Protein polymer and production process thereof

Abstract

The present invention belongs to the field of biotechnology and discloses a protein polymer and its production process. The protein polymer is obtained by stimulating MSCs, followed by lysis, isolation, and purification. The protein polymer has strong oxidative damage repair capabilities, can reduce the extent of cerebral infarction, improve neurological function, inhibit neuroinflammation, and increase the number of surviving neurons.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a protein polymer and a production process thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, and urine. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and easy collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN), and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, and immune maturation. They can be used for immune regulation, tissue repair, and the treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0003] Mesenchymal stem cells (MSCs) cultured under different stimulation conditions can produce different stress proteins. These stress protein aggregates have complex physiological activities. How to use MSCs to produce protein aggregates with specific biological activities is a very challenging task. Summary of the Invention

[0004] The object of the present invention is to overcome at least one deficiency of the prior art and to provide a protein polymer and a production process thereof.

[0005] The technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides:

[0007] A protein polymer, the production process of which comprises:

[0008] S1) Cultivating mesenchymal stem cells and using ultraviolet irradiation to create a stress environment;

[0009] S2) lysing the mesenchymal stem cells, isolating and purifying to obtain protein polymers;

[0010] The protein polymer satisfies at least one of the following characteristics:

[0011] 1) During size exclusion chromatography, under the conditions of a flow rate of 0.1-0.3 ml / min and PBS as the eluent, the elution volume of the first component peak was 12-13.2 ml, the elution volume of the second component peak was 15.2-17 ml, the elution volume of the third component peak was 17-20 ml, the elution volume of the fourth component peak was 29-31 ml, and the elution volume of the fifth component peak was 31-34 ml;

[0012] 2) During SDS-PAGE, 4-20% precast gel was used for sample separation and detection. The sample bands were mainly distributed between 11KD and 100KD. Among them, the first band was located between 75KD and 100KD, and the second band was located between 63KD and 75KD.

[0013] 3) During reverse-phase HPLC detection, under the reverse-phase HPLC conditions of a sample loading volume of 60 to 80 μL, a column temperature of 25 to 40°C, a flow rate of 0.5 to 1 mL / min, a detection wavelength of 220 to 280 nm, mobile phase A consisting of a TFA aqueous solution, mobile phase B consisting of a TFA acetonitrile solution, and an elution time of 6 to 150 min, the peak elution time after sample separation was between 10 and 40 min, of which the peak elution time of characteristic peak 1 was 13 to 17 min, the peak elution time of the second group of sample components was 2 to 5 min, and the peak elution time of characteristic peak 2 was 20 to 22 min.

[0014] In some examples of protein polymers, they include at least the following proteins:

[0015] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0016] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0017] Preferably, the mass of the above two proteins accounts for more than 40% of the total mass of the protein polymer;

[0018] Preferably, it further comprises at least one of the following proteins:

[0019] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0020] sp|P62736|ACTA_HUMANActin,aortic smooth muscle OS=Homo sapiens;

[0021] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0022] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0023] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0024] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0025] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0026] sp|P21333|FLNA_HUMAN Filamin-AOS=Homo sapiens;

[0027] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0028] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0029] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0030] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0031] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0032] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens。

[0033] In some examples of protein polymers, the ultraviolet irradiation stimulation time for mesenchymal stem cells is 1 to 30 hours, preferably, the irradiation time is 10 to 30 hours, more preferably 6 to 18 hours;

[0034] Preferably, the intensity of ultraviolet irradiation stimulation is 10 to 100 μW / cm 2 , the wavelength of ultraviolet light is preferably 290 to 340 nm;

[0035] Preferably, the culture medium used during ultraviolet irradiation stimulation is a serum-free MSCs culture medium.

[0036] In some examples of protein aggregates, size exclusion chromatography procedures include:

[0037] After balancing the Superdex 150 molecular sieve 8×500 molecular sieve chromatography column, load the sample and elute with PBS. The elution rate is preferably 0.2-0.4 mL / min. Start collecting from the 280 nm UV absorbance value of 4 mAU, and collect five components with elution volumes of 12-13.2 ml, 15.2-17 ml, 17-20 ml, 29-31 ml, and 31-34 ml.

[0038] In some examples of protein polymers, the mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.

[0039] The above features can be combined arbitrarily unless they conflict with each other.

[0040] The second aspect of the present invention provides:

[0041] A production process for a protein polymer comprises the following steps: amplifying MSCs, irradiating MSCs in a culture medium with ultraviolet light, performing stress treatment, collecting the MSCs after stress treatment, performing lysis treatment, and separating and purifying proteins to obtain the protein polymer.

[0042] In some examples of production processes, the ultraviolet irradiation stimulation time of mesenchymal stem cells is 1 to 30 hours, preferably, the irradiation time is 10 to 30 hours, more preferably 6 to 18 hours;

[0043] Preferably, the intensity of ultraviolet irradiation stimulation is 10 to 100 μW / cm 2 , the wavelength of ultraviolet light is preferably 290 to 340 nm;

[0044] Preferably, the culture medium used during ultraviolet irradiation stimulation is a serum-free MSCs culture medium.

[0045] In some examples of production processes, molecular sieve exclusion chromatography is used to separate and purify the protein. Preferably, the operation of the molecular sieve exclusion chromatography includes:

[0046] After balancing the Superdex 150 molecular sieve 8×500 molecular sieve chromatography column, load the sample and elute with PBS. The elution rate is preferably 0.2-0.4 mL / min. Start collecting from the 280 nm UV absorbance value of 4 mAU, and collect five components with elution volumes of 12-13.2 ml, 15.2-17 ml, 17-20 ml, 29-31 ml, and 31-34 ml.

[0047] In some examples of production processes, HPLC-SEC separation and purification of proteins involves:

[0048] A reversed-phase liquid chromatography column was used, with mobile phase A consisting of 0.1% TFA in water and mobile phase B consisting of 0.075% TFA in 71.4% acetonitrile. The chromatographic conditions were:

[0049] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 72 28 1.0 75 0 100 1.0 81 0 100 1.0 135 72 28 1.0 145 72 28 1.0

[0050] Or the chromatographic conditions are:

[0051] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 72 28 1.0 75 0 100 1.0 81 0 100 1.0 81.1 72 28 1.0 90 72 28 1.0

[0052] Or the chromatographic conditions are:

[0053] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (ml / min) 0 65 35 1.0 3 65 35 1.0 13 50 50 1.0 27 43 57 1.0 51 20 80 1.0 53 0 100 1.0 53.1 65 35 1.0 60 65 35 1.0

[0054] The above features can be combined arbitrarily unless they conflict with each other.

[0055] The third aspect of the present invention provides:

[0056] The use of the protein polymer described in the first aspect of the present invention includes use in preparing a medicament for treating neurodegenerative diseases or stroke. Furthermore, the neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and various types of spinocerebellar ataxia (SCA).

[0057] The beneficial effects of the present invention are:

[0058] The protein polymers of some examples of the present invention have good cell damage repair effects and are expected to be used to treat neurodegenerative diseases and stroke. In particular, the neurodegenerative diseases include but are not limited to Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and different types of spinocerebellar ataxia (SCA).

[0059] The production processes of some examples of the present invention can effectively overcome the differences between different batches of MSCs, obtain more stable MSCs with small batch differences, and greatly ensure the quality and yield of protein polymers.

[0060] The production processes of some examples of the present invention can better ensure the activity of umbilical cord-derived MSCs and BMSCs, and are beneficial to increasing the initial yield of MSCs.

[0061] According to the production process of some embodiments of the present invention, the freezing and resuscitation rates of MSCs are high.

[0062] The production processes of some examples of the present invention can well separate and purify protein aggregates. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a photo of the growth status of MSCs after culture in Tangyi 3D culture medium in culture experiment 1-1.

[0064] Figure 2 This is a photograph of the cell status after 8 hours of ultraviolet irradiation in culture experiment 1-1.

[0065] Figure 3 This is the SDS-PAGE result of the protein harvested from culture experiment 1-1.

[0066] Figure 4 This is a photograph of the cell status of MSCs before ultraviolet irradiation in culture experiment 1-2.

[0067] Figure 5 This is a photograph of the cell status of MSCs in culture experiment 1-2 after 6 hours of weak-intensity ultraviolet irradiation.

[0068] Figure 6 This is the SDS-PAGE result of the proteins harvested from culture experiments 1-2.

[0069] Figure 7 This is the elution curve of Experiment 2.

[0070] Figure 8 This is the SDS-PAGE electrophoresis diagram of sample No. 12 (pure-12#) after purification in Experiment 3.

[0071] Figure 9 is the gradient screening result.

[0072] Figure 10 is the elution curve of HPLC-SEC.

[0073] Figure 11 and Figure 12 The effect of different treatments on the scope of cerebral infarction in rats.

[0074] Figure 13 The effects of different treatments on the neurological function of rats.

[0075] Figure 14The effects of intrathecal combined intravenous administration of aritol on neurons. DETAILED DESCRIPTION

[0076] The technical solution of the present invention is further illustrated below in conjunction with experimental examples.

[0077] Experiment 1: Effects of different treatments on protein expression:

[0078] 1-1 Culture Test 1

[0079] HUC-MSCs were cultured in 2 L of HK-G050 (PRF) 3D culture medium from Tangyi Huike Biotechnology, with a total cell count of approximately 5 × 10 8 The microcarriers were filled with cells. Figure 1 shown.

[0080] LED ultraviolet irradiation of cells, irradiation conditions: 60μW / cm 2 At 0 h, sample 8 ml and allow to settle. Filter the supernatant through a 0.22 μm filter and store at 4°C. Add 20 ml of pure water to swell the cells for 10 min, filter through a 0.22 μm filter, and store at 4°C. Samples were then collected from different bottles at 8, 12, 16, 18, 24, and 30 h to collect only intracellular protein for protein concentration measurement.

[0081] Taking UV irradiation for 8 hours as an example, the cell status after irradiation is as follows Figure 2 shown.

[0082] After irradiation, cells were harvested at different time points. The culture was filtered through a 300-mesh filter bag to retain the microcarriers. The supernatant was centrifuged at 1200 rpm for 6 minutes. The cells were stored at 4°C. The cell pellet was washed three times with 150 ml of normal saline, lysed with 14 ml of pure water, and filtered through a 0.22 μm filter membrane. The harvested intracellular proteins were frozen at -80°C. The harvested intracellular proteins were subjected to gel electrophoresis analysis, and the SDS-PAGE results were as follows: Figure 3 shown.

[0083] 1-2 Culture Experiment 2

[0084] Resuscitate a small crystal of P8 HUC-MSCs and place them in 9 T25 culture flasks. Add 2.5 ml of Huakai Mesenchymal Stem Cell Serum-Free Medium to each flask.

[0085] Four vials of cells were irradiated with two different intensities of 300nm LED UV light for 6, 12, 18, and 24 hours. The cells were carefully decanted, washed twice with 1ml of saline, and lysed by repeated pipetting in 660μl of pure water for 10 minutes. The cells were then filtered through a 0.22μm filter and stored at 4°C.

[0086] UV irradiation conditions are as follows:

[0087]

[0088] Cell status before UV irradiation Figure 4 As shown, taking weak intensity irradiation for 6 hours as an example, the cell state after irradiation is as follows Figure 5 As shown, UV irradiation affects cell morphology and creates stress for the cells, which in turn causes them to produce stress proteins under this stressful environment. The concentration and volume of the harvested proteins are shown in Table 1.

[0089] Table 1

[0090] Irradiation time (h) 6 12 18 24 Strong protein concentration (mg / ml) 0.670 0.155 0.165 0.082 Weak protein concentration (mg / ml) 0.718 0.343 0.215 0.165 Volume (ml) 500 μl 500 μl 500 μl 500 μl

[0091] The SDS-PAGE results of the harvested proteins are as follows Figure 6 shown.

[0092] From Table 1 and Figure 6 It can be seen that weak ultraviolet light can effectively promote the expression of target proteins, but it may require longer irradiation time.

[0093] Experiment 2: Molecular sieve purification and activity detection of protein aggregates

[0094] 2-1 Molecular sieve purification of protein aggregates

[0095] Instrument: AKTAexplorer

[0096] Chromatography column: Nanomicro Superdex 150 molecular sieve 8×500, column volume is about 30mL

[0097] Reagents: 0.1 M NaOH, 20% ethanol, 1× PBS, purified water

[0098] UV absorption wavelength: 280nm, 260nm as reference

[0099] Equilibrate the columns in the following order:

[0100] The chromatography column was first rinsed with 2CV of purified water, and then equilibrated with 1×PBS for 2CV, and the ultraviolet absorption value at 280nm was returned to zero.

[0101] Sample preparation: Take the sample from Experiment 1 that was irradiated with weak UV light for 6 hours, approximately 20 mL in volume. Use an ultrafiltration concentrator with a molecular weight cutoff of 3 kD to reduce the 20 mL sample to approximately 600 μL.

[0102] Experimental procedure: After balancing the column, select a 500 μL sample loop to load the sample, the sample flow rate is 0.4 mL / min, and the 1× PBS elution flow rate is 0.2 mL / min, eluting to the peak. Collect the protein starting from the UV absorbance value of 4 mAU. The elution curve is detailed in Figure 7 .Pick Figure 7The proteins at positions 2, 9, 12, 16, and 18 were freeze-dried and stored for activity detection.

[0103] 2-2 Bioactivity Detection of Protein Aggregates

[0104] Day 1: Cell plating: Complete medium (5% FBS + DMEM) was used to dilute PC12 poorly differentiated cells, and 6,000 cells were plated per well in a 96-well plate. The cells were incubated at 37° C. with 5% CO 2 overnight.

[0105] The next day: the lyophilized sample and the sample before purification were diluted with DMEM+5% FBS medium (about 800ug / ml).

[0106] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0107] Hydrogen peroxide treatment: discard 80 μL / well culture supernatant, add 50 μL / well diluted hydrogen peroxide to the corresponding cultured cells, and leave at room temperature for 25 minutes.

[0108] Untreated control: 50 μL / well of DMEM+5% FBS medium was added as the injury treatment control.

[0109] 50 μL / well of the diluted sample was added to the wells treated with hydrogen peroxide.

[0110] The supernatant of the untreated wells was discarded, and 100 μL / well of complete culture medium was added as a cell growth control (PC).

[0111] Incubate at 37°C for 2 days.

[0112] Day 5: Discard the culture supernatant and add 100 μL / well of two complete culture media. Set up a culture medium blank control well. Add 10 μL / well of CCK8 and incubate at 37℃ for 3.5 hours. OD 450 Read the value and calculate after deducting the culture medium blank.

[0113] The experimental results are shown in Table 2.

[0114] Table 2 Biological activity experimental data of different samples

[0115]

[0116]

[0117] As shown in Table 2, in the PC12 cell oxidative damage model, samples 2#, 12#, and 16# all have strong oxidative damage repair capabilities, and their repair capabilities are better than those of the unpurified sample. The repair ability of sample 18# is lower than that of the unpurified sample.

[0118] The purified 12# sample was subjected to SDS-PAGE electrophoresis analysis. The electrophoresis results were as follows: Figure 8 As shown in the figure, the sample bands are mainly distributed between 11KD and 100KD. Among them, the molecular weight is from large to small. The first band is between 75KD and 100KD; the second band is between 63KD and 75KD. Further analysis shows that the purified protein aggregates include the following proteins:

[0119] sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens;

[0120] sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens.

[0121] The above two proteins are the main proteins, accounting for more than 40% of the total mass of protein polymer. Other proteins include:

[0122] sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens;

[0123] sp|P62736|ACTA_HUMANActin,aortic smooth muscle OS=Homo sapiens;

[0124] sp|P01009|A1AT_HUMANAlpha-1-antitrypsin OS=Homo sapiens;

[0125] sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens;

[0126] sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens;

[0127] sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens;

[0128] sp|P09493|TPM1_HUMAN Tropomyosin alpha-1chain OS=Homo sapiens;

[0129] sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens;

[0130] sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1heavy chain OS=Homosapiens;

[0131] sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens;

[0132] sp|P01023|A2MG_HUMANAlpha-2-macroglobulin OS=Homo sapiens;

[0133] sp|P60709|ACTB_HUMANActin,cytoplasmic 1OS=Homo sapiens;

[0134] sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1OS=Homo sapiens;

[0135] sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

[0136] Experiment 3: HPLC-SEC purification and protein aggregation activity detection

[0137] Based on differences in hydrophobicity, reversed-phase chromatography can be used to separate the stimulated protein aggregates obtained in the present invention. Under initial conditions, the concentration of organic components in the mobile phase is low, resulting in a strong hydrophobic interaction between the protein aggregate and the stationary phase, leading to near-complete adsorption. When the organic components in the mobile phase reach a specific concentration, the protein aggregate is completely eluted from the stationary phase and no longer interacts with it. Therefore, even minimal changes in the organic components of the mobile phase can significantly affect the reversed-phase retention behavior of the protein aggregate.

[0138] Sample information: Protein polymer stock solution (ALEETO stock solution) prepared by the method of Experiment 1-1.

[0139] 3-1 Solution Preparation

[0140] Mobile phase A (0.1% TFA in water): Take 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix well and then ultrasonicate to obtain the mobile phase.

[0141] Mobile phase B (0.075% TFA in 71.4% acetonitrile): Take 286 ml of ultrapure water, add 714 ml of acetonitrile and 0.75 ml of trifluoroacetic acid, mix well and sonicate.

[0142] ALEETO sample: Weigh the ALEETO stock solution, dilute with PBS pH 7.2 buffer, and mix thoroughly to prepare a concentration of 1 mg / mL.

[0143] 3-2 Chromatographic conditions

[0144] Chromatographic conditions: For the sake of convenience, the chromatographic column used in chromatographic conditions 1 to 6 is XBridgeProtein BEH C4. 3.5μm, 4.6mm*150mm, column temperature 40℃, mobile phase A: 0.1% TFA in water; mobile phase B: 0.075% TFA in 71.4% acetonitrile, detector at 220nm.

[0145] Chromatographic conditions 1

[0146]

[0147] Chromatographic condition 2

[0148]

[0149] Chromatographic conditions 3

[0150]

[0151]

[0152] Chromatographic conditions 4

[0153]

[0154] Chromatographic conditions 5

[0155]

[0156] Chromatographic conditions 6

[0157]

[0158]

[0159] 3-3 Analysis Results

[0160] The results were as follows: Figure 9 As shown in Tables 3 and 4.

[0161] Table 3 Purity statistics of six gradient screening results

[0162] name Peak 1 purity (%) Peak 2 purity (%) Peak 3 purity (%) Peak 4 purity (%) Chromatographic conditions: 1-145 min-60 μg 0.05 3.28 95.64 1.04 Chromatographic conditions: 2-90min-60μg 0.04 3.18 95.65 1.14 Chromatographic conditions: 3-60min-80μg 0.04 3.23 95.89 0.84 Chromatographic conditions: 4-100min-60μg 0.04 3.45 95.20 1.31 Chromatographic conditions 5-105min-60μg 0.04 3.43 95.32 1.21 Chromatographic conditions 6-115min-60μg 0.04 3.32 95.47 1.17 RSD (%) N / A 3.3 0.3 N / A

[0163] Table 4 Peak area statistics of six gradient screening results

[0164]

[0165] from Figure 9 As shown in Tables 3 and 4, the peak areas and purities of the four parts of the sample peaks are not much different when comparing the six gradient screening results.

[0166] from Figure 9 As can be seen from Tables 3 and 4, the peak areas and purities of Peak 1 and Peak 4 are basically consistent, and the peak shapes are basically consistent. However, since these two parts account for a relatively low proportion of the overall purity, the RSD value is relatively large; the overall peak area and purity of Peak 2 are basically consistent, and the different elution gradient times lead to obvious differences in peak shapes. Overall, the peak shapes of chromatographic conditions 1 to 3 are acceptable, while the peak shapes of chromatographic conditions 4 to 6 are poor.

[0167] from Figure 9 As can be seen from Table 3 and Table 4, the overall peak area and purity of peak 3 are basically the same, and the different elution gradient times lead to obvious differences in peak shape. Overall, the peak shape separation of chromatographic conditions 2 and chromatographic conditions 4 to chromatographic conditions 6 is poor and unstable. The peak shape of chromatographic conditions 1 and chromatographic conditions 3 is acceptable, and the elution time of gradient screening 1 is greater than that of chromatographic conditions 3, so chromatographic conditions 3 are preferred.

[0168] 3-4 Activity Detection

[0169] Sample information: Protein polymer stock solution (ALEETO stock solution) prepared by the method of Experiment 1-1.

[0170] Mobile phase: PBS

[0171] Detection conditions: injection volume 100 μL, column temperature 25°C, flow rate 0.4 ml / min, wavelength 280 nm, 260 nm; time: 48 min, repeated loading twice.

[0172] The collection time range is approximately: 1#13.3~14.5min; 2#21.5~22min; 3#22~22.8min; 4#23~23.7min; 5#26.8~27.8min; 6#27.8~28.7min; 7#28.7~30min; 8#30~30.6min; 9#30.6~32 (no obvious peak of A280)min; 10#33.8~34.8min.

[0173] Elution curve Figure 10 As shown, the proteins at positions 1-10 shown in the figure were taken and freeze-dried for storage in preparation for activity detection.

[0174] Day 1: Cell plating: Complete culture medium (5% FBS + DMEM) was used to dilute PC12 poorly differentiated cells, and 6,000 cells were plated per well in a 96-well plate. The cells were incubated at 37° C. with 5% CO 2 overnight.

[0175] The next day: the lyophilized sample and the sample before purification were diluted with DMEM+5% FBS medium (about 800ug / ml).

[0176] Take 30% hydrogen peroxide and dilute it 15,000 times with DMEM + 5% FBS.

[0177] Hydrogen peroxide treatment: discard 80 μL / well culture supernatant, add 50 μL / well diluted hydrogen peroxide to the corresponding cultured cells, and leave at room temperature for 25 minutes.

[0178] Untreated control: 50 μL / well of DMEM+5% FBS medium was added as the injury treatment control.

[0179] 50 μL / well of the diluted sample was added to the wells treated with hydrogen peroxide.

[0180] The supernatant of the untreated wells was discarded, and 100 μL / well of complete culture medium was added as a cell growth control (PC).

[0181] Incubate at 37°C for 2 days.

[0182] Day 5: Discard the culture supernatant and add 100 μL / well of two complete culture media. Set up a culture medium blank control well. Add 10 μL / well of CCK8 and incubate at 37℃ for 3.5 hours. OD 450 Read the value and calculate after deducting the culture medium blank.

[0183] The biological activity results are shown in Table 5.

[0184] Table 5 Biological activity experimental data of different samples

[0185]

[0186] Table 5 shows that in the PC12 cell oxidative damage model, samples 1#, 2#, 7#, 8#, and 10# all have strong oxidative damage repair capabilities. Their repair capabilities are superior to those of the unpurified sample. Sample 5# has a lower repair capacity than the unpurified sample.

[0187] Experiment 4: Application of ALT in Stroke Treatment

[0188] Next, the protein polymer obtained in Example 1 of the present invention (also referred to as Aleeto or ALT) was used to further study its biological activity.

[0189] After 7 days of drug administration, TTC staining was used to observe the cerebral infarction of MCAO rats. The cerebral infarction range of rats in the 36μg / kg alitol intrathecal combined with intravenous administration group was significantly reduced (p<0.05), while the administration of butylphthalide and edaravone and dextroborneol failed to significantly reduce the cerebral infarction range ( Figure 11 and Figure 12 ).

[0190] The neurological function of the model mice was evaluated in a blinded manner before and after medication, and the results showed:

[0191] After 5-7 days of medication, the neurological function of rats in the alitol intrathecal combined with intravenous administration group was better than that of the control group, while the neurological function of rats in the butylphthalide and edaravone / dexamethasone administration groups had no significant difference from that of the control group ( Figure 13 ).

[0192] Immunofluorescence was used to detect neuroinflammation and neuronal markers in the rat brain. The results showed that intrathecal administration of alituo combined with intravenous administration could inhibit the level of neuroinflammation and increase the number of surviving neurons ( Figure 14 ).

[0193] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions that do not depart from the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A protein polymer, characterized in that Its production process includes: S1) Cultivate mesenchymal stem cells and create a stress environment using ultraviolet irradiation. The duration of ultraviolet irradiation stimulation of mesenchymal stem cells is 1 to 30 hours, and the intensity of ultraviolet irradiation stimulation is 10 to 60 μW / cm 2 , the wavelength of ultraviolet light is 290~340nm; S2) lysing the mesenchymal stem cells, isolating and purifying to obtain the protein polymer; When the protein polymer meets the requirements of SDS-PAGE detection, 4-20% precast gel is used for sample separation and detection. The sample bands are mainly distributed in the range of 11KD to 100KD, wherein the molecular weight is from large to small, the first band is located between 75KD and 100KD; the second band is located between 63KD and 75KD; 1) During size exclusion chromatography, at a flow rate of 0.2 ml / min and PBS as the eluent, the elution volume of the first component peak was 12-13.2 ml, the elution volume of the second component peak was 15.2-17 ml, the elution volume of the third component peak was 17-20 ml, the elution volume of the fourth component peak was 29-31 ml, and the elution volume of the fifth component peak was 31-34 ml; 2) During reversed-phase HPLC detection, under the conditions of a sample loading volume of 60-80 µL, a column temperature of 25-40°C, a flow rate of 0.5-1 mL / min, a detection wavelength of 220-280 nm, mobile phase A consisting of a TFA aqueous solution, mobile phase B consisting of a TFA acetonitrile solution, and an elution time of 6-150 min, the peak elution time after sample separation is between 10-40 min, wherein the peak elution time of characteristic peak 1 is 13-17 min, the peak elution time of the second group of sample components is 2-5 min, and the peak elution time of characteristic peak 2 is 20-22 min; the protein polymer comprises at least the following proteins: sp|P02768|ALBU_HUMAN Serum albumin OS=Homo sapiens; sp|P02787|TRFE_HUMAN Serotransferrin OS=Homo sapiens; The mass of the above two proteins accounts for more than 40% of the total mass of protein polymers.

2. The protein polymer according to claim 1, characterized in that Further comprising at least one of the following proteins: sp|P51884|LUM_HUMAN Lumican OS=Homo sapiens; sp|P62736|ACTA_HUMAN Actin, aortic smooth muscle OS=Homo sapiens; sp|P01009|A1AT_HUMAN Alpha-1-antitrypsin OS=Homo sapiens; sp|P07951|TPM2_HUMAN Tropomyosin beta chain OS=Homo sapiens; sp|P08670|VIME_HUMAN Vimentin OS=Homo sapiens; sp|P02751|FINC_HUMAN Fibronectin OS=Homo sapiens; sp|P09493|TPM1 _HUMAN Tropomyosin alpha-1 chain OS=Homo sapiens; sp|P21333|FLNA_HUMAN Filamin-A OS=Homo sapiens; sp|P0DOX5|IGG1_HUMAN Immunoglobulin gamma-1 heavy chain OS=Homo sapiens; sp|P24821|TENA_HUMAN Tenascin OS=Homo sapiens; sp|P01023|A2MG_HUMAN Alpha-2-macroglobulin OS=Homo sapiens; sp|P60709|ACTB_HUMAN Actin, cytoplasmic 1 OS=Homo sapiens; sp|P69891|HBG1_HUMAN Hemoglobin subunit gamma-1 OS=Homo sapiens; sp|P01024|C3 HUMAN Complement C3 OS=Homo sapiens.

3. The protein polymer according to claim 1, characterized in that The time for ultraviolet irradiation to stimulate mesenchymal stem cells is 10 to 30 hours.

4. The protein polymer according to claim 3, characterized in that The time for ultraviolet irradiation to stimulate mesenchymal stem cells is 6 to 18 hours.

5. The protein polymer according to claim 1, characterized in that The culture medium used during ultraviolet irradiation stimulation was serum-free MSCs culture medium.

6. The protein polymer according to claim 1, characterized in that The operation of molecular sieve exclusion chromatography includes: After balancing the superdex 150 molecular sieve 8×500 molecular sieve chromatography column, load the sample and elute with PBS at an elution rate of 0.2-0.4 mL / min. Start collecting from the 280 nm UV absorbance value of 4 mAU, and collect five components with elution volumes of 12-13.2 ml, 15.2-17 ml, 17-20 ml, 29-31 ml, and 31-34 ml.

7. The protein polymer according to any one of claims 1 to 6, characterized in that The mesenchymal stem cells are selected from umbilical cord-derived human mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and human placenta-derived mesenchymal stem cells.

8. A process for producing a protein polymer, comprising expanding MSCs, irradiating MSCs in a culture medium with ultraviolet light, subjecting them to stress treatment, collecting the stressed MSCs, lysing them, and separating and purifying the protein to obtain the protein polymer, wherein the protein separation and purification steps include: A reversed-phase liquid chromatography column was used, mobile phase A was 0.1% TFA in water, mobile phase B was 0.075% TFA in 71.4% acetonitrile, and the chromatographic conditions were: ， Or the chromatographic conditions are: ， Or the chromatographic conditions are: ， The ultraviolet irradiation stimulation time of mesenchymal stem cells is 1 to 30 hours, and the intensity of ultraviolet irradiation stimulation is 10 to 60 μW / cm 2 The wavelength of ultraviolet light is 290 to 340 nm.

9. The production process according to claim 8, characterized in that: The time for ultraviolet irradiation to stimulate mesenchymal stem cells is 10 to 30 hours.

10. The production process according to claim 9, characterized in that: The time for ultraviolet irradiation to stimulate mesenchymal stem cells is 6 to 18 hours.

11. The production process according to any one of claims 8 to 10, characterized in that: The culture medium used during ultraviolet irradiation stimulation was serum-free MSCs culture medium.

12. Use of the protein polymer according to any one of claims 1 to 7, wherein the use is selected from the group consisting of preparation of drugs for treating neurodegenerative diseases and stroke.

13. The use according to claim 12, characterized in that The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and different types of spinocerebellar ataxia.

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