A long-acting heat-stable recombinant human fibronectin and its preparation method

Abstract

The present application discloses a long-acting heat-stable recombinant human fibronectin and a preparation method thereof, which 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 the skin. The long-acting heat-stable recombinant human fibronectin has an amino acid sequence of the recombinant human fibronectin as shown in SEQ ID No. 3, is derived from natural fibronectin, and contains a human fibronectin binding region as shown in SEQ ID No. 1 and four repeated cell binding regions as shown in SEQ ID No. 2.

Description

Technical Field

[0001] This application relates to the technical field of recombinant proteins, and particularly relates to a long-acting heat-stable recombinant human fibronectin and a preparation method thereof. Background Art

[0002] Fibronectin (FN) is an important protein in the extracellular matrix and exists in human tissues and body fluids. It exists in a soluble form in plasma and an insoluble form in the extracellular matrix. It consists of two subunits, each with a molecular weight of 220 - 250 kDa, in 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, promoting 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 the healing time and reduce scarring; in skin care, it binds to 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, is prone to aggregation and precipitation, which limits its application. Heat-resistant fibronectin variants are obtained through genetic engineering and directed evolution, but may have disadvantages in biocompatibility and safety.

[0004] Recombinant fibronectin on the market does not consider the problem of enzymatic hydrolysis in the skin and is easily hydrolyzed and inactivated by matrix metalloproteinases, affecting the persistence of its efficacy. Summary of the Invention

[0005] This application provides a long-acting heat-stable recombinant human fibronectin and a preparation method thereof, aiming to solve the technical problem that existing recombinant fibronectin is easily hydrolyzed and inactivated in the skin.

[0006] On the one hand, to solve the above technical problem, an embodiment of this application provides: a long-acting heat-stable recombinant human fibronectin, the amino acid sequence of the recombinant human fibronectin is as shown in SEQ ID No.3, derived from natural fibronectin, and contains a human 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 alternative embodiments of this application, the amino acid sequence of the recombinant human fibronectin avoids the main cleavage site sequence recognized by MMPs for fibronectin.

[0008] As some alternative embodiments of this application, the recombinant human fibronectin contains only two aggregation regions, and the aggregation trend scores are 0.397 and 0.416 respectively.

[0009] In some alternative embodiments of the present application, the gene sequence of the recombinant human fibronectin is as shown in SEQ ID No. 4.

[0010] On the other hand, the embodiments of the present application also provide: a method for preparing the long-acting heat-stable recombinant human fibronectin as described above, comprising the following steps:

[0011] After optimizing the codon preference design of the gene sequence of the recombinant human fibronectin as shown in SEQ ID No. 4, at the 5' end of its base sequence, add the base sequence AAAAAGA, and insert the synthesized gene fragment into the pPICZαA plasmid through the Xho Ⅰ and Not Ⅰ restriction enzyme sites to obtain a recombinant human fibronectin plasmid;

[0012] After linearizing and digesting the recombinant human fibronectin plasmid with QuickCut Sac Ⅰ, a linearized plasmid is obtained; after electrotransforming the linearized plasmid into GS115 competent cells and screening for positive transformants, a recombinant human fibronectin yeast engineering strain with high expression is obtained;

[0013] Purify the recombinant human fibronectin yeast engineering strain with high expression by anion exchange chromatography to obtain a recombinant human fibronectin stock solution.

[0014] In some alternative embodiments of the present application, the temperature of the linearizing digestion treatment is 37 °C, and the time of the linearizing digestion treatment is 5 h;

[0015] The system of the linearizing digestion treatment includes: 20 μg of the recombinant human fibronectin plasmid, 10 μL of QuickCut Sac Ⅰ, 5 μL of 10× Quick Cut buffer, and the total volume is 50 μL.

[0016] In some alternative embodiments of the present application, after electrotransforming the linearized plasmid into GS115 competent cells and screening for positive transformants, obtaining a recombinant human fibronectin yeast engineering strain with high expression includes:

[0017] Mix the yeast competent cells with the linearized plasmid, transfer them into an electroporation cuvette and perform ice bath; select the yeast mode of the electroporator for electroporation; add pre-cooled sorbitol to the electroporation cuvette, mix well and transfer to a sterile EP tube, and place it in an incubator for static incubation for 1-2 h to obtain a bacterial solution; spread the bacterial solution on a YPD plate, invert it in an incubator for cultivation until single colonies appear;

[0018] Pick the single colony and transfer it to the first 96-well culture plate containing YPD medium for continued culture. After culturing for 45 h - 50 h, blow the bacterial solution evenly, and transfer 5 μL - 15 μL from each well to the second 96-well culture plate containing YPD medium for continued culture. After culturing for 20 h - 28 h, blow the bacterial solution evenly, and transfer 5 μL - 15 μL from each well to the third 96-well culture plate containing YPD medium for continued culture.

[0019] After 20 h - 28 h, blow the bacterial solution in the third 96-well culture plate containing YPD medium evenly to obtain transformants. Then, pipette 1 μL of the transformants and spot them on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin respectively for continued culture.

[0020] Screen for transformants that can grow on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin simultaneously, and use them as positive transformants, that is, the recombinant human fibronectin yeast engineering strains with high expression.

[0021] As some alternative embodiments of the present application, the positive transformants are identified through the following steps:

[0022] Pick the single colony, resuspend it with sodium hydroxide solution, boil it in a boiling water bath and then centrifuge, take the supernatant as the PCR template, and perform PCR amplification to obtain the PCR amplification product.

[0023] Take the PCR amplification product for 1.0% agarose gel electrophoresis to identify whether the gene fragment of the expected size is amplified.

[0024] As some alternative embodiments of the present application, before purifying the recombinant human fibronectin yeast engineering strains with high expression by anion exchange chromatography to obtain the recombinant human fibronectin stock solution, it further includes:

[0025] Pick a single colony and inoculate it into BMGY medium, culture at 30 °C and 220 rpm for 24 h until OD600nm reaches 2 - 6.

[0026] Adjust the volume of the BMGY culture bacterial solution according to the measured OD value, collect the bacterial cells at 3000 rpm for 10 min, and resuspend the bacterial cells with BMMY medium with the same volume as BMGY to make the initial OD value 2.0.

[0027] Continue to culture at 30 °C and 220 rpm, add 0.5% methanol to the medium every 24 h, and take the centrifuged supernatant samples at the 72nd h after induction.

[0028] Analyze the molecular weight and expression of the target protein by SDS-PAGE electrophoresis to determine whether it is consistent with the theoretical value of the molecular weight of recombinant human fibrinogen-like protein. If it is consistent, the expression is successful.

[0029] As some alternative embodiments of the present application, the anion exchange chromatography method is used to purify the highly expressed recombinant human fibrinogen-like protein yeast engineering strain to obtain the recombinant human fibrinogen-like protein stock solution, including:

[0030] Centrifuge to collect the supernatant of the fermentation broth of the highly expressed recombinant human fibrinogen-like protein yeast engineering strain, and use a solid-liquid separation system to separate and obtain the fermentation supernatant and the bacterial cells;

[0031] Pass the fermentation supernatant through a Sephadex G-25 desalting column, and equilibrate the chromatography column with 20 mM phosphate buffer until the conductivity value and the A280 absorbance value remain unchanged. Set the sample loading flow rate at 40 cm / h, detect the ultraviolet A280 absorbance value, and start sampling when it rises;

[0032] After the sample loading is completed, equilibrate the desalting column with 20 mM phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change, and stop sampling;

[0033] The desalted product is then passed through an anion exchange column, and the chromatography column is equilibrated with 20 mM phosphate buffer until the conductivity value and the A280 absorbance value remain unchanged. Set the sample loading flow rate at 40 cm / h;

[0034] After the sample loading is completed, equilibrate the anion chromatography column with 20 mM phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change;

[0035] Finally, elute and collect the corresponding protein with 20 mM phosphate buffer containing 300 mM NaCl, and dilute the elution product with an equal volume of 20 mM phosphate buffer to obtain the recombinant human fibrinogen-like protein stock solution.

[0036] Compared with the prior art, the present application provides a recombinant human fibronectin amino acid sequence (as shown in SEQ ID No. 3). This sequence avoids the main cleavage sites recognized by matrix metalloproteinases, preventing or reducing the proteolytic hydrolysis of recombinant human fibronectin in the skin, thereby ensuring its long-term effectiveness in the body. This sequence is a human fibronectin sequence, without amino acid sequences of expression vectors or tag proteins that are not fibronectin, and is completely derived from the natural fibronectin amino acid sequence. Therefore, it has more advantages in biosafety and biocompatibility. In addition, this sequence is a repeat sequence of the activity-related region, minimizing sequences unrelated to activity or structural stability. Thus, the expressed recombinant human fibronectin has a higher specific activity of promoting cell adhesion. At the same time, this sequence is also obtained through molecular dynamics simulation and screening, avoiding structural regions prone to aggregation and solving the problem of poor thermal stability and easy precipitation of fibronectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale.

[0038] Figure 1 It is the predicted result diagram of the fibronectin structure Alphafold involved in the embodiment of the present application;

[0039] Figure 2 It is the analysis diagram of the fibronectin aggregation region Discovery studio involved in the embodiment of the present application;

[0040] Figure 3 It is the PCR identification result diagram of the recombinant human fibronectin transformant involved in the embodiment of the present application;

[0041] Figure 4 It is the SDS-PAGE analysis result diagram of the expression of the recombinant human fibronectin multi-copy transformant involved in the embodiment of the present application;

[0042] Figure 5 It is the SDS-PAGE result diagram of the purified sample of the recombinant human fibronectin involved in the embodiment of the present application;

[0043] Figure 6 It is the appearance change diagram of the recombinant human fibronectin stock solution after heating at 100 °C for 30 min involved in the embodiment of the present application;

[0044] Figure 7Purity change graph of the recombinant human fibronectin stock solution involved in the embodiments of the present application after heat treatment at 100 °C for 30 min;

[0045] Figure 8 Skin pharmacokinetic result graph of the recombinant human fibronectin involved in the embodiments of the present application.

[0046] The realization, functional features and advantages of the purpose of the present application will be further described in conjunction with the embodiments with reference to the accompanying drawings. Detailed implementation manners

[0047] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present application.

[0048] Fibronectin (FN) is an important extracellular matrix protein that widely exists in various tissues and body fluids of the human body. It mainly exists in a soluble form in plasma and in an insoluble form in the extracellular matrix. Fibronectin is formed by cross-linking of two subunits through a disulfide bond at the C-terminus. The molecular weight of each subunit is 220-250 kDa, and the whole molecule is in a V shape. Each subunit of fibronectin has several domains, including: three repeated modules of FNⅠ, FNⅡ, and FNⅢ, as well as 2 alternative splicing sites EDA and EDB and 1 variable region V. These domains are mainly divided into: heparin-binding domain, fibronectin-binding domain, human 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 integrin through the RGD motif on FNⅢ10, thereby promoting cell adhesion, migration, proliferation, etc.

[0049] In the life activities of the body, fibronectin is involved in cell migration, adhesion, proliferation, hemostasis, tissue repair and embryonic development. In recent years, there have been more and more studies on the application value and fields of fibronectin. In skin wound repair and healing, fibronectin can shorten the wound healing time and reduce wound scars; in skin care, especially in anti-aging and skin repair, fibronectin, together with other structural proteins such as human fibronectin and elastin, provides a support network for the skin, helping to improve the skin texture and appearance.

[0050] Although fibronectin has many advantages, there are also some disadvantages. As a high-molecular-weight glycoprotein, its physicochemical properties are not very stable, and it is prone to aggregation and protein precipitation at room temperature and normal pressure, which to a certain extent limits its further development and application. Currently, the heat-resistant fibronectin available on the market is mainly obtained by genetic engineering and directed evolution methods. Some amino acid residues in the protein that affect its thermal stability are replaced with amino acid residues (such as proline) that are beneficial to protein stability, and a mutant library is established and screened to obtain a heat-resistant fibronectin variant. However, the heat-resistant fibronectin variant is a kind of human-like fibronectin rather than natural fibronectin and human-derived recombinant fibronectin. Compared with natural fibronectin and human-derived recombinant fibronectin, it may have disadvantages in biocompatibility and potential risks in biosafety.

[0051] In addition, the recombinant fibronectin available or reported on the market currently does not consider the problem of enzymatic hydrolysis of proteins in the skin during construction and expression. The recombinant fibronectin containing the matrix metalloproteinase cleavage site sequence is easily hydrolyzed and inactivated in the skin, so it is difficult to exert the efficacy of recombinant fibronectin for a long time.

[0052] Therefore, to solve the above-mentioned disadvantages of the existing recombinant fibronectin, the present application provides a recombinant human-derived recombinant fibronectin amino acid sequence (SEQ ID No.3). This sequence avoids the main cleavage sites recognized by matrix metalloproteinases, avoids or reduces the protease hydrolysis of recombinant human-derived recombinant fibronectin in the skin, and thus ensures its long-term effectiveness in vivo; this sequence is a human-derived recombinant fibronectin sequence, does not contain amino acid sequences of non-fibronectin such as expression vectors or tag proteins, and is completely derived from the natural fibronectin amino acid sequence, so it has more advantages in biosafety and biocompatibility; in addition, this sequence is a repeat sequence of the activity-related region, minimizing the sequences unrelated to activity or structural stability as much as possible, so the expressed recombinant human-derived recombinant fibronectin has a higher specific activity of promoting cell adhesion; at the same time, this sequence is also obtained through molecular dynamics simulation and screening, and avoids the structural regions prone to aggregation, solving the problem of poor thermal stability and easy precipitation of fibronectin.

[0053] For the convenience of those skilled in the art to understand, the sequences involved in the technical solution of the present application are listed below:

[0054] SEQ ID No.1:

[0055] AAHEEICTTNEGVM;

[0056] SEQ ID No.2:

[0057] VPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID;

[0058] SEQ ID No.3:

[0059] AAHEEICTTNEGVMVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID;

[0060] SEQ ID No.4:

[0061]

[0062] The technical solutions of the present application will be described in detail below in conjunction with specific embodiments:

[0063] Example 1

[0064] Construction, identification and preparation of engineered bacteria

[0065] 1. Protein sequence selection and design

[0066] The amino acid sequence of the recombinant human-derived fibrinogen-like protein described in the present application (390 aa, SEQ ID No. 3) is derived from natural fibronectin (UniProt: P02751), and contains a human-derived fibrinogen-like protein binding region (14 aa, SEQ ID No. 1) and four repeated cell-binding regions (94 aa, SEQ ID No. 2). The amino acid sequence avoids the main cleavage site sequences recognized by matrix metalloproteinases for fibronectin.

[0067] The matrix metalloproteinases (MMPs) are zinc- and calcium-dependent endopeptidases that can target and cleave extracellular matrix proteins such as fibronectin, human-derived fibrinogen-like protein, 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, etc. Therefore, when selecting the amino acid sequence of the recombinant human-derived fibrinogen-like protein described in the present application, these MMP cleavage sites are avoided as much as possible, which can reduce the hydrolysis of the recombinant human-derived fibrinogen-like protein by MMPs, and thus play its long-term effect in the extracellular matrix.

[0068] In addition to avoiding the main cleavage site sequences recognized by MMPs, the amino acid sequence of the recombinant human-derived fibrinogen-like protein (SEQ ID No. 3) also avoids the structural regions prone to aggregation through molecular dynamics simulation and screening. The specific method includes the following steps:

[0069] Intercept different structural regions from natural fibronectin and use the Alphafold tool for protein structure prediction and analysis. Then use the Discovery studio software to analyze the protein aggregation regions of this structure. The force field is selected as CHARMm Polar H, and the analysis radius is set to 10 Å. Analyze the number of protein aggregation regions and the aggregation trend scores of each region. The larger the score, the more prone the region is to aggregation. The amino acid sequence of the recombinant human-derived fibrinogen-like protein (SEQ ID No. 3) avoids some of the structural regions prone to aggregation during sequence design.

[0070] In addition to analyzing the aggregation regions of the amino acid sequence of recombinant human fibroconnectin (SEQ ID No. 3), the aggregation regions of fibronectin were also compared. The results showed that fibronectin contains multiple aggregation regions, among which two aggregation regions have relatively high aggregation trend scores (>2) and are prone to aggregation. In contrast, the recombinant human fibroconnectin of the present application contains only two aggregation regions, and the maximum aggregation trend score is only 0.416 (as shown in Table 1, Figure 1 and Figure 2 shown).

[0071] The results of molecular dynamics simulation showed that the recombinant human fibroconnectin of the present application has better stability and solubility than its counterparts and is not easily precipitated.

[0072] Table 1. Analysis results of fibronectin aggregation regions by Discovery studio

[0073]

[0074] A terminator was added to the C-terminus, and the gene sequence (1173 bp, SEQ ID No. 4) encoding recombinant human fibroconnectin was optimized according to the codon preference of Pichia pastoris to improve the expression efficiency of the recombinant protein in Pichia pastoris.

[0075] At the 5'-end of the optimized base sequence of recombinant human fibroconnectin, that is, Xho a base sequence AAAAAGA was added between Ⅰ and the target gene sequence, and GenScript Biotech Corporation was commissioned to synthesize the gene fragment. The synthesized gene fragment was inserted into the pPICZαA plasmid through the restriction enzyme sites of Xho Ⅰ and Not Ⅰ to obtain the recombinant human fibroconnectin plasmid.

[0076] 2. Construction and screening of recombinant engineering bacteria

[0077] 2.1 Obtaining recombinant plasmids

[0078] The optimized base sequence of recombinant human fibroconnectin mentioned above was commissioned to GenScript Biotech Corporation for gene fragment synthesis. After sequencing verification, the corresponding recombinant plasmid and stab culture were provided. After large-scale culture, high-concentration recombinant plasmid was extracted for standby.

[0079] 2.2 Linearization of recombinant human fibroconnectin plasmid

[0080] QuickCut Sac Ⅰ was used to linearly digest 20 μg of the recombinant human fibroconnectin plasmid extracted in 2.1. The digestion conditions were 37 °C for 5 h. The digestion system is shown in Table 2:

[0081] Table 2 Digestion system

[0082]

[0083] After electrophoresis verification, add 0.1 volume of 3 M NaAc (pH 5.2) and 2.5 volumes of absolute ethanol, and place at -20 °C overnight. Centrifuge at 13,000 rpm for 20 min at 4 °C, discard the supernatant; add 700 μL of 75% ethanol for rinsing, centrifuge at 13,000 rpm for 20 min, discard the supernatant, and repeat once; Invert the Ep tube on the absorbent paper in the laminar flow hood for about 10 min to remove moisture and residual ethanol as much as possible, dissolve the plasmid with 20 μL of ddH2O, take 1 μL and dilute it 10 times, and detect the nucleic acid concentration with one-drop.

[0084] 2.3 Preparation of GS115 Competent Cells

[0085] After streaking the GS115 strain on a plate, pick a single colony and inoculate it into 20 mL of YPD, and culture at 30 °C and 225 rpm for 24 h; Transfer inoculum at a ratio of 1:1000 to 50 mL of YPD liquid medium, and culture 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 3000 rpm for 5 min at 4 °C, discard the supernatant and collect the bacterial cells; Resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water, centrifuge at 3000 rpm for 5 min at 4 °C; After discarding the supernatant, resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water; Centrifuge at 3000 rpm for 5 min at 4 °C, after discarding the supernatant, resuspend the cell pellet with 40 mL of pre-cooled sterile 1 M sorbitol; Centrifuge at 3000 rpm for 5 min at 4 °C, after discarding the supernatant, resuspend the cell pellet with 100 - 150 μL of pre-cooled sterile 1 M sorbitol, gently rotate and mix evenly, and place on ice for later use.

[0086] 2.4 Electroporation into GS115 Competent Cells

[0087] Take 100 μL of yeast competent cells and mix with 10 μL of linearized plasmid, transfer them into a pre-cooled electroporation cuvette, and immediately ice-bath for 5 min. Select the yeast mode of the electroporator for electroporation. Then immediately add 1 mL of pre-cooled 1 M sorbitol to the electroporation cuvette, mix well and transfer the mixture to a sterile EP tube, and incubate in a 30 °C incubator for 1 h - 2 h. Take 100 μL - 200 μL of the 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 incubate it inverted in a 30 °C incubator for about 2 d - 5 d until single colonies appear.

[0088] 2.5 Screening of positive transformants

[0089] Pick single colonies from the YPD plate and transfer them to a 96-well culture plate containing 200 μL of YPD medium (containing 0.5 mg / mL Zeocin), and continue culturing at 30 °C. After 48 h, resuspend the bacterial solution, and transfer 10 μL from each well to a new 96-well plate (containing 190 μL of YPD), and repeat the above steps after culturing for another 24 h. After 24 h, resuspend the bacterial solution in the third 96-well plate, and pipette 1 μL and spot it on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin respectively, and continue culturing. If the transformant can grow on the plate containing 3 mg / mL of high-concentration Zeocin, it indicates that the transformant contains multiple copies of the expression cassette. Through this step of screening, a recombinant human fibronectin yeast engineering strain with high expression can be obtained.

[0090] 2.6 Identification of positive transformants

[0091] Pick a small amount of single colonies from the plate with 3 mg / mL Zeocin in step 2.4 above, resuspend them with 20 μL of 20 mM sodium hydroxide solution, boil them in a boiling water bath for 30 min, centrifuge at 12,000 rpm for 5 min, and take the supernatant as the PCR template to identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0092] Forward primer: 5’AOX1 (5’-GCAGCGCACGAGGAGATATG-3’)

[0093] Reverse primer: 3'AOX1 (5’-TCAGTCTATTTCAGTTCGGTAAT-3’)

[0094] PCR conditions: Pre-denaturation at 95 °C for 5 min, hot denaturation at 95 °C for 50 s, annealing at 60 °C for 30 s, extension at 72 °C for 60 s, 35 cycles; final extension at 72 °C for 10 min.

[0095] Take the PCR amplification product for 1.0% agarose gel electrophoresis to identify whether the gene fragment of the expected size is amplified. Lanes 1-3 are the PCR products of different recombinant human fibronectin transformants. The results show that the size of the PCR nucleic acid bands of the recombinant human fibronectin transformants is consistent with the theoretical value (1173 bp), and the identification results are all positive (as Figure 3 shown).

[0096] 3. Expression of recombinant human fibronectin

[0097] The formula of the medium used is as follows:

[0098] 1) YPD complete medium:

[0099] 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (solid medium contains 2% agar);

[0100] 2) MD solid medium:

[0101] 13.4 g / L YNB without amino acid nitrogen source, 0.4 mg / L biotin, 20 g / L glucose, (solid medium contains 2% agar);

[0102] 3) BMGY yeast growth medium:

[0103] 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L K2HPO4, add water to 890 mL, sterilize at 121 °C for 20 min, then after the temperature drops to 60 °C, add 100 mL of 10×YNB (13.4 g / L), 2 mL of 500×biotin (4×10 -4 g / L), 10 mL of glycerol in the laminar flow hood, with a total volume of 1 L.

[0104] 4) BMMY yeast induction medium:

[0105] 10 g / L yeast extract, 20 g / L peptone, 3 g / L K2HPO4, 11.8 g / L K2HPO4, add water to 895 mL, sterilize at 121 °C for 20 min, then after the temperature drops to 60 °C, add 100 mL of 100×YNB (13.4 g / L), 2 mL of 500×biotin (4×10 -4 g / L), 5 mL of methanol, with a total volume of 1 L.

[0106] The specific operation is as follows: Pick a single colony and inoculate it into BMGY medium, culture at 30 °C, 220 rpm for 24 h until OD 600nm 2 - 6. Adjust the volume of the BMGY culture broth according to the measured OD value, collect the cells at 3000 rpm for 10 min, resuspend the cells with BMMY medium of the same volume as BMGY to make the initial OD value 2.0. Continue to culture at 30 °C, 220 rpm, add 0.5% methanol to the medium every 24 h, and take the centrifuged supernatant samples at 72 h after induction. Analyze the molecular weight and expression of the target protein by SDS-PAGE electrophoresis. The results show that an obvious protein band is detected at 45 kDa, which is consistent with the theoretical value of the molecular weight of recombinant human fibronectin (about 45 kDa, with about 4 kDa of glycosylation modification), indicating successful initial expression, and the expression level of the multi-copy transformant 2 is higher (as Figure 4 shown).

[0107] 4. Purification by anion exchange chromatography

[0108] Centrifuge to collect the supernatant of the culture medium of the engineered bacteria fermentation broth, and use a solid-liquid separation system to separate the fermentation supernatant and the bacterial cells. The fermentation supernatant is passed through a Sephadex G-25 desalting column, and the chromatography column is equilibrated with 20 mM phosphate buffer (pH 7.6) until the conductivity value and the A280 absorbance value remain unchanged. Set the sample loading flow rate at 40 cm / h, detect the ultraviolet A280 absorbance value, and start sampling when it rises. After the sample loading is completed, the desalting column is equilibrated again with 20 mM phosphate buffer (pH 7.6) until the ultraviolet and conductivity drop to the lowest and no longer change, and then stop sampling. The desalted product is passed through an anion exchange column (the chromatography packing is Q Purose 6 HighPerformance produced by Chunchun, loaded on a GE AKTA chromatography system), and the chromatography column is equilibrated with 20 mM phosphate buffer (pH 7.6) until the conductivity value and the A280 absorbance value remain unchanged. Set the sample loading flow rate at 40 cm / h. After the sample loading is completed, the anion chromatography column is equilibrated again with 20 mM phosphate buffer (pH 7.6) until the ultraviolet and conductivity drop to the lowest and no longer change. Finally, elute and collect the corresponding protein with 20 mM phosphate buffer (pH 7.6) containing 300 mM NaCl, and the eluted product is diluted with an equal volume of 20 mM phosphate buffer (pH 7.6) to obtain the recombinant human fibrinogen-like protein stock solution. The purity of the recombinant human fibrinogen-like protein stock solution is detected by SDS-PAGE electrophoresis. The results show that the purity of the recombinant human fibrinogen-like protein reaches the electrophoresis purity, and there is almost no loss of the target protein in the flow-through solution, and the recovery rate of the purification method is high (as Figure 5 shown).

[0109] Example 2: Heat resistance test of recombinant human fibrinogen-like protein

[0110] High temperature will destroy the secondary bonds such as hydrogen bonds and ionic bonds of proteins, causing changes in the spatial structure of proteins and irreversible denaturation. The spatial structure of proteins is crucial for their functions. Once irreversible denaturation occurs, the proteins lose their original biological activities. High temperature will also increase the intermolecular interaction forces of proteins, leading to protein aggregation and the formation of protein precipitates. Generally, when the environmental temperature exceeds 60 °C, proteins begin to undergo irreversible denaturation and protein precipitation phenomena. The heat resistance test of recombinant human fibrinogen-like protein verifies the thermal stability of recombinant human fibrinogen-like protein by the method of treating proteins in a high-temperature water bath and then comparing and analyzing the changes in the appearance, purity and activity of proteins before and after treatment.

[0111] 1. Effects of high-temperature treatment on the appearance and purity of the recombinant human fibrinogen-like protein stock solution

[0112] 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 packed in a vial, sealed, and no protein stabilizer is added), put it into boiling water and heat for 30 minutes, then take out the sample to observe the appearance change. There is no precipitation of any protein in the recombinant human fibronectin stock solution, and there is no color change ( Figure 6 ); Take a sample for SDS-PAGE electrophoresis detection. The recombinant human fibronectin stock solution sample has no degradation phenomenon after being boiled at high temperature ( Figure 7 ).

[0113] The results of this experiment show that heating at 100°C for 30 minutes has no effect on the appearance and purity of the recombinant human fibronectin stock solution.

[0114] 2. Effect of high-temperature treatment on the cell adhesion-promoting activity of recombinant human fibronectin stock solution

[0115] 2.1 Experimental materials:

[0116] Complete cell culture medium: 1640 medium (containing double antibodies) added with 10% fetal bovine serum, stored at 4°C.

[0117] Serum-free medium: 1640 medium (containing double antibodies), stored at 4°C.

[0118] Digestive solution: 0.25% trypsin.

[0119] PBS buffer solution: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up the volume to 1000 mL, and sterilize at 121°C for 15 minutes under high pressure.

[0120] MDBK cells (purchased from ATCC).

[0121] BSA (purchased from Sigma).

[0122] 2.2 Specific implementation method:

[0123] The purified stock solution of recombinant human fibronectin and the same type of fibronectin before and after heating at 100 °C for 30 min were diluted to 0.5 μg / mL with PBS buffer, respectively. Then, the diluted samples were taken and serially diluted 2-fold in a 96-well cell culture plate, with a total of 8 dilution factors, 50 μL per well. A negative control (50 μL of PBS buffer added) was set up and incubated overnight at 4 °C; the liquid in the cell culture plate was discarded, and each well was washed with 100 μL of PBS buffer, for a total of 3 washes; after the washing was completed, each well was blocked with 100 μL of 30 μg / μL BSA and incubated in an incubator at 37 °C for 1 h; the liquid in the cell culture plate was discarded, each well was washed 3 times with 100 μL of PBS buffer, and then fibroblast cell suspension was added. The cell seeding density was 1.0×10 5 cells / mL, 100 μL was seeded in 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.

[0124] Five points were selected under a 200-fold microscope to count the number of adherent cells except at the edges. According to the counting results, the titer was obtained using a four-parameter fitting curve. The results of each group are shown in Table 3. The results showed that recombinant human fibronectin had higher cell adhesion-promoting activity than commercially available fibronectin, and after treatment with heating at 100 °C for 30 min, it still had an activity recovery rate of 94.9%, while the activity recovery rate of commercially available fibronectin was only 13.9%.

[0125] The results indicated that the cell adhesion-promoting activity of recombinant human fibronectin was heat-resistant to heating at 100 °C for 30 min.

[0126] Table 3 Effects of high temperature on the cell adhesion-promoting activity of recombinant human fibronectin

[0127]

[0128] Example 3: Analysis of the long-acting effect of recombinant human fibronectin in vivo

[0129] The analysis of the long-acting effect of recombinant human fibronectin in vivo was mainly based on the results of a rat skin pharmacokinetic experiment. The specific implementation method was as follows:

[0130] 1. Test materials:

[0131] Total protein extraction kit (strong): Solarbio.

[0132] Propofol: purchased from sigma, product number Y0000016.

[0133] Digestive solution: 0.25% trypsin.

[0134] Fibronectin ELISA kit: purchased from R&D company, product number DFBN10.

[0135] Recombinant human fibronectin: Prepared in Example 1.

[0136] 2. Specific implementation method:

[0137] Randomly divide female SD rats (weighing 190 - 210 g) into a control group of homologous fibronectin and a recombinant human fibronectin group. After anesthetizing the rats intravenously with propofol, shave the hair on their backs. The following mechanical injury model was established. Place the rats in the prone position and draw a circle (diameter 2.5 cm) around the spine. Use a sterile scalpel to make three 0.5-cm-long scratches horizontally in the marked area of each rat. These rats were kept in a cage and fed freely. For single-dose topical administration, immediately after modeling, PBS (0.2 mL), commercially available fibronectin, and recombinant human fibronectin were evenly applied to the damaged areas of the rats (application dose: 30 mg). The rats were euthanized under anesthesia at specified times during the experiment. The rat skin was collected at 0.5 h, 2 h, 4 h, 8 h, 12 h, 18 h, and 24 h after administration. Weigh the skin tissue, then cut it into pieces approximately 1 mm 3 in size, put them into centrifuge tubes, and store them in liquid nitrogen for later use.

[0138] Use a total protein extraction kit (strong) to extract the total protein in the skin tissue according to the operation instructions in the kit. Then, according to the operation requirements of the ELISA kit, perform corresponding processing and detection on the samples for pharmacokinetic analysis. The results are as Figure 8 shown. For commercially available fibronectin, T max was at 0.5 h after administration, and the C max value was 1297 pg / mL. Fibronectin was not detected in the skin at 8 h after administration; while for the recombinant human fibronectin prepared in Example 1, T max was at 2 h after administration, and the C max value was 1565 pg / mL. Fibronectin was not detected in the skin at 24 h after administration, which is more long-acting than commercially available fibronectin.

[0139] Thus, compared with the prior art, the main beneficial effects of the recombinant human fibronectin described in this application are as follows:

[0140] More heat-resistant and stable. The long-acting heat-resistant and stable recombinant human fibronectin stock solution (without protein protectant) prepared in this application still has good stability and biological activity after being heated at 100 °C for 30 min, and there is no precipitation or degradation; better biological activity. The cell adhesion-promoting activity of the long-acting heat-resistant and stable recombinant human fibronectin prepared in this application reaches 1×10 5Above U / mg, which is one order of magnitude higher than commercially available fibronectin; longer retention time on the skin surface. The skin pharmacokinetic results show that the long-acting heat-stable recombinant humanized fibronectin prepared in this application has better long-acting properties than commercially available fibronectin.

[0141] The above are only the preferred embodiments of this application, and do not limit the patent scope of this application accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of this application, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of this application.

Claims

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

2.

2. A method for preparing the long-acting heat-stable recombinant human fibronectin as described in claim 1, characterized in that, It includes the following steps: After optimizing the codon preference of the gene sequence of recombinant human fibrinogen-like protein shown in SEQ ID No. 4, at the 5' end of its base sequence, the base sequence AAAAAGA was added, and the synthesized gene fragment was passed through Xho Ⅰ and Not The restriction enzyme sites of Ⅰ were inserted into the pPICZαA plasmid to obtain the recombinant human fibrinogen-like protein plasmid; After linearizing and digesting the recombinant human fibronectin plasmid with QuickCut Sac Ⅰ, a linearized plasmid is obtained; After electrotransforming the linearized plasmid into GS115 competent cells and screening for positive transformants, a recombinant human fibronectin yeast engineering strain with high expression is obtained; The recombinant human fibronectin yeast engineering strain with high expression is purified by anion exchange chromatography to obtain the recombinant human fibronectin stock solution.

3. The preparation method of the long-acting heat-resistant and stable recombinant human fibronectin according to claim 2, characterized in that, The temperature of the linearizing digestion treatment is 37 °C, and the time of the linearizing digestion treatment is 5 h; The system of the linearizing digestion treatment includes: 20 μg of recombinant human fibronectin plasmid, 10 μL of QuickCut SacⅠ, 5 μL of 10× Quick Cut buffer, and the total volume is 50 μL.

4. The preparation method of the long-acting heat-resistant and stable recombinant human fibronectin according to claim 2, wherein After electrotransforming the linearized plasmid into GS115 competent cells and screening for positive transformants, obtaining a recombinant human fibronectin yeast engineering strain with high expression includes: Mix the yeast competent cells with the linearized plasmid, transfer them into an electroporation cuvette for ice bath; select the yeast mode of the electroporator for electroporation; add pre-cooled sorbitol to the electroporation cuvette, mix well and transfer to a sterile EP tube, and place it in an incubator for static incubation for 1 - 2 h to obtain a bacterial solution; spread the bacterial solution on a YPD plate, invert it in an incubator for culture until single colonies appear; Pick the single colonies, transfer them to the first 96-well culture plate containing YPD medium and continue culturing; after culturing for 45 h - 50 h, blow the bacterial solution evenly, and transfer 5 μL - 15 μL from each well to the second 96-well culture plate containing YPD medium and continue culturing; after culturing for 20 h - 28 h, blow the bacterial solution evenly, and transfer 5 μL - 15 μL from each well to the third 96-well culture plate containing YPD medium and continue culturing; After 20 h - 28 h, blow the bacterial solution in the third 96-well culture plate containing YPD medium evenly to obtain transformants; and transfer 1 μL of the transformants and spot them on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin respectively and continue culturing; Screen for transformants that can grow on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL Zeocin simultaneously as positive transformants, that is, recombinant human fibronectin yeast engineering strains with high expression.

5. The preparation method of the long-acting heat-stable recombinant human fibronectin according to claim 4, characterized in that, The positive transformants are identified through the following steps: Pick the single colonies, resuspend them with sodium hydroxide solution, boil them in a boiling water bath and then centrifuge, take the supernatant as the PCR template, and perform PCR amplification to obtain the PCR amplification product; Take the PCR amplification product for 1.0% agarose gel electrophoresis to identify whether the gene fragment of the expected size is amplified.

6. The preparation method of the long-acting heat-stable recombinant human fibronectin according to claim 2, characterized in that, Before the purification treatment of the highly expressed recombinant human fibronectin yeast engineering strain by anion exchange chromatography to obtain the recombinant human fibronectin stock solution, it also includes: Pick a single colony and inoculate it into BMGY medium, culture at 30 °C and 220 rpm for 24 h until OD600nm is 2 - 6; Adjust the volume of the BMGY culture broth according to the measured OD value, collect the cells at 3000 rpm for 10 min, resuspend the cells with BMMY medium of the same volume as BMGY to make the initial OD value 2.0; Continue to culture at 30 °C and 220 rpm, add 0.5% methanol to the medium every 24 h, and take the centrifuged supernatant samples at 72 h after induction; Analyze the molecular weight and expression of the target protein by SDS-PAGE electrophoresis to judge whether it is consistent with the theoretical value of the molecular weight of recombinant human fibronectin. If it is consistent, the expression is successful.

7. The preparation method of the long-acting heat-resistant and stable recombinant human fibronectin according to claim 2, characterized in that, The purification treatment of the highly expressed recombinant human fibronectin yeast engineering strain by anion exchange chromatography to obtain the recombinant human fibronectin stock solution includes: Centrifuge to collect the supernatant of the fermentation broth of the highly expressed recombinant human fibronectin yeast engineering strain, and use a solid-liquid separation system to separate and obtain the fermentation supernatant and cells; Pass the fermentation supernatant through a Sephadex G-25 desalting column, balance the chromatography column with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remain unchanged, set the sample loading flow rate at 40 cm / h, detect the ultraviolet A280 absorbance value, and start sampling when it rises; After the sample loading is completed, balance the desalting column with 20 mM phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change, and stop sampling; The desalted product is passed through an anion exchange column again, balance the chromatography column with 20 mM phosphate buffer until the conductivity value and A280 absorbance value remain unchanged, and set the sample loading flow rate at 40 cm / h; After the sample loading is completed, balance the anion chromatography column with 20 mM phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change; Finally, elute and collect the corresponding protein with 20 mM phosphate buffer containing 300 mM NaCl, and dilute the elution product with 20 mM phosphate buffer of the same volume to obtain the recombinant human fibronectin stock solution.

Images