CN113735980A - Self-fusion tandem protein modification method and application thereof - Google Patents

Abstract

The invention discloses a simple but universal protein modification method, namely a self-fused tandem (SEC) protein modification method, so as to improve the biological activity and the pharmaceutical property of a protein. The invention obtains a group of GFP monomers, GFP dimers and GFP trimers by a genetic engineering technology. GFP multimers can significantly increase the in vitro bioactivity and thermal stability of GFP monomers and the residence time of mouse tumors, and the bioactivity, thermal stability and tumor residence time of proteins are positively correlated with the number of self-fused tandem proteins. In addition, the invention synthesizes IFN monomer, IFN dimer and IFN trimer by SEC technology, and the IFN dimer can obviously improve the in vitro bioactivity, in vivo half-life period and anti-tumor effect of the IFN monomer. The results show that SEC can be used as an option to replace the existing PEG or albumin fusion technology and widely applied to other protein or small peptide drugs to improve the pharmacological properties of the drugs and successfully design long-acting protein or polypeptide drugs.

Description

Self-fusion tandem protein modification method and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to development of a self-fusion tandem protein modification method and application of the self-fusion tandem protein modification method in protein modification.

Background

Compared with small molecule drugs, proteins and polypeptides have high specificity and low toxicity, and have great medical treatment potential clinically. At present, various FDA approved therapeutic drugs (such as etanercept, insulin glargine, pefloxacin, bivalirudin, cyclosporine, octreotide and the like) are applied in the fields of tumors, immunity, viral diseases, endocrine diseases and the like. However, proteins and polypeptides have problems of poor stability, strong immunogenicity, short half-life, and the like.

Many strategies have been developed to partially address the above problems and to improve the efficiency of protein/polypeptide delivery. For example, covalent attachment of the nontoxic polymer polyethylene glycol (PEG) to proteins can be effective in extending the circulating half-life and increasing protein stability, a process known as PEGylation. At present, a plurality of PEGylated protein drugs, such as PEG-interferon alpha-2 a, PEG-L-asparaginase, PEG-adenosine deaminase, PEG-uricase, PEG-tumor necrosis factor alpha, PEG-erythropoietin receptor activator and the like, are applied clinically, and show better drug characteristics compared with unmodified protein. Another albumin fusion technique has been successfully used for the modification of IFN α, antihemophilic factor (AHF), recombinant factor IX (F9), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and anti-vascular endothelial growth factor (anti-VEGF) and effectively extending the circulating half-life in vivo by fusing the protein to serum albumin (HSA) or Fc fragment with a long half-life. However, both the well-established pegylation technology and HSA and Fc fusion require the introduction of foreign macromolecules for modification, which may lead to immunogenicity, reduced protein bioactivity, and other potential toxic effects.

Disclosure of Invention

The technical problem to be solved by the invention is how to prolong the half-life period and/or increase the activity retention rate of the protein in an organism without introducing exogenous molecules.

First, the present invention provides a method for modifying a self-fusing tandem protein, which comprises the steps of expressing genes encoding proteins in tandem in a protein expression system, and purifying the expressed multimer.

Wherein, the protein expression system includes but is not limited to prokaryotic expression vector, yeast expression vector, plant expression vector, mammal expression vector, insect expression vector.

Wherein the protein is fluorescent protein, and the fluorescent protein is selected from green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein and derivatives thereof.

Wherein the protein is interferon, and the interferon is one or more selected from interferon alpha, interferon beta, interferon gamma and interferon lambda.

Wherein the protein is a therapeutic protein including, but not limited to, insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, Tumor Necrosis Factor (TNF) and enzymes. Specific examples include, but are not limited to: asparaginase, glutaminase, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, Epidermal Growth Factor (EGF), insulin-like growth factor (IGF), Transforming Growth Factor (TGF), Nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), Bone Morphogenetic Protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, Colony Stimulating Factor (CSF), blood clotting factors, tumor necrosis factors, interferons, interleukins, gastrointestinal peptides, Vasoactive Intestinal Peptide (VIP), intestinal tryptic peptide (CCK), gastrin, secretin, erythropoietin, hormones, antidiuretic hormones, octreotide, pancreatic enzymes, Superoxide dismutase, thyroid stimulating hormone releasing hormone (TRH), thyroid stimulating hormone; luteinizing hormone, Luteinizing Hormone Releasing Hormone (LHRH), tissue plasminogen activator, interleukin-1, interleukin-15, receptor antagonist (IL-1RA), glucagon-like peptide-1 (GLP-1), leptin, auxin, granulocyte colony stimulating factor (GM-CSF), interleukin-2 (IL-2), adenosine deaminase, uricase, asparaginase, human growth hormone, asparaginase; macrophage activation; chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vectors, relaxin; cyclosporine, oxytocin, vaccines, monoclonal antibodies, single chain antibodies, ankyrin repeat proteins, affibodies and the like.

The invention also provides a self-fusion tandem protein polymer prepared by the modification method.

The invention also provides the application of the self-fusion tandem protein polymer in preparing protein drugs for improving stability, prolonging in vivo half-life period and further improving treatment effect.

The invention develops a novel and universal protein modification method, namely self-fused cascade (SEC), so as to improve the biological activity of the protein, prolong the half-life period in vivo and effectively exert the treatment efficacy. The invention firstly selects Green Fluorescent Protein (GFP) as a model protein, designs a group of polymers by utilizing the protein fusion technology, and systematically studies the influence of SEC modification on the biological activity and stability of GFP. Research results show that the serial GFP is significantly higher than a GFP monomer in the aspects of bioactivity, in-vitro thermal stability and in-vivo tumor retention, and the results are positively correlated with the number of the serial proteins. Further, the invention selects the pharmaceutical protein Interferon (IFN) as an example, synthesizes a series of IFN polymers, and proves that the IFN polymers have more excellent pharmaceutical properties than monomers by systematically researching the bioactivity, in vivo half-life period and anti-tumor effect of SEC modification on the IFN. Based on the above results, it can be proved that SEC is possible to be a novel protein modification method, and the pharmaceutical properties of the protein are improved without introducing other macromolecules.

Drawings

FIG. 1 shows the physicochemical properties of GFP multimers. a) Schematic representation of GFP concatemers designed and synthesized by SEC. b) SDS-PAGE analysis of GFP multimers. The left panel shows the expression of GFP multimers, lanes 1-3 correspond to crude E.coli lysates for GFP1, GFP2 and GFP3, respectively. The right panel shows purified GFP multimers, lanes 1-3 correspond to purified GFP1, GFP2 and GFP3, respectively. c) GFP polymer liquid chromatography-electrospray mass spectrometry. d) GFP multimer dynamic light Scattering analysis. e) Circular dichroism data for GFP multimers.

FIG. 2 shows the fluorescence of GFP polymers at an excitation wavelength of 460 nm. a) Fluorescence spectra of GFP multimers at the same mass concentration. b) Retention of fluorescence per unit of multimer of GFP. Data are shown as mean ± standard deviation (n-3, GFP3 vs GFP1 statistical analysis P < 0.05). c) Fluorescence spectra of GFP multimers at the same molar concentration. d) The relative amount of GFP per unit protein multimer. Data are shown as mean ± sd (n-3, GFP3 vs GFP1, GFP2 vs GFP1 statistical analysis P < 0.05).

FIG. 3 shows the thermal stability of GFP multimers after denaturation at 90 ℃ for 2 min. a) The fluorescence recovery of GFP varies with time. Data are shown as mean ± standard deviation (n ═ 3, GFP2, GFP3 and GFP1 statistical analysis P < 0.0001). Fluorescence recovery of GFP polymers after denaturation and 2h at ambient temperature. Data are shown as mean ± standard deviation (n 3, GFP2, GFP3 and GFP1 statistical analysis, P <0.01 and P < 0.0001).

FIG. 4 shows fluorescence retention of GFP multimers in tumors. a) Dynamic visualization of fluorescence imaging of GFP multimers within mouse tumors. b) The fluorescence of GFP polymers varies with time in mice. Data are shown as mean ± standard deviation (n 4, statistical analysis of GFP3 and GFP2 at different time points P <0.05 and P <0.01, statistical analysis of GFP2 and GFP1 at different time points P <0.01 and P < 0.0001).

Figure 5 is IFN polymer physicochemical properties. a) SDS-PAGE analysis of IFN multimers. Lanes 1-3 correspond to purified IFN1, IFN2 and IFN3, respectively. b) IFN polymer liquid chromatography-electrospray mass spectrometry. c) IFN multimer dynamic light scattering analysis. d) Circular dichroism data of IFN multimers.

Figure 6 IFN polymer in vitro biological activity analysis. a) IFN multimer bioactivity at the same mass concentration. b) IC50 values for IFN multimers at the same mass concentration (IFN 2, IFN3 and IFN1 statistical analysis P <0.05, respectively). c) IFN multimer bioactivity at the same molar concentration. d) IC50 values for IFN multimers at the same molar concentration (IFN 3 and IFN1 statistical analysis P <0.05, respectively). Data are shown as mean ± standard deviation (n ═ 3).

Figure 7 IFN polymer pharmacokinetics. Data are shown as mean ± standard deviation (n ═ 3).

FIG. 8 shows the in vivo antitumor effect of IFN multimers. a) IFN multimers inhibited tumor growth (IFN 2 vs IFN1, IFN3 vs IFN2 were statistically analyzed as P <0.01, respectively). Data are shown as mean ± standard deviation (n ═ 3). b) Survival curves for IFN multimers. Data are shown as mean ± standard deviation (n ═ 3). c) Tumor, heart, liver and kidney HE staining after dosing.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The plasmid pET-25b (+) in the following examples is a product of Industrial bioengineering (Shanghai) Inc.

The TB medium in the following examples was prepared as follows: adding peptone 12g, yeast extract 24g and glycerol 4mL into 900mL water, fully dissolving, autoclaving at 121 deg.C for 15min, cooling the sterilized mixture to 60 deg.C, and adding 100mL sterilized 170mmol/LKH₂PO₄And 0.72mol/L K₂HPO₄An aqueous solution of (a).

Mouse colon cancer cells (C26), human Burkitt's B lymphoma cells (Daudi B), and human ovarian cancer cells (OVCAR-3) in the examples described below were purchased from the tumor cell bank of Chinese academy of sciences.

The RMPI-1640 medium in the following examples is a Hyclone.

The female athymic (Nude) Nude mice in the following examples are products of experimental animal technology ltd, viton, beijing. Female athymic (Nude) Nude mice are hereinafter abbreviated Nude mice.

Example 1 biosynthesis and physicochemical Properties of GFP multimers

Genes encoding GFP monomer GFP1, dimer GFP2 and trimer GFP3 (containing a 6 XHis tag) were synthesized by biological engineering (Shanghai, China) and successfully cloned into the pET-25b (+) vector. The sequence of GFP protein is shown in SEQ ID No. 1. The multimeric GFP subunits are separated by the flexible linker GGGGS. After confirmation of gene sequencing, the plasmid was transformed into E.coli BL21(DE3) strain for expression. Before large-scale expression, the transformed monoclonal bacteria were inoculated in 50mL of TB medium (containing 100. mu.g/mL ampicillin) and cultured overnight at 37 ℃ with shaking at 250 rpm. The next day, the cells were transferred into 1L of fresh TB medium (contained in 2L flasks at an ampicillin concentration of 100. mu.g/mL) for large-scale culture and expression was induced. The method comprises the following specific steps: first, shake culture was carried out at 37 ℃ and 200rpm for 5 hours, then the culture temperature was set to 25 ℃ and isopropyl-. beta. -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM, and after 16 hours of culture, centrifugation was carried out at 4000 Xg for 15 minutes, and the cells were collected and suspended in 10mM PBS, pH 7.4. After lysis with a continuous flow cell disruptor (JNBIO, guangzhou, china), the supernatant was harvested by centrifugation at 16000 × g for 30 minutes, while the cells in the pellet were discarded. To the collected supernatant was added 2mL of polyethyleneimine (PEI, 10%), and centrifuged again for 15 minutes in order to remove nucleic acids and other negatively charged materials from the cell lysate. The resulting supernatant was filtered through a 0.45 μm filter and purified by a nickel affinity column (HisTrap HP 5mL) on an AKTA protein purification system (AKTA Purifier 10, GE). 100mL of equilibration buffer (50mM Tris, 500mM NaCl, 10% glycerol and 5mM imidazole) was added first followed by 50mL of wash buffer (50mM Tris, 500mM NaCl, 10% glycerol and 30mM imidazole). Finally, the 6 × His-tagged protein was eluted with 5mL of elution buffer (50mM Tris, 500mM sodium chloride and 500mM imidazole). The eluted protein was subjected to Desalting column (HiPrep26/10 desaling) to remove imidazole while substituting 50mM Tris. HCl, 150mM NaCl, pH 7.4, and concentrated by ultrafiltration. Purified samples were tested for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE analysis samples are prepared by Laemmli sample buffer solution containing 5% beta-mercaptoethanol, the concentration is 1mg/mL, after heating for 5min at 95 ℃, 10 mu L of samples are loaded into a prefabricated 10% SDS-PAGE gel, and vertical electrophoresis is carried out for 90min under the voltage of 80-100V (electrophoresis solution is 25mM Tris, 250mM Glycine and 0.1% SDS). The gel was stained with Coomassie blue G-250 and the band positions were observed. Bovine Serum Albumin (BSA) was used as a protein standard, and the concentration of the protein was determined and evaluated according to the BCA kit (petunia, shanghai, china) instructions. The sequence of GFP protein is shown in SEQ ID No. 1.

Liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) was used to measure the molecular weight of GFP1, GFP2 and GFP 3. The instrument used was a SYNAPT-G2-Si mass spectrometer (Waters, USA) and the system used was ACQUITY UPLC. The sample was eluted through a gradient of 10 minutes at a flow rate of 0.5 mL/min. Mobile phase a consisted of 0.1% aqueous formic acid and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The analytical column was a silica capillary column (inner diameter 2.1mm, length 100mm, manufactured by Ireland) of protein BEH C4 using C-4resin (C-4 resin: (C-4 resin)

1.7 μm, Waters, USA). 2 μ L of analyte was loaded into an autosampler for nanoelectrospray ionization. Samples were optimized for high quality protein analysis by a Q-TOF mass spectrometer (SYNAPT G2-Si; Waters, USA) instrument, measured in 3000V capillaries, and data collected over the expected m/z range. Once the original native electrospray mass spectrum is obtained, the original spectrum can be deconvoluted by MaxEnt 1(Waters, USA) to generate one spectrum (relative intensity/mass) in which all charge state peaks of a single species are folded into a single (zero charge) peak.

Dynamic Light Scattering (DLS) determination the hydration radius of a protein can be measured by a Malvern Zeta sizer Nano ZS90 particle sizer at a laser wavelength of 633nm and a scattering angle of 90.

And (5) performing circular dichroism chromatographic analysis to obtain the secondary structure of the protein to be detected. Protein samples were diluted with deionized water (GFP 1, GFP2 and GFP3 concentrations were approximately 0.18, 0.25 and 0.28mg/mL, respectively) and analyzed by UV scanning using a Pistar π -180(Applied Photophysics, Inc., UK) over a wavelength range of 195-255nm, with an optical path of 1 mm.

The protein UV absorption spectrum (230nm-600nm) was analyzed by a Varioskan Flash microplate reader (Thermo Scientific, USA). The sample was diluted to 2.4mg/mL for determination of the same mass concentration of UV absorption spectrum and to 25. mu.M for determination of the same molar concentration of UV absorption spectrum.

As shown in FIG. 1a, the present invention constructs three recombinant plasmids capable of expressing green fluorescent protein monomer (GFP 1), dimer (GFP 2) and trimer (GFP 3) and successfully expresses them in Escherichia coli. After nickel column affinity purification, the target protein polymer with purity of 95% was successfully obtained by SDS-PAGE analysis (FIG. 1 b). LC-ESI-MS further confirmed that the molecular weights of GFP1, GFP2 and GFP3 were 28915.0, 56237.0 and 83559.0Da, respectively, in agreement with the theoretical molecular weights of 28915.3, 56237.0 and 83558.7Da, respectively (FIG. 1 c). The above data indicate that the present invention successfully achieves high quality GFP monomers, dimers and trimers. Hydrodynamic radius (R) of GFP2 and GFP3 by DLS analysis_h) 6.85 and 9.62nm, respectively, which are 1.83 and 2.57 times as high as GFP 1(3.75nm) (FIG. 1 d). By circular dichroism spectrum analysis, the GFP polymer spectrum in the 'far ultraviolet' region (195-255nm) is consistent with GFP1, the minimum peak is detected at 217-218nm, the maximum peak is detected at 195-198nm (FIG. 1e), and the secondary structure shows typical beta-barrel characteristics. This result indicates that the self-fused tandem modification has no significant effect on the conformation of GFP. The uv-vis absorption spectra of GFP1, GFP2 and GFP3 at the same mass (fig. 1f) or molar (fig. 1g) concentration overlap and show two absorption peaks at 280 and 478nm, indicating that the photophysical properties of GFP are well preserved.

Example 2 analysis of biological Activity of GFP multimers

The protein fluorescence spectrum (480nm-570nm) was analyzed by a Varioskan Flash enzyme reader (Thermo Scientific, USA) and the excitation wavelength was 460 nm. The fluorescence of the protein was measured at an excitation wavelength of 460nm and an emission wavelength of 507 nm. At the same mass concentration (protein test concentration of 0.25mg/mL), the retention of fluorescence intensity per unit GFP was calculated by the formula: the retention of fluorescence per unit GFP in the multimer ═ x 100% of the fluorescence of the multimer/fluorescence of GFP1, where the retention of GFP fluorescence intensity per unit in GFP1 was 100%. At the same molar concentration (protein test concentration of 10. mu.M), the relative amount of GFP per concatemer was calculated by the formula: the number of GFP contained per unit multimer is equal to the fluorescence of multimer/fluorescence of GFP1, where GFP1 means 1 GFP contained per unit GFP 1.

As shown in FIG. 2, the fluorescence spectra of GFP multimers (GFP 2 and GFP 3) and monomeric GFP (GFP 1) were identical to each other, with an emission wavelength maximum of 507 nm. The monomeric GFP showed the highest fluorescence intensity per unit GFP, whereas due to steric hindrance of the GFP subunits, the retention of fluorescence in the multimers decreased with increasing number of concatemers (FIG. 2a), and the retention of activity per unit GFP in GFP2 and GFP3 was 83.21% and 66.57%, respectively (FIG. 2 b). In contrast, the fluorescence intensity of the protein multimers increased significantly with the increase in the number of GFP concatemers (fig. 2 c). By calculation, the amount of GFP in the same molar amounts of GFP2 and GFP3 corresponded to 1.71-fold and 2.24-fold, respectively, of GFP1 (FIG. 2 d). The above results indicate that the increase in activity due to the amount of fused GFP is superior to the decrease in activity due to steric hindrance, thereby achieving an increase in the biological activity of the multimer. Prior art PEGylation, HSA and Fc fusion is the introduction of inactive macromolecules (e.g.non-toxic polymers or long half-life proteins) at the protein surface, resulting in a significant reduction in biological activity, sometimes even up to 1% compared to unmodified proteins. Taken together, the results indicate that SEC can significantly enhance the in vitro biological activity of proteins.

Example 3 in vitro thermal stability analysis of GFP multimers

The in vitro thermostability of the proteins was tested by heat denaturation of the proteins at 90 ℃ for 2 minutes and renaturation at room temperature. Before the assay, the fluorescence concentration of the samples was adjusted to the same value (final protein fluorescence value of 2500 by microplate reader, corresponding to protein concentrations of GFP1, GFP2 and GFP3 of 10, 7 and 5. mu.M), and the assay was performed as in example 2. After heating at high temperature for 2 minutes, the fluorescence value of the protein was measured at given times (1,5, 10, 15, 20, 25, 30, 45, 60 and 120 min).

As shown in FIG. 3, GFP3 recovered fluorescence faster than GFP2, and GFP2 recovered fluorescence significantly faster than GFP1 (FIG. 3 a). After standing at room temperature for 2h, the recovered fluorescence of GFP3 (53%) and GFP2 (22%) was 4.2-fold and 1.8-fold higher than that of GFP1 (12%), respectively (FIG. 3 b). The above data indicate that the thermostability of GFP multimers is positively correlated with the number of tandem proteins.

Example 4 analysis of tumor Retention in vivo of GFP multimers

All animal experiments used in this study were subject to strict approval by the animal care and use committee of the Beijing coordination and Hospital institution. C26 cell culture in RPMI-1640 complete Medium containing 10% fetal bovine serum, 4.5g/L D-glucose, 10mM HEPES, 1mM sodium pyruvate, 1mM non-essential amino acid (NEAA) and 1% penicillin/streptomycin, the incubator was maintained at 37 ℃ and 5% CO₂The aeration environment is continuously conducted. Cells were harvested by trypsinization, washed and resuspended in fresh empty RPMI-1640 medium. C26 cells are inoculated on the dorsal subcutaneous part of the left hind limb femur of a nude mouse, and after 15 to 20 days of culture, solid tumor masses 100-150mm are formed³Thus establishing a nude mouse tumor model. The tumor volume is calculated as volume (width x width) x length)/2. Mice were randomized into 3 groups and intratumorally injected with 50 μ L GFP1, GFP2 and GFP 3. Protein fluorescence concentrations were adjusted to be consistent (fluorescence values of 3500, protein concentrations corresponding to GFP1, GFP2 and GFP3, 15, 9 and 7 μ M, respectively) prior to protein injection, and the fluorescence of proteins in tumors was tested using the IVIS luminea II in vivo imaging system (Caliper Life Sciences, USA) at given times (1,5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 2, 3, 4, 6, 8, 12 hours). Images were analyzed by Living Image 4.2 software.

As shown in FIG. 4a, GFP1 cleared rapidly from the tumor, with little detectable fluorescence after 2 hours, whereas GFP2 cleared more slowly, with fluorescence falling low until 4 hours. In contrast, the fluorescence of GFP3 was significantly prolonged, and fluorescence was detectable even after 6 hours. In particular, GFP2 (2.01X 10) in tumors 2h after protein injection¹⁰Photon) and GFP3 (2.67X 10)¹⁰Photons) were GFP1 (4.52X 10), respectively⁹Photon) 4.45 and 5.91 times, GFP 2(1.38 × 10) 4h after injection¹⁰Photon) and GFP3 (2.39X 10)¹⁰Photons) were 220 times and 381 times as much as GFP1, respectively. In summary, the above data show that the dimensional and thermal stability is increased due to the fluid mechanicsAdditionally, increasing the molecular weight of the GFP multimer significantly increased the tumor residence time and increased the stability of GFP within the tumor.

Example 5 biosynthesis and physicochemical Properties of IFN multimers

Genes encoding IFN monomer IFN1, dimer IFN2 and trimer IFN3 (containing 6 XHis tag) were synthesized by biological technology (Shanghai, China) and successfully cloned into pET-25b (+) vector. IFN multimer expression, purification, storage, LC-ESI-MS mass spectrometry, DLS analysis and all methods were consistent with those of GFP multimer in example 1. The sequence of the IFN protein is shown in SEQ ID No. 2.

CD analysis measures the secondary structure of the protein. IFN multimers were diluted to 0.12mg/mL with deionized water and analyzed by UV scanning at a wavelength range of 190-260nm using a Pistar π -180(Applied Photophysics, Inc., UK) with an optical path of 1 mm.

The invention constructs three recombinant plasmids capable of expressing interferon (IFN 1), dimer (IFN 2) and trimer (IFN 3) and successfully expresses the three recombinant plasmids in escherichia coli. After nickel column affinity purification, the target protein polymer with purity of 95% was successfully obtained by SDS-PAGE analysis (FIG. 5 a). LC-ESI-MS further confirmed that IFN1, IFN2 and IFN3 had molecular weights of 20092.0, 40930.0 and 60470.0Da, respectively, consistent with theoretical molecular weights of 20092.0, 40930.6 and 60470.0Da, respectively (FIG. 5 b). The above data indicate that the present invention successfully achieves high quality IFN monomers, dimers and trimers. Hydrodynamic radius (R) of IFN2 and IFN3 by DLS analysis_h) 4.61 and 6.86nm, respectively, 1.67 and 2.49 times larger than IFN 1(2.76nm), respectively (FIG. 5 c). By circular dichroism spectroscopy, the IFN multimer spectra all exhibited the same 209/219nm bimodal curve and overlapped well with the IFN1 curve, indicating that SEC modification had no significant effect on the secondary structure of the protein molecule (fig. 5 d).

Example 6 analysis of the biological Activity of IFN multimers

The biological activity of the IFN multimer of the invention is the determination of anti-cell proliferative activity by the MTT method. We chose human Burkitt's B lymphoma cells (Daudi B) because of the higher sensitivity of the cells to IFN-. alpha.2. DaudiAfter B cells were cultured in RMPI-1640 containing 15% FBS, 50U/mL penicillin and 50. mu.g/mL streptomycin for a certain period of time, a cell suspension (50. mu.L/well, 10. mu.L/well) was seeded in a 96-well plate at a certain concentration⁴Individual cells), IFN multimer samples were serially diluted (tested at the same mass concentration in a concentration gradient of 200000, 20000, 2000, 1000, 400, 200, 100, 40, 20, 10, 4pg/mL, tested at the same molar concentration in a concentration gradient of 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, 0.125, 0.0625pM), each 50. mu.L was added to a 96-well plate, and a negative control (no IFN) and a blank control (medium only) were set at 37 ℃, 5% CO₂Culturing for 72-96 h, adding 20 mu L/hole of MTT solution, measuring the absorption value of 490nm wavelength of each hole by using an enzyme-labeling instrument after 3h, and comparing the cell proliferation degree after different samples are treated. The IFN multimer IC50 is calculated as: IC50 (%) ═ 100% (sample uv absorbance-blank)/(negative control-blank).

As shown in FIGS. 6a and 6b, IC50 for IFN1, IFN2 and IFN3 was 20.82pg/mL, 29.36pg/mL and 49.98pg/mL, respectively, at the same mass concentration, with the retention of activity for IFN2 and IFN3 being 70.91% and 41.66%, respectively. As shown in figures 6c and 6d, at the same molarity, IC50 for IFN1, IFN2 and IFN3 was 1.19pM, 0.74pM and 0.52pM, respectively, with IFN2 and IFN3 having 1.61 and 2.29 times greater biological activity than IFN 1. The results show that after SEC modification, the activity increase caused by the fused IFN quantity is better than the activity reduction caused by steric hindrance, and the biological activity of the IFN polymer can be well preserved, thereby providing a basis for in vivo antitumor activity tests.

Example 7 pharmacokinetic testing of IFN multimers

In the invention, after the nude mouse is injected with the IFN polymer through tail vein, the change of the IFN concentration in blood along with time is measured, and the DAS software is used for data analysis. Prior to the drug treatment period, 9 female nude mice were randomly divided into 3 groups after a period of observation. Injecting IFN polymer into tail vein at 1mg/kg body weight, collecting 20 μ L (heparin sodium (product of Wolfram Biochemical medicine Co., Ltd.) of blood from tail vein at set time point (1,5,15,30min,1,3,6,24 and 48h), standing at room temperature for 1h, centrifuging at 4 deg.C and 3000 Xg to collect upper layer plasma, and storing in low temperature refrigerator at-80 deg.C. IFN-. alpha.2 content in serum was determined using a human IFN-. alpha.2 ELISA kit (PBL interferon source) according to the instructions. The pharmacokinetic parameters of the IFN multimers were calculated using DAS 3.0 pharmacokinetic analysis software.

Figure 7 and table 1 shows the IFN multimers in nude mice drug metabolism kinetics. IFN1 distribution half-life (t1/2 alpha) elimination half-life (t1/2 beta) is 0.411h and 1.26h respectively, 5 minutes after administration, the interferon concentration in blood rapidly drops to 50% of the initial dose, and the residual concentration after 24 hours is less than 0.02% of the initial concentration. IFN2 in vivo concentration is gradually reduced, its initial half-life and elimination half-life is 0.618h and 6.05h, respectively, is 1.50 times and 4.80 times of IFN 1. IFN3 in vivo concentration decrease the slowest, its initial half-life and elimination half-life is 0.854h and 13.3h, respectively, IFN1 2.08 times and 10.6 times. IFN2 and IFN3 drug time curve area (AUC0- ∞) is 0.474 u g/L x h and 1.36 u g/L x h, respectively, IFN 1(0.213 u g/L x h) 2.23 times and 6.38 times. The above results show that, compared with unmodified IFN1, the elimination half-life and in vivo average retention time of IFN polymer are obviously prolonged, the curve area is obviously increased during drug administration, and the clearance rate is obviously reduced.

TABLE 1

Example 8 in vivo antitumor Activity test of IFN multimers

In the present invention, the antitumor activity of IFN multimers in vivo was measured using nude mice transplanted with ovarian cancer cells. Human ovarian carcinoma cells (OVCAR-3) were cultured in RMPI-1640 medium containing 10% FBS, 50U/mL penicillin and 50. mu.g/mL streptomycin for a certain period of time, then detached by trypsinization, washed with PBS, and resuspended in 100. mu.L (5X 10) of empty RMPI-1640 medium without the above additives⁶Individual cell) is inoculated to the dorsal subcutaneous part of the left hind limb femur of a nude mouse, and 100-200mm is formed after 30 days of culture³Solid tumor masses of size. Nude mice were randomly divided into 4 groups, and physiological saline, IFN1, IFN2 and IFN3 were injected into nude mice by tail vein injection at a dose of 1mg/kg body weight. The injection was performed every three days until all of the mice in the saline group died. The death of the nude mice in this experiment included natural death and euthanasia, which means that the tumor growth of the nude mice exceeded 1000mm³Or weight loss of more than 15% by injection of barbiturate. The survival status and the tumor growth status of the nude mice were observed every week, and the changes of the nude mice body weight and the tumor volume with time were dynamically measured. To detect the toxicity of IFN multimers, mice were sacrificed after dosing, tumors, hearts, livers and kidneys were collected, fixed in 4% formaldehyde solution, sectioned and HE stained by standard methods to observe the histological morphology of organs.

As shown in FIGS. 8a and 8b, the experimental results showed that the tumor volume of the mice in the saline group rapidly increased during the experiment, and the tumor volume of the mice exceeded 1000mm on the 39 th day of injection³Median survival was only 36 days; the tumor volume of the IFN1 group nude mice gradually increased, and the tumor volume of the IFN1 injection day 42 group nude mice also exceeded 1000mm³The median survival time is 40.5 days, and no obvious antitumor activity is embodied; IFN2 tumor volume gradually increased, injection day 63 nude mice tumor volume also exceeded 1000mm³The median survival time is 58.5 days, and the compound has certain anti-tumor activity; while the tumor volume of the IFN3 group of nude mice slowly increased, 3 mice had their tumors disappeared, the remaining 5 mice had their tumors slowly increased until the tumor volume exceeded 1000mm after 87 days³Median survival was 85.5 days. The data show that the IFN polymer modified by SEC can effectively inhibit the growth of tumors, has very good in-vivo anti-tumor activity, and the anti-tumor effect is positively correlated with the serial number of IFN.

FIG. 8c is a histological section of tumor, heart, liver and kidney of mice after administration, and it can be seen that, after IFN multimer was injected, cavities appeared in the tumor cell gaps of the mice, cytoplasm and nuclear morphology were not obvious, cells were necrotic, large cell membranes were exfoliated, and the difference from the control group was obvious. Meanwhile, the cell morphology of the heart, the liver and the kidney is complete, no obvious cell necrosis exists, and no obvious difference exists between the cell morphology and the histological morphology of the control group. The above data indicate that the IFN multimers do not cause significant toxicity to the organs in nude mice.

The method has the advantages of high synthesis technology yield, simple purification, easy industrialization, high preservation rate of the bioactivity of the prepared protein polymer and obviously improved pharmaceutical properties. The SEC technique is expected to be a very effective new method for modifying protein drugs to improve drug stability, improve pharmacokinetics and enhance therapeutic efficacy.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing coordination hospital of Chinese academy of medical sciences

<120> self-fusion tandem protein modification method and application thereof

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Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys

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Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu

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Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg

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His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

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Claims (7)

1. A self-fusion tandem protein modification method is characterized in that genes encoding proteins are expressed in a protein expression system after being connected in series, and expressed polymer proteins are purified.

2. The modification method of claim 1, wherein the protein expression system includes, but is not limited to, prokaryotic expression vectors, yeast expression vectors, plant expression vectors, mammalian expression vectors, insect expression vectors.

3. The method of claim 1, wherein the protein is a fluorescent protein selected from the group consisting of green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein, and derivatives thereof.

4. The modification method according to claim 1, wherein the protein is an interferon selected from one or more of interferon alpha, interferon beta, interferon gamma, and interferon lambda.

5. The modification process of claim 1 wherein the protein is a therapeutic protein including but not limited to insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, Tumor Necrosis Factor (TNF) and enzymes.

6. The self-fusing tandem protein multimer prepared by the modification method of any one of claims 1-5.

7. Use of the self-fusing tandem protein multimer of claim 6 for the preparation of a protein drug that increases stability and extends half-life in vivo to improve therapeutic efficacy.

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