Long-acting heat-resistant stable recombinant human chemical fibronectin and preparation method thereof

Abstract

The invention discloses a long-acting heat-resistant stable recombinant human fibronectin and a preparation method thereof, relates to the technical field of recombinant proteins, and aims to solve the technical problem that existing recombinant fibronectin is easily hydrolyzed and inactivated in skin. According to the long-acting heat-resistant stable recombinant human chemical fibronectin, the amino acid sequence of the recombinant human chemical fibronectin is shown as SEQ ID No.3, the recombinant human chemical fibronectin is derived from natural fibronectin, and the recombinant human chemical fibronectin comprises a human chemical fibronectin binding region shown as SEQ ID No.1 and a four-segment repeated cell binding region shown as SEQ ID No.2.

Description

Technical Field

[0001] The present application relates to the field of recombinant protein technology, and in particular to a long-acting, heat-resistant and stable recombinant humanized fibronectin and a preparation method thereof. Background Art

[0002] Fibronectin (FN) is an important protein of the extracellular matrix, present in human tissues and body fluids. It exists in plasma in a soluble form and in the extracellular matrix in an insoluble form. It consists of two subunits, each with a molecular weight of 220~250kDa and a V-shape. Fibronectin contains multiple domains, such as FNⅠ, FNⅡ, FNⅢ, as well as alternative splicing sites and variable regions. These domains are divided into multiple functional regions, such as heparin binding domain, fibronectin binding domain, etc. Among them, the eukaryotic cell binding domain binds to integrin through the RGD motif to promote cell adhesion, migration, and proliferation.

[0003] Fibronectin is involved in cell migration, adhesion, proliferation, hemostasis, tissue repair and embryonic development. In skin repair, it can shorten healing time and reduce scars; in skin care, it combines with other structural proteins such as elastin to improve skin texture and appearance. However, as a high molecular weight glycoprotein, fibronectin has unstable physical and chemical properties and is prone to aggregation and precipitation, which limits its application. Heat-resistant fibronectin variants have been obtained through genetic engineering and directed evolution, but may have disadvantages in biocompatibility and safety.

[0004] The recombinant fibronectin on the market does not take into account the problem of enzymatic hydrolysis in the skin and is easily hydrolyzed and inactivated by matrix metalloproteinases, affecting the durability of its efficacy. Summary of the invention

[0005] The present application provides a long-acting, heat-resistant and stable recombinant humanized fibronectin and a preparation method thereof, aiming to solve the technical problem that the existing recombinant fibronectin is easily inactivated by hydrolysis in the skin.

[0006] On the one hand, in order to solve the above-mentioned technical problems, the embodiments of the present application provide: a long-acting, heat-resistant and stable recombinant humanized fibronectin, the amino acid sequence of the recombinant humanized fibronectin is as shown in SEQ ID No.3, which is derived from natural fibronectin and comprises a humanized fibronectin binding region as shown in SEQ ID No.1 and four repeated cell binding regions as shown in SEQ ID No.2.

[0007] As some optional embodiments of the present application, the amino acid sequence of the recombinant humanized fibronectin avoids the main enzyme cleavage site sequence of fibronectin recognized by MMPs.

[0008] As some optional embodiments of the present application, the recombinant humanized fibronectin contains only two aggregation regions, and the aggregation tendency scores are 0.397 and 0.416 respectively.

[0009] As some optional embodiments of the present application, the gene sequence of the recombinant humanized fibronectin is shown as SEQ ID No.4.

[0010] On the other hand, the present application also provides: a method for preparing the long-acting heat-stable recombinant humanized fibronectin as described above, comprising the following steps: After optimizing the Pichia pastoris codon preference of the gene sequence of the recombinant humanized fibronectin as shown in SEQ ID No. 4, the base sequence AAAAGA was added to the 5' end of the base sequence, and the synthesized gene fragment was synthesized by XOt Ⅰ and Not The restriction site of Ⅰ was inserted into the pPICZαA plasmid to obtain the recombinant humanized fibronectin plasmid; The recombinant humanized fibronectin plasmid was linearized by using QuickCut Sac I to obtain a linearized plasmid; the linearized plasmid was electrotransformed into GS115 competent cells and positive transformants were screened to obtain a yeast strain that efficiently expressed the recombinant humanized fibronectin; The highly efficiently expressed recombinant humanized fibronectin yeast engineering strain is purified by anion exchange chromatography to obtain a recombinant humanized fibronectin stock solution.

[0011] As some optional embodiments of the present application, the temperature of the linearization enzyme treatment is 37° C., and the time of the linearization enzyme treatment is 5 h; The linearized enzyme digestion system includes: 20 μg recombinant human fibronectin plasmid, 10 μL QuickCutSac Ⅰ, 5 μL 10× Quick Cut buffer, and the total volume is 50 μL.

[0012] As some optional embodiments of the present application, the linearized plasmid is electro-transformed into GS115 competent cells and the positive transformants are screened to obtain a highly efficient expression of recombinant humanized fibronectin yeast engineering strain, including: After mixing the yeast competent cells with the linearized plasmid, transfer them into an electroporation cup for ice bath; select the yeast mode of the electroporator for electroporation; add pre-cooled sorbitol to the electroporation cup, mix well, transfer to a sterile EP tube, and place in an incubator for 1-2 h to obtain a bacterial solution; take the bacterial solution and spread it on a YPD plate, invert it in an incubator for culture, until a single colony appears; Pick up the single colony, transfer it to the first 96-well culture plate containing YPD medium, and continue to culture; after culturing for 45h-50h, blow the bacterial solution evenly, draw 5μL-15μL from each well and transfer it to the second 96-well culture plate containing YPD medium, and continue to culture; after culturing for 20h-28h, blow the bacterial solution evenly, draw 5μL-15μL from each well and transfer it to the third 96-well culture plate containing YPD medium, and continue to culture; After 20h-28h, the bacterial solution in the third 96-well culture plate containing YPD medium was blown evenly to obtain transformants; and 1 μL of the transformants were respectively spotted on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL Zeocin for further culture; Transformants that can grow on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL Zeocin at the same time were screened and obtained as positive transformants, namely, engineered yeast strains that efficiently express recombinant humanized fibronectin.

[0013] As some optional embodiments of the present application, the positive transformants are identified by the following steps: Pick the single colony, resuspend it in a sodium hydroxide solution, boil it in a boiling water bath and then centrifuge it, take the supernatant as a PCR template, perform PCR amplification, and obtain a PCR amplification product; The PCR amplification product was subjected to 1.0% agarose gel electrophoresis to identify whether a gene fragment of the expected size was amplified.

[0014] As some optional embodiments of the present application, before the highly expressed recombinant humanized fibronectin yeast engineering strain is purified by anion exchange chromatography to obtain the recombinant humanized fibronectin stock solution, the process further includes: Pick a single colony and inoculate it into BMGY medium, culture it at 30 °C, 220 rpm for 24 h until OD600nm is 2-6; Adjust the volume of BMGY culture solution according to the measured OD value, collect the bacteria at 3000 rpm for 10 min, and resuspend the bacteria with BMMY culture medium of the same volume as BMGY to make the initial OD value 2.0; The culture was continued at 30 °C and 220 rpm, 0.5% methanol was added to the culture medium every 24 h, and the supernatant samples were collected at 72 h after induction; The molecular weight and expression of the target protein were analyzed by SDS-PAGE electrophoresis to determine whether it was consistent with the theoretical value of the molecular weight of recombinant humanized fibronectin. If so, the expression was successful.

[0015] As some optional embodiments of the present application, the anion exchange chromatography is used to purify the highly expressed recombinant humanized fibronectin yeast engineering strain to obtain the recombinant humanized fibronectin stock solution, including: Collecting the supernatant of the fermentation broth of the yeast engineering strain that efficiently expresses the recombinant humanized fibronectin by centrifugation, and separating the fermentation supernatant and the bacterial cells using a solid-liquid separation system; The fermentation supernatant was passed through a Sephadex G-25 desalting column, and the column was equilibrated with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 40 cm / h, and the ultraviolet A280 absorbance value was detected. When it increased, the sample was started; After the sample loading is completed, the desalting column is equilibrated with 20 mM phosphate buffer until the UV and conductivity drop to the minimum and no longer change, and then the sample loading is stopped; The desalted product was then passed through an anion exchange column, and the column was equilibrated with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remained unchanged, and the sample loading flow rate was set to 40 cm / h; After the sample loading is completed, the anion chromatography column is equilibrated with 20 mM phosphate buffer until the UV and conductivity are reduced to the minimum and no longer change; Finally, the corresponding protein was eluted with 20 mM phosphate buffer containing 300 mM NaCl and collected. The eluted product was diluted with an equal volume of 20 mM phosphate buffer to obtain a recombinant humanized fibronectin stock solution.

[0016] Compared with the prior art, the present application provides a recombinant humanized fibronectin amino acid sequence (as shown in SEQ ID No. 3), which avoids the main cleavage sites recognized by matrix metalloproteinases, avoids or reduces the hydrolysis of recombinant humanized fibronectin by proteases in the skin, and thus ensures its long-term effectiveness in the body; the sequence is a humanized fibronectin sequence, does not contain non-fibronectin amino acid sequences such as expression vectors or tag proteins, and is completely derived from natural fibronectin amino acid sequences, and thus has more advantages in biosafety and biocompatibility; in addition, the sequence is a repeated sequence of activity-related regions, and minimizes sequences that are not related to activity or structural stability, so that the expressed recombinant humanized fibronectin has a higher specific activity in promoting cell adhesion; at the same time, the sequence is obtained through molecular dynamics simulation and screening, avoiding structural regions that are prone to aggregation, and solving the problem of poor thermal stability and easy precipitation of fibronectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the prior art description. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0018] Figure 1 This is a diagram of the Alphafold prediction results of the fibronectin structure involved in the embodiments of the present application; Figure 2 This is a Discovery Studio analysis diagram of the fibronectin aggregation area involved in the examples of this application; Figure 3 This is a diagram showing the PCR identification results of the recombinant humanized fibronectin transformant involved in the examples of the present application; Figure 4 This is a diagram showing the results of SDS-PAGE analysis of the expression of multiple copies of the recombinant humanized fibronectin transformants involved in the examples of the present application; Figure 5 This is a diagram showing the SDS-PAGE results of the purified recombinant humanized fibronectin sample involved in the examples of the present application; Figure 6 This is a diagram showing the appearance changes of the recombinant humanized fibronectin stock solution involved in the examples of the present application after being heated at 100°C for 30 minutes; Figure 7 This is a graph showing the purity change of the recombinant humanized fibronectin stock solution involved in the examples of the present application after being heated at 100°C for 30 minutes; Figure 8 This is a graph showing the skin pharmacokinetics of the recombinant humanized fibronectin involved in the examples of this application.

[0019] 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

[0020] 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.

[0021] Fibronectin (FN) is an important extracellular matrix protein that is widely present in various tissues and body fluids of the human body. It mainly exists in soluble form in plasma and in insoluble form in the extracellular matrix. Fibronectin is formed by two subunits cross-linked by disulfide bonds at the C-terminus. The molecular weight of each subunit is 220~250kDa, and the whole molecule is V-shaped. Each subunit of fibronectin has several domains, including three repeated modules of FNⅠ, FNⅡ and FNⅢ, two alternative splicing sites EDA and EDB, and a variable region V. These domains are mainly divided into heparin binding domain, fibronectin binding domain, humanized fibronectin binding domain, eukaryotic cell binding domain, prokaryotic cell binding domain and DNA binding domain according to their functions. Among them, the eukaryotic cell binding domain mainly recognizes and binds to integrins through the RGD motif on FNⅢ10, thereby promoting cell adhesion, migration, proliferation, etc.

[0022] Fibronectin is involved in the body's life activities, including cell migration, adhesion, proliferation, hemostasis, tissue repair and embryonic development. In recent years, there has been more and more research on the application value and field of fibronectin. In skin wound repair and healing, fibronectin can shorten wound healing time and reduce wound scars; in skin care, especially in anti-aging and skin repair, fibronectin and other structural proteins such as humanized fibronectin and elastin provide a support network for the skin, helping to improve skin texture and appearance.

[0023] Although fibronectin has many advantages, it also has some disadvantages. As a high molecular weight glycoprotein, its physical and chemical properties are not very stable, and it is easy to aggregate and precipitate at room temperature and normal pressure, which limits its further development and application to a certain extent. The heat-resistant fibronectin currently available on the market is mainly obtained by replacing some amino acid residues in the protein that affect its thermal stability with amino acid residues that are beneficial to protein stability (such as proline) through genetic engineering and directed evolution methods, and establishing and screening mutant libraries to obtain a heat-resistant fibronectin variant. However, the heat-resistant fibronectin variant is a human-like fibronectin rather than natural fibronectin and humanized fibronectin. Compared with natural fibronectin and humanized fibronectin, it may have disadvantages in biocompatibility and risks in biosafety.

[0024] In addition, the recombinant fibronectin currently available or reported on the market does not take into account the problem of enzymatic hydrolysis of the protein in the skin during its construction and expression. The recombinant fibronectin containing matrix metalloproteinase cleavage site sequences is easily hydrolyzed and inactivated in the skin, making it difficult for the recombinant fibronectin to exert its efficacy for a long time.

[0025] Therefore, in order to solve the above-mentioned shortcomings of existing recombinant fibronectin, the present application provides a recombinant humanized fibronectin amino acid sequence (SEQ ID No. 3), which avoids the main cleavage sites recognized by matrix metalloproteinases, avoids or reduces the hydrolysis of recombinant humanized fibronectin by proteases in the skin, and thus ensures its long-term effectiveness in the body; the sequence is a humanized fibronectin sequence, does not contain non-fibronectin amino acid sequences such as expression vectors or tag proteins, and is completely derived from natural fibronectin amino acid sequences, and therefore has more advantages in biosafety and biocompatibility; in addition, the sequence is a repetitive sequence of activity-related regions, and minimizes sequences that are not related to activity or structural stability, so that the expressed recombinant humanized fibronectin has a higher cell adhesion-promoting specific activity; at the same time, the sequence is obtained through molecular dynamics simulation and screening, and avoids structural regions that are prone to aggregation, solving the problem of poor thermal stability and easy precipitation of fibronectin.

[0026] To facilitate understanding by those skilled in the art, the sequences involved in the technical solution described in this application are listed below: SEQ ID No.1: AAHEICTTNEGVM; SEQ ID No.2: VPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID; SEQ ID No.3: AAHEEICTTNEGVMVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISI NYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID; SEQ ID No.4:

[0027] The technical solution described in this application is described in detail below in conjunction with specific implementation methods: Example 1 Construction, identification and preparation of engineered bacteria 1. Protein sequence selection and design The recombinant humanized fibronectin amino acid sequence (390aa, SEQ ID No. 3) described in the present application is derived from natural fibronectin (UniProt: P02751), comprising a humanized fibronectin binding region (14aa, SEQ ID No. 1) and four repeated cell binding regions (94aa, SEQ ID No. 2). The amino acid sequence avoids the main cleavage site sequence of fibronectin recognized by matrix metalloproteinases.

[0028] The matrix metalloproteinase (MMPs) is a zinc and calcium-dependent endopeptidase that can target and cut extracellular matrix proteins such as fibronectin, humanized fibronectin, gelatin, and elastin. MMPs that can hydrolyze fibronectin in the extracellular matrix include MMP-16, MMP-25, MMP-26, MMP-10, MMP-13, MMP-12, and MMP-19. Therefore, when selecting the amino acid sequence of the recombinant humanized fibronectin described in the present application, these MMPs cleavage sites are avoided as much as possible, which can reduce the hydrolysis of the recombinant humanized fibronectin by MMPs, thereby exerting its long-term effect in the extracellular matrix.

[0029] In addition to avoiding the main restriction site sequence recognized by MMPs, the recombinant humanized fibronectin amino acid sequence (SEQ ID No. 3) also avoids the structural region that tends to aggregate through molecular dynamics simulation and screening. The specific method includes the following steps: Different structural regions were extracted from natural fibronectin and the Alphafold tool was used to predict the protein structure. The Discovery studio software was then used to analyze the protein aggregation regions of the structure. The force field was CHARMm Polar H and the analysis radius was set to 10Å. The number of protein aggregation regions and the aggregation trend scores of each region were analyzed. The larger the score, the more likely the region was to aggregate. The recombinant humanized fibronectin amino acid sequence (SEQ ID No. 3) avoided some of the structural regions that tended to aggregate during sequence design.

[0030] In addition to analyzing the aggregation region of the recombinant humanized fibronectin amino acid sequence (SEQ ID No. 3), the aggregation region results of fibronectin were also compared. The results showed that fibronectin contained multiple aggregation regions, two of which had large aggregation tendency scores (>2) and were prone to aggregation; while the recombinant humanized fibronectin of the present application contained only two aggregation regions, and the maximum aggregation tendency score was only 0.416 (as shown in Table 1, Figure 1 and Figure 2 as shown).

[0031] The results of molecular dynamics simulation show that the recombinant humanized fibronectin of the present application has better stability and solubility than similar fibronectins and is not easy to precipitate.

[0032] Table 1. Fibronectin aggregation area Discovery studio analysis results

[0033] A terminator was added to the C-terminus, and the gene sequence encoding the recombinant humanized fibronectin (1173 bp, SEQ ID No. 4) was optimized for Pichia pastoris codon preference to improve the expression efficiency of the recombinant protein in Pichia pastoris.

[0034] The 5' end of the optimized base sequence of recombinant humanized fibronectin, XOt The base sequence AAAAGA was added between Ⅰ and the target gene sequence, and GenScript Biotech Co., Ltd. was commissioned to synthesize the gene fragment. XOt Ⅰ and Not The restriction site of Ⅰ was inserted into the pPICZαA plasmid to obtain the recombinant human fibronectin plasmid.

[0035] 2. Construction and screening of recombinant engineered bacteria 2.1 Obtaining recombinant plasmid The optimized recombinant humanized fibronectin base sequence was entrusted to GenScript Biotech Co., Ltd. for gene fragment synthesis. After sequencing verification, the corresponding recombinant plasmid and puncture bacteria were provided. After expansion culture, high-concentration recombinant plasmid was extracted for standby use.

[0036] 2.2 Linearization of recombinant human fibronectin plasmid Using QuickCut Sac Ⅰ Linearize the 20 μg recombinant human fibronectin particles extracted in 2.1 under the conditions of 37°C for 5 h. The enzyme digestion system is shown in Table 2: Table 2 Enzyme digestion system

[0037] After electrophoresis verification, add 0.1 times the volume of 3 M NaAc (pH 5.2) and 2.5 times the volume of anhydrous ethanol, and place at -20 °C overnight. Centrifuge at 4 °C, 13000 rpm for 20 min, discard the supernatant; add 700 μL 75% ethanol for rinsing, centrifuge at 13000 rpm for 20 min, discard the supernatant, and repeat; invert the Ep tube on the clean bench absorbent paper for about 10 min to remove water and residual ethanol as much as possible, and 20 μL ddH 2 O to redissolve the plasmid, take 1 μL and dilute it 10 times, and use one-drop to detect the nucleic acid concentration.

[0038] 2.3 Preparation of GS115 competent cells After streaking the GS115 plate, a single colony was picked and inoculated into 20 mL YPD and cultured at 30 °C and 225 rpm for 24 h; the colony was transferred to 50 mL YPD liquid medium at a ratio of 1:1000 and cultured at 30 °C and 225 rpm until OD 600 1.3-1.5; Transfer the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 4 ℃, 3000 rpm for 5 min, discard the supernatant and collect the bacteria; resuspend the bacterial pellet with 50 mL pre-cooled sterile ultrapure water, centrifuge at 4 ℃, 3000 rpm for 5 min; discard the supernatant, and resuspend the bacterial pellet with 50 mL pre-cooled sterile ultrapure water; centrifuge at 4 ℃, 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 40 mL pre-cooled sterile 1 M sorbitol; centrifuge at 4 ℃, 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 100-150 μL pre-cooled sterile 1 M sorbitol, gently rotate to mix, and place on ice for use.

[0039] 2.4 Electrotransformation into GS115 competent cells Take 100 μL of yeast competent cells and mix with 10 μL of linearized plasmid, transfer to the pre-cooled electroporation cup, and immediately place on ice for 5 min. Select the yeast mode of the electroporator and perform electroporation. Then immediately add 1 mL of pre-cooled 1 M sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate in a 30 ℃ incubator for 1 h~2 h. Take 100 μL~200 μL of bacterial solution and spread it on a YPD plate containing 0.25 mg / mL Zeocin, let it stand at room temperature for 10 min, and invert it in a 30 ℃ incubator for about 2 d~5 d until a single colony appears.

[0040] 2.5 Screening of positive transformants Pick a single colony from the YPD plate and transfer it to a 96-well culture plate containing 200 μL YPD medium (containing 0.5 mg / mL Zeocin), and continue to culture at 30°C. After 48 hours, blow the bacterial solution evenly, transfer 10 μL from each well to a new 96-well culture plate (containing 190 μL YPD), and continue to culture for 24 hours before repeating the above steps. After 24 hours, blow the bacterial solution in the third 96-well plate evenly, and draw 1 μL and continue to culture on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin. If the transformant can grow on a plate containing 3 mg / mL high-concentration Zeocin, it means that the transformant contains multiple copies of the expression cassette. After this step of screening, a highly efficient expression of recombinant humanized fibronectin yeast engineering strain can be obtained.

[0041] 2.6 Identification of positive transformants Pick a small amount of single colonies from the plate with 3 mg / mL Zeocin in step 2.4 above, resuspend them in 20 μL of 20 mM sodium hydroxide solution, boil them in a boiling water bath for 30 min, centrifuge them at 12,000 rpm for 5 min, take the supernatant as a PCR template, and identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0042] Upstream primer: 5'AOX1 (5'-GCAGCGCACGAGGAGATATG-3') Downstream primer: 3'AOX1 (5'-TCAGTCTATTTCAGTTCGGTAAT-3') PCR conditions: 95°C pre-denaturation for 5 min, 95°C thermal denaturation for 50 s, 60°C annealing for 30 s, 72°C extension for 60 s, 35 cycles; 72°C annealing for 10 min.

[0043] The PCR amplification products were subjected to 1.0% agarose gel electrophoresis to identify whether the expected size of the gene fragment was amplified. Lanes 1-3 are PCR products of different recombinant humanized fibronectin transformants. The results show that the size of the PCR nucleic acid band of the recombinant humanized fibronectin transformant is consistent with the theoretical value (1173bp), and the identification results are all positive (such as Figure 3 as shown).

[0044] 3. Expression of recombinant humanized fibronectin The culture medium formula used is as follows: 1) YPD complete medium: Yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L (solid medium contains 2% agar); 2) MD solid medium: YNB without amino acids nitrogen source 13.4 g / L, 0.4 mg / L biotin, 20 g / L glucose, (solid medium contains 2% agar); 3) BMGY yeast growth medium: Yeast extract 10 g / L, peptone 20 g / L, 3g / LK 2 HPO 4 , 11.8g / LK 2 HPO 4 , add water to 890mL, sterilize at 121℃ for 20min, then add 10×YNB 100mL (13.4 g / L), 500×biotin 2mL (4×10 -4 g / L), glycerol 10mL, total volume 1L.

[0045] 4) BMMY yeast induction medium: Yeast extract 10 g / L, peptone 20 g / L, 3g / LK 2 HPO 4 , 11.8g / LK 2 HPO 4 , add water to 895mL, sterilize at 121℃ for 20min, then add 100×YNB 100mL (13.4 g / L), 500×biotin 2mL (4×10 -4 g / L), methanol 5mL, total volume 1L.

[0046] The specific operation is as follows: pick a single colony and inoculate it into BMGY medium, and culture it at 30 °C and 220 rpm for 24 h until OD 600nm 2~6. Adjust the volume of BMGY culture solution according to the measured OD value, collect the bacteria at 3000 rpm for 10 min, and resuspend the bacteria with an equal volume of BMMY culture medium to make the initial OD value 2.0. Continue to culture at 30 ℃ and 220 rpm, add 0.5% methanol to the culture medium every 24 h, and take the centrifugal supernatant samples at 72 h after induction. The molecular weight and expression of the target protein were analyzed by SDS-PAGE electrophoresis. The results showed that a clear protein band was detected at 45 kDa, which is consistent with the theoretical molecular weight of recombinant humanized fibronectin (about 45 kDa, of which glycosylation modification is about 4 kDa), indicating that the initial expression was successful, and the expression level of multi-copy transformant 2 was higher (such as Figure 4 as shown).

[0047] 4. Purification by anion exchange chromatography The culture supernatant of the engineered bacteria fermentation broth was collected by centrifugation, and the fermentation supernatant and the bacteria were separated using a solid-liquid separation system. The fermentation supernatant was passed through a Sephadex G-25 desalting column, and the column was equilibrated with 20mM phosphate buffer (pH 7.6) until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 40cm / h, and the UV A280 absorbance value was detected. When it rose, the sample was started. After the sample loading was completed, the desalting column was equilibrated with 20mM phosphate buffer (pH 7.6) again until the UV and conductivity dropped to the minimum and no longer changed, and the sample was stopped. The desalted product was then passed through an anion exchange column (the chromatography filler was Qianchun Product Q Purose 6 HighPerformance, loaded on a GE AKTA chromatography system), and the column was equilibrated with 20mM phosphate buffer (pH 7.6) until the conductivity value and A280 absorbance value remained unchanged, and the sample loading flow rate was set to 40cm / h. After the loading is completed, the anion chromatography column is equilibrated with 20mM phosphate buffer (pH 7.6) until the UV and conductivity are reduced to the minimum and no longer change. Finally, the corresponding protein is eluted with 20mM phosphate buffer (pH 7.6) containing 300mM NaCl and collected. The eluted product is diluted with an equal volume of 20mM phosphate buffer (pH 7.6) to obtain a recombinant humanized fibronectin stock solution. The purity of the recombinant humanized fibronectin stock solution was detected by SDS-PAGE electrophoresis. The results showed that the purity of the recombinant humanized fibronectin reached the electrophoretic purity, and there was almost no loss of the target protein in the flow-through. The purification method had a high recovery rate (such as Figure 5 as shown).

[0048] Example 2: Heat resistance test of recombinant humanized fibronectin High temperature will destroy the secondary bonds of proteins, such as hydrogen bonds and ionic bonds, causing changes in the spatial structure of proteins and irreversible denaturation. The spatial structure of proteins is crucial to their functions. Once irreversible denaturation occurs, the protein loses its original biological activity. High temperature will also increase the interaction between protein molecules, causing protein aggregation and the formation of protein precipitation. Generally, when the ambient temperature exceeds 60°C, proteins begin to undergo irreversible denaturation and protein precipitation. The heat resistance test of recombinant humanized fibronectin verifies the thermal stability of recombinant humanized fibronectin by treating the protein in a high-temperature water bath and then comparing and analyzing the changes in the appearance, purity and activity of the protein before and after the treatment.

[0049] 1. Effect of high temperature treatment on the appearance and purity of recombinant human fibronectin stock solution Specific implementation method: Heat the water bath to 100°C, take out the recombinant human fibronectin stock solution sample to be tested from the 4°C refrigerator (the sample is in a vial, sealed, and no protein stabilizer is added), put it into boiling water and heat it for 30 minutes, then take out the sample and observe the appearance change. The recombinant human fibronectin stock solution has no protein precipitation and no color change ( Figure 6 ); Sampling was performed for SDS-PAGE electrophoresis, and the recombinant human fibronectin stock solution sample showed no degradation after high-temperature boiling ( Figure 7 ).

[0050] The test results show that heating at 100°C for 30 minutes has no effect on the appearance and purity of the recombinant human fibronectin stock solution.

[0051] 2. Effect of high temperature treatment on the cell adhesion activity of recombinant human fibronectin stock solution 2.1 Test materials: Complete cell culture medium: Add 10% fetal bovine serum to 1640 culture medium (containing double antibody) and store at 4°C.

[0052] Serum-free culture medium: 1640 culture medium (containing double antibodies), stored at 4°C.

[0053] Digestion solution: 0.25% trypsin.

[0054] 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.

[0055] MDBK cells (purchased from ATCC).

[0056] BSA (purchased from Sigma).

[0057] 2.2 Specific implementation methods: The purified stock solution of recombinant humanized fibronectin and similar fibronectin before and after heating at 100°C for 30 minutes were diluted to 0.5 μg / mL with PBS buffer, and then the diluted samples were taken for 2-fold gradient dilution in 96-well cell culture plates, with a total of 8 dilutions, 50 μL per well, and a negative control (50 μL of PBS buffer) was set up, and incubated at 4°C overnight; the liquid in the cell culture plate was discarded, and 100 μL of PBS buffer was added to each well for washing, and washed 3 times in total; after washing, 100 μL of 30 μg / μL BSA was added to each well for blocking, and incubated in a 37°C incubator for 1 h; the liquid in the cell culture plate was discarded, and 100 μL of PBS buffer was added to each well for washing 3 times, and then the fibroblast suspension was added, and the cell seeding density was 1.0×10 5cells / mL, 100 μL was inoculated into each well and incubated in an incubator for 5 h; the incubated cell culture plate was washed 3 times with PBS buffer and the cell adhesion was observed under a microscope.

[0058] The number of adherent cells was counted at five points except the edge under a 200x microscope, and the titer was obtained using a four-parameter fitting curve based on the counting results. The results of each group are shown in Table 3. The results showed that recombinant humanized fibronectin had higher cell adhesion promoting activity than commercially available fibronectin, and it still had an activity recovery rate of 94.9% after heating at 100°C for 30 minutes, while the activity recovery rate of commercially available fibronectin was only 13.9%.

[0059] The results showed that the cell adhesion promoting activity of recombinant human fibronectin could withstand heating at 100℃ for 30min.

[0060] Table 3 Effect of high temperature on the cell adhesion activity of recombinant human fibronectin

[0061] Example 3: Analysis of the long-term effects of recombinant humanized fibronectin in vivo The analysis of the long-term effect of recombinant humanized fibronectin in vivo is mainly based on the results of rat skin pharmacokinetic tests. The specific implementation method is as follows: 1. Test materials: Total Protein Extraction Kit (Strong): Solebol.

[0062] Propofol: purchased from Sigma, product number Y0000016.

[0063] Digestion solution: 0.25% trypsin.

[0064] Fibronectin ELISA kit: purchased from R&D Company, catalog number DFBN10.

[0065] Recombinant humanized fibronectin: prepared as in Example 1.

[0066] 2. Specific implementation methods: SD female rats (weight 190-210g) were randomly divided into a control group of the same type of fibronectin and a recombinant humanized fibronectin group. The rats in each group were anesthetized by intravenous injection of propofol and then shaved of their back hair. The following mechanical injury model was established. The rats were placed in a prone position and a circle (2.5cm in diameter) was drawn around the spine. Three 0.5cm long scratches were horizontally made on the marked area of ​​each rat with a sterile scalpel. The rats were kept in a cage and fed freely. After a single back application, PBS (0.2mL), commercial fibronectin and recombinant humanized fibronectin were evenly applied to the injured area of ​​the rats in the commercial fibronectin group and the recombinant humanized fibronectin group immediately after modeling (the application dose was 30mg). The rats were euthanized under anesthesia at the designated time during the experiment. The skin of the rats was collected at 0.5h, 2h, 4h, 8h, 12h, 18h and 24h after administration. The skin tissue was weighed and then cut into approximately 1mm 3 The size of the samples was put into centrifuge tubes and stored in liquid nitrogen for later use.

[0067] The total protein extraction kit (strong) was used to extract the total protein from the skin tissue according to the operating instructions of the kit, and then the samples were processed and tested according to the operating requirements of the ELISA kit for pharmacokinetic analysis. Figure 8 As shown, commercially available fibronectin T max 0.5h after administration, C max The value was 1297 pg / mL, and no fibronectin was detected in the skin 8 hours after administration; while the recombinant humanized fibronectin T prepared in Example 1 max 2h after administration, C max The value was 1565pg / mL. No fibronectin was detected in the skin 24 hours after administration, which is more long-lasting than commercially available fibronectin.

[0068] It can be seen that compared with the prior art, the recombinant humanized fibronectin described in this application has the following main beneficial effects: The long-acting heat-stable recombinant humanized fibronectin stock solution (without protein protective agent) prepared in this application still has good stability and biological activity after being heated at 100°C for 30 minutes, and there is no precipitation and no degradation; the long-acting heat-stable recombinant humanized fibronectin prepared in this application has better biological activity, and the cell adhesion promoting activity reaches 1×10 5 U / mg, which is one order of magnitude higher than the commercially available fibronectin; the longer storage time on the skin surface, the skin pharmacokinetic results show that the long-acting heat-stable recombinant humanized fibronectin prepared in the present application has better long-term effectiveness than the commercially available fibronectin.

[0069] The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A long-acting heat-stable recombinant humanized fibronectin, characterized in that: The amino acid sequence of the recombinant humanized fibronectin is shown in SEQ ID No.3, and comprises a humanized fibronectin binding region shown in SEQ ID No.1 and four repeated cell binding regions shown in SEQ ID No.

2.

2. The long-acting heat-stable recombinant humanized fibronectin according to claim 1, characterized in that: The amino acid sequence of the recombinant humanized fibronectin avoids the main enzyme cleavage site sequence of fibronectin recognized by MMPs.

3. The long-acting heat-stable recombinant humanized fibronectin according to claim 1, characterized in that: The recombinant humanized fibronectin contains only two aggregation regions, and the aggregation tendency scores are 0.397 and 0.416 respectively.

4. The long-acting heat-stable recombinant humanized fibronectin according to claim 1, characterized in that: The gene sequence of the recombinant humanized fibronectin is shown in SEQ ID No.

4.

5. A method for preparing the long-acting heat-stable recombinant humanized fibronectin according to any one of claims 1 to 4, characterized in that: The following steps are involved: After optimizing the Pichia pastoris codon preference of the gene sequence of the recombinant humanized fibronectin as shown in SEQ ID No. 4, the base sequence AAAAGA was added to the 5' end of the base sequence, and the synthesized gene fragment was synthesized by XOt Ⅰ and Not The restriction site of Ⅰ was inserted into the pPICZαA plasmid to obtain the recombinant humanized fibronectin plasmid; After linearization restriction digestion of the recombinant humanized fibronectin plasmid using QuickCut Sac Ⅰ, a linearized plasmid was obtained; The linearized plasmid is electro-transformed into GS115 competent cells and positive transformants are screened to obtain a yeast engineering strain that efficiently expresses recombinant humanized fibronectin; The highly efficiently expressed recombinant humanized fibronectin yeast engineering strain is purified by anion exchange chromatography to obtain a recombinant humanized fibronectin stock solution.

6. The method for preparing the long-acting heat-stable recombinant humanized fibronectin according to claim 5, characterized in that: The temperature of the linearization enzyme digestion treatment is 37°C, and the time of the linearization enzyme digestion treatment is 5h; The linearized enzyme digestion system includes: 20 μg recombinant human fibronectin plasmid, 10 μL QuickCut SacⅠ, 5 μL 10× Quick Cut buffer, and the total volume is 50 μL.

7. The method for preparing the long-acting heat-stable recombinant humanized fibronectin according to claim 5, characterized in that: The linearized plasmid is electrotransformed into GS115 competent cells and positive transformants are screened to obtain a highly efficient expression of recombinant humanized fibronectin yeast engineering strain, including: After mixing the yeast competent cells with the linearized plasmid, transfer them into an electroporation cup for ice bath; select the yeast mode of the electroporator for electroporation; add pre-cooled sorbitol to the electroporation cup, mix well, transfer to a sterile EP tube, and place in an incubator for 1-2 h to obtain a bacterial solution; take the bacterial solution and spread it on a YPD plate, invert it in an incubator for culture, until a single colony appears; Pick up the single colony, transfer it to the first 96-well culture plate containing YPD medium, and continue to culture; after culturing for 45h-50h, blow the bacterial solution evenly, draw 5μL-15μL from each well and transfer it to the second 96-well culture plate containing YPD medium, and continue to culture; after culturing for 20h-28h, blow the bacterial solution evenly, draw 5μL-15μL from each well and transfer it to the third 96-well culture plate containing YPD medium, and continue to culture; After 20h-28h, the bacterial solution in the third 96-well culture plate containing YPD medium was blown evenly to obtain transformants; and 1 μL of the transformants were respectively spotted on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL Zeocin for further culture; Transformants that can grow on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL Zeocin at the same time were screened and obtained as positive transformants, namely, engineered yeast strains that efficiently express recombinant humanized fibronectin.

8. The method for preparing the long-acting heat-stable recombinant humanized fibronectin according to claim 7, characterized in that: The positive transformants were identified by the following steps: Pick the single colony, resuspend it in a sodium hydroxide solution, boil it in a boiling water bath and then centrifuge it, take the supernatant as a PCR template, perform PCR amplification, and obtain a PCR amplification product; The PCR amplification product was subjected to 1.0% agarose gel electrophoresis to identify whether a gene fragment of the expected size was amplified.

9. The method for preparing the long-acting heat-stable recombinant humanized fibronectin according to claim 5, characterized in that: Before the anion exchange chromatography is used to purify the highly expressed recombinant humanized fibronectin yeast engineering strain to obtain the recombinant humanized fibronectin stock solution, the method further comprises: Pick a single colony and inoculate it into BMGY medium, culture it at 30 °C, 220 rpm for 24 h until OD600nm is 2-6; Adjust the volume of BMGY culture solution according to the measured OD value, collect the bacteria at 3000 rpm for 10 min, and resuspend the bacteria with BMMY culture medium of the same volume as BMGY to make the initial OD value 2.0; The culture was continued at 30 °C and 220 rpm, 0.5% methanol was added to the culture medium every 24 h, and the supernatant samples were collected at 72 h after induction; The molecular weight and expression of the target protein were analyzed by SDS-PAGE electrophoresis to determine whether it was consistent with the theoretical value of the molecular weight of recombinant humanized fibronectin. If so, the expression was successful.

10. The method for preparing the long-acting heat-stable recombinant humanized fibronectin according to claim 5, characterized in that: The method of purifying the highly efficiently expressed recombinant humanized fibronectin yeast strain by anion exchange chromatography to obtain a recombinant humanized fibronectin stock solution comprises: Collecting the supernatant of the fermentation broth of the yeast engineering strain that efficiently expresses the recombinant humanized fibronectin by centrifugation, and separating the fermentation supernatant and the bacterial cells using a solid-liquid separation system; The fermentation supernatant was passed through a Sephadex G-25 desalting column, and the column was equilibrated with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 40 cm / h, and the ultraviolet A280 absorbance value was detected. When it increased, the sample was started; After the sample loading is completed, the desalting column is equilibrated with 20 mM phosphate buffer until the UV and conductivity drop to the minimum and no longer change, and then the sample loading is stopped; The desalted product was then passed through an anion exchange column, and the column was equilibrated with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remained unchanged, and the sample loading flow rate was set to 40 cm / h; After the sample loading is completed, the anion chromatography column is equilibrated with 20 mM phosphate buffer until the UV and conductivity are reduced to the minimum and no longer change; Finally, the corresponding protein was eluted with 20 mM phosphate buffer containing 300 mM NaCl and collected. The eluted product was diluted with an equal volume of 20 mM phosphate buffer to obtain a recombinant humanized fibronectin stock solution.

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