesis method according to claim 7, characterized in that Repeat steps (4) to (5) in sequence to obtain pPIC9KM-C3A1-III, pPIC9KM-C3A1-IV, and pPIC9KM-C3A1-V.

9. The synthesis method according to claim 8, characterized in that By repeating (6) to (10) in sequence, high molecular weight recombinant collagen C3A1-III, C3A1-IV and C3A1-V can be obtained.

10. The synthesis method according to claim 7, characterized in that: The Pichia pastoris includes any one of Pichia pastoris X33, GS115, KM71, KM71H, SMD1163, SMD1165, SMD1168 and SMD1168H.

11. Use of the high molecular weight recombinant humanized collagen as claimed in any one of claims 1 to 6 as a tissue filling material in the field of medical devices.

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