Preparation method of recombinant protein for improving oxidative stress of retina

Abstract

The invention provides a preparation method of a recombinant protein for improving retinal oxidative stress. The recombinant protein comprises an immunoglobulin Fc region and a functional structure domain, and the amino acid sequence of the functional structure domain is as shown in SEQ ID NO. 1. The preparation method comprises the following steps: connecting polynucleotide for coding the recombinant protein to an expression vector to construct a recombinant expression vector; and transferring the recombinant expression vector into an expression cell, and carrying out induced expression, separation and purification to obtain the recombinant protein. According to the preparation method, the recombinant protein with high soluble expression and enhanced stability can be obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine. Specifically, the present application relates to a preparation method of a recombinant protein for improving retinal oxidative stress. BACKGROUND

[0002] Glaucoma and Retinitis pigmentosa (RP) are the major retinal degenerative diseases leading to irreversible vision loss worldwide. Despite different initial causes, both of them share the common pathological endpoint of progressive apoptosis of retinal neurons (such as photoreceptor cells and retinal ganglion cells RGCs). Studies have shown that oxidative stress plays a key role in this process, which is an important hub connecting various pathogenic mechanisms and ultimately leading to cell death. However, current clinical treatment methods mainly target the disease symptoms (such as reducing intraocular pressure), and lack of therapies that can effectively resist oxidative stress and fundamentally block neuronal apoptosis.

[0003] The core pathological mechanism of glaucoma involves RGC apoptosis induced by factors such as high intraocular pressure. Studies have confirmed that mechanical compression and ischemia-hypoxia of the optic nerve during the course of glaucoma can lead to uncoupling of mitochondrial oxidative phosphorylation, producing excess ROS, causing oxidative damage to RGCs and their axons. Persistent oxidative stress not only directly damages cells, but also exacerbates inflammation, forming a vicious cycle. Current treatment strategies centered on controlling intraocular pressure cannot reverse the oxidative damage and RGC loss that has already occurred. The decrease in the expression of Brn3a, a specific nuclear transcription factor for RGC health, is closely related to the increase in oxidative stress level.

[0004] In RP, PDE6B gene mutation is an important cause. This mutation leads to impaired cGMP hydrolysis in photoreceptor cells, causing abnormal accumulation of cGMP and calcium ions. Calcium overload induces mitochondrial dysfunction, producing a burst of reactive oxygen species (ROS), triggering severe oxidative stress, and ultimately activating the apoptosis pathway. Existing treatment strategies for PDE6B mutant RP, such as gene therapy, small molecule drugs or protein replacement therapy, all have difficulty overcoming delivery barriers or protein function defects, and none of them directly address the oxidative stress damage derived therefrom.

[0005] Macromolecular biological agents such as neurotrophic factors have neuroprotective potential, but they have limitations such as large molecular weight, difficulty in penetrating the blood-retinal barrier (BRB), poor in vivo stability, and inability to precisely respond to oxidative stress microenvironment. Therefore, there is an urgent need in the art to develop a new type of small-sized protein molecule with excellent antioxidant activity, good stability, and the ability to target delivery to the retina, to neutralize ROS and block the oxidative stress pathway from the source, and to provide a fundamental treatment strategy for related diseases with both neuroprotection and antioxidant repair effects. SUMMARY

[0006] In view of the problems in the prior art, the inventors of the present application have made an in-depth study on the protein sequence in view of the problems that the natural PDE6B (UniProt P35913) needs to rely on the activation of regulatory subunit, the intracellular activity is limited, and the full-length PDE protein is difficult to express and easy to degrade, and by deleting the N-terminal regulatory domain and introducing a stability enhancement module, a recombinant protein for improving retinal oxidative stress is provided, and a preparation method of the recombinant protein is provided.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions: In a first aspect, the present application provides a preparation method of a recombinant protein for improving retinal oxidative stress, comprising the following steps: Step 1, linking a polynucleotide encoding the recombinant protein to an expression vector to construct a recombinant expression vector; Step 2, transforming the recombinant expression vector into an expression cell, inducing expression and separating and purifying to obtain the recombinant protein.

[0008] According to some embodiments of the present application, the recombinant protein comprises an immunoglobulin Fc region and a functional domain, the amino terminus of the functional domain is connected to the carboxy terminus of the immunoglobulin Fc region; wherein the amino acid sequence of the functional domain is selected from: (i) the amino acid sequence as shown in SEQ ID NO. 1; (ii) an amino acid sequence having one or more, for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions compared to the amino acid sequence shown in SEQ ID NO. 1; (iii) an amino acid sequence chemically modified from the amino acid sequence shown in SEQ ID NO. 1; or (iv) an amino acid sequence having at least 90% homology, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher homology with the amino acid sequence shown in SEQ ID NO. 1.

[0009] In the present application, the amino acid sequence shown in SEQ ID NO. 1 is as follows: APCRKSCFRAVRAPPPYDMQDGDMRPEIWIAQELRRIGDEFNGSTPPTMSERPTEDEELDTSKALRQRQIRGFSDS According to some embodiments of the application, the amino acid sequence of the immunoglobulin Fc region is selected from the group consisting of: (i) the amino acid sequence as shown in SEQ ID NO. 2; (ii) an amino acid sequence having one or more, e.g. 1, 2, 3, 4 or 5, substitutions, deletions, or additions compared to the amino acid sequence as shown in SEQ ID NO. 2; (iii) an amino acid sequence which is chemically modified of the amino acid sequence as shown in SEQ ID NO. 2; or (iv) an amino acid sequence having at least 90% homology, e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more homology to the amino acid sequence as shown in SEQ ID NO. 2.

[0010] In the present application, the amino acid sequence as shown in SEQ ID NO. 2 is as follows: EAAAKRRKTYFRSDSLPDLQKVVEKLRGQNLLVHECLFEVGEAYLVMELVKGGDLFDAITSTSLTKTERRIMDDFEACLRLHDSLHPGPGPLHRIMDDFEACFYKLDTLLKSVRSLPTFQKLLHQLFHGHRMSLIDLGLAFKK According to some embodiments of the application, the amino terminus of the functional domain is connected to the carboxy terminus of the immunoglobulin Fc region via a linker.

[0011] Preferably, the amino acid sequence of the linker consists of 1 to 3 amino acid sequences as shown in SEQ ID NO. 3.

[0012] More preferably, the amino acid sequence of the linker is as shown in SEQ ID NO. 3.

[0013] In the present application, the amino acid sequence as shown in SEQ ID NO. 3 is GGGGS.

[0014] According to some embodiments of the application, the amino terminus of the immunoglobulin Fc region is further connected, in this order, to a hinge region and a signal peptide.

[0015] According to a preferred embodiment of the application, the amino acid sequence of the hinge region comprises or is as shown in SEQ ID NO. 4.

[0016] In the present application, the amino acid sequence set forth in SEQ ID NO. 4 is as follows: VQLTISPKTGVVREKIGADIVLVNDKLGEGGFGIVYLCNPRSESNTPPT According to another preferred embodiment of the present application, the amino acid sequence of the signal peptide comprises SEQ ID NO. 5 or is as set forth in SEQ ID NO. 5.

[0017] In the present application, the amino acid sequence set forth in SEQ ID NO. 5 is as follows: MEPRLLLALLLGSTPAVGS According to some embodiments of the present application, the carboxy terminus of the functional domain is further linked to a sIgA mimic tail.

[0018] Preferably, the amino acid sequence of the sIgA mimic tail comprises SEQ ID NO. 6 or is as set forth in SEQ ID NO. 6.

[0019] In the present application, the amino acid sequence set forth in SEQ ID NO. 6 is SCLLL.

[0020] According to one preferred embodiment of the present application, the amino acid sequence of the recombinant protein is selected from: (i) the amino acid sequence as set forth in SEQ ID NO. 7; (ii) an amino acid sequence having one or more, e.g. 1, 2, 3, 4 or 5, substitutions, deletions, or additions compared to the amino acid sequence as set forth in SEQ ID NO. 7; (iii) an amino acid sequence which is chemically modified from the amino acid sequence as set forth in SEQ ID NO. 7; or (iv) an amino acid sequence which has at least 90% homology, e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more, to the amino acid sequence as set forth in SEQ ID NO. 7.

[0021] According to one particularly preferred embodiment of the present application, the amino acid sequence of the recombinant protein is as set forth in SEQ ID NO. 7.

[0022] In the present application, the amino acid sequence set forth in SEQ ID NO. 7 is as follows: MEPRLLLALLLGSTPAVGSVQLTISPKTGVVREKIGADIVLVNDKLGEGGFGIVYLCNPRSESNTPPTEAAAKRRKTYFRSDSLPDLQKVVEKLRGQNLLVHECLFEVGEAYLVMELVKGGDLFDAITSTSLTKTERRIMDDFEACLRLHDSLHPGPGPLHRIMDDFEACFYKLDTLLKSVRSLPTFQKLLHQLFHGHRMSLIDLGLAFKKGGGGSAPCRKSCFRAVRAPPPYDMQDGDMRPEIWIAQELRRIGDEFNGSTPPTMSERPTEDEELDTSKALRQRQIRGFSDSSCLLL The amino acid sequence as shown in SEQ ID NO. 7 is a particularly preferred recombinant protein obtained by the inventors through in-depth research. The functions of each region in the recombinant protein are shown in Table 1.

[0023]

[0024] It should be noted that, in order to efficiently and massively produce the recombinant protein for improving retinal oxidative stress of the present application, a secretion signal peptide such as KLAT (SEQ ID NO. 12) or KLAAT (SEQ ID NO. 13) can be connected to the N-terminus thereof. In addition, in order to be able to rapidly, efficiently and at low cost purify the recombinant protein of the present application, a "6xHis tag" (HHHHHH, SEQ ID NO. 14) can be added to the C-terminus thereof. Of course, after purification, the "6xHis tag" can be retained or removed as needed.

[0025] According to some embodiments of the present application, in step 1, the two ends of the polynucleotide respectively comprise a HindIII restriction site and a BamHI restriction site.

[0026] Preferably, the step 1 further comprises the following steps: Step 101, using HindIII endonuclease and BamHI endonuclease to respectively digest the polynucleotide and the expression vector; Step 102, connecting the digested polynucleotide and the expression vector to obtain a ligation product; Step 103, transforming the ligation product into a host cell, and then culturing on a culture medium containing ampicillin; Step 104, screening positive clones, thereby obtaining a recombinant expression vector.

[0027] According to some embodiments of the application, the nucleotide sequence of the polynucleotide comprises SEQ ID NO: 8 or as set forth in SEQ ID NO: 8.

[0028] SEQ ID NO: 8: atggagcccagactgctgctggccctgctgctgggctccacccccgccgtgggcagcgtgcagctgaccatcagccccaagaccggcgtggtgagagagaagatcggcgccgacatcgtgctggtgaacgacaagctgggcgagggcggcttcggcatcgtgtacctgtgcaaccccaggagcgagagcaacaccccccccactgaggccgccgccaagcggagaaagacctacttcagatccgactccctgcccgacctgcagaaggtggtggagaagctgaggggccagaacctgctggtgcacgagtgtctgttcgaggtgggcgaggcctacctcgtgatggagctggtgaagggcggggacctgtttgacgccatcacctccaccagcctgaccaagaccgagcggcggatcatggacgacttcgaggcctgcctgaggctgcacgactccctgcaccccggccccggccccctgcaccggatcatggacgacttcgaggcctgcttctacaagctggataccctgctgaagagcgtgcggagcctgcccaccttccagaagctgctgcatcagctgtttcatggacacagaatgagcctgatcgacctgggcctggcgtttaagaagggaggcggcggtagtgctccttgtaggaaatcttgttttcgggccgtgagagctcctcccccctatgatatgcaggatggcgatatgagacctgagatatggatcgctcaggaattgagaagaattggagatgagtttaatggcagcacacctcctacaatgagcgaaagacctactgaggatgaggagctggatactagcaaggccctgagacagagacagattagaggctttagtgattcttcttgcctgctgctg According to some embodiments of the application, the nucleotide sequence of the polynucleotide comprises SEQ ID NO: 15 or is as set forth in SEQ ID NO: 15.

[0029] SEQ ID NO: 15: aagcttgccaccatggagcccagactgctgctggccctgctgctgggctccacccccgccgtgggcagcgtgcagctgaccatcagccccaagaccggcgtggtgagagagaagatcggcgccgacatcgtgctggtgaacgacaagctgggcgagggcggcttcggcatcgtgtacctgtgcaaccccaggagcgagagcaacaccccccccactgaggccgccgccaagcggagaaagacctacttcagatccgactccctgcccgacctgcagaaggtggtggagaagctgaggggccagaacctgctggtgcacgagtgtctgttcgaggtgggcgaggcctacctcgtgatggagctggtgaagggcggggacctgtttgacgccatcacctccaccagcctgaccaagaccgagcggcggatcatggacgacttcgaggcctgcctgaggctgcacgactccctgcaccccggccccggccccctgcaccggatcatggacgacttcgaggcctgcttctacaagctggataccctgctgaagagcgtgcggagcctgcccaccttccagaagctgctgcatcagctgtttcatggacacagaatgagcctgatcgacctgggcctggcgtttaagaagggaggcggcggtagtgctccttgtaggaaatcttgttttcgggccgtgagagctcctcccccctatgatatgcaggatggcgatatgagacctgagatatggatcgctcaggaattgagaagaattggagatgagtttaatggcagcacacctcctacaatgagcgaaagacctactgaggatgaggagctggatactagcaaggccctgagacagagacagattagaggctttagtgattcttcttgcctgctgctgcatcatcaccaccaccactaaggatcc According to some embodiments of the application, the expression vector is a eukaryotic expression vector.

[0030] Preferably, the expression vector is pcDNA3.1 vector.

[0031] According to some embodiments of the present application, the host cell is a prokaryotic host cell.

[0032] Preferably, the host cell is E. coli, for example, E. coli TOP10.

[0033] According to some embodiments of the present application, in step 2, the expression cell is a mammalian cell line.

[0034] Preferably, the expression cell is Chinese hamster ovary cell (CHO).

[0035] According to some embodiments of the present application, in step 2, the separation and purification comprises: ultrasonic disruption of the expression cell; removing cell debris by centrifugation, collecting supernatant; chromatography of the supernatant.

[0036] Preferably, the chromatography comprises, in sequence, affinity chromatography, ion exchange chromatography and molecular sieve chromatography.

[0037] From the above technical solutions, the technical solutions of the present application have at least the following beneficial effects: The present application first obtains a recombinant protein with independent catalytic domain and autonomous activity by deleting the N-terminal regulatory domain of PDE protein and introducing a stability-enhancing module. The recombinant protein of the present application has the advantage of high soluble expression, and its targeting is optimized and can pass through the blood-retinal barrier. In addition, the half-life of the recombinant protein of the present application is prolonged, which can reduce the frequency of administration. Moreover, the stability of the recombinant protein of the present application is enhanced, which can resist enzymatic hydrolysis and denaturation.

[0038] Through experimental verification, the preparation method of the present application can simply, quickly and efficiently prepare the recombinant protein of the present application, which has correct molecular weight, is safe, non-toxic and high-purity.

[0039] In addition, cell experiments verify that the recombinant protein of the present application can significantly reverse the retinal cell damage caused by oxidative stress, showing strong protective or therapeutic effect. Animal experiments using RD mice verify that the recombinant protein of the present application can effectively inhibit the loss of photoreceptor cells caused by light damage, protect the retinal morphology, increase the thickness of the retinal photoreceptor cell layer, play an anti-damage / protective role, and can significantly improve the visual function of the model mice. In addition, animal experiments using DBA / 2J glaucoma mice verify the preventive and therapeutic effects of the recombinant protein of the present application on glaucoma and the mechanism of action from the molecular level, the retinal cell ultrastructure level and the tissue level. BRIEF DESCRIPTION OF DRAWINGS

[0040] Embodiments of the present application will be described in detail below with reference to the drawings, in which: Figure 1 Restriction enzyme identification results of the recombinant plasmid constructed in Example 1 of the present application are shown, wherein lane 1: original plasmid, lane 2: product after double enzyme digestion of the recombinant plasmid with HindIII / BamHI, lane 3: marker.

[0041] Figure 2 A map of the recombinant plasmid constructed in Example 1 of the present application is shown.

[0042] Figure 3 SDS-PAGE detection results of the GCNM01 protein expressed in Example 1 of the present application are shown.

[0043] Figure 4 Cell viability of different experimental groups of cells in Example 2 of the present application is shown, wherein, p < 0.01, ns: not significant.

[0044] Figure 5 The drug administration process of preventive intervention in Example 3 of the present application is shown.

[0045] Figure 6 HE staining results of Example 3 of the present application are shown.

[0046] Figure 7 Photoreceptor cell layer thickness results of Example 3 of the present application are shown. Compared with the control group: p <0.01; compared with the control group: p <0.01; compared with the model group: # p <0.05.

[0047] Figure 8 A comparison chart of ERG waveforms of mice in each group in Example 3 of the present application is shown.

[0048] Figure 9 WB detection results of Brn3a protein expression in Example 4 of the present application are shown, wherein A: model group, B: blank control group, C: positive drug control group, D: drug group. p <0.01, ## p <0.01 p <0.05.

[0049] Figure 10 Retinal cell ultrastructure of the model group in Example 4 of the present application is shown.

[0050] Figure 11 The retinal cell ultrastructure of the blank control group in Example 4 of the present application is shown.

[0051] Figure 12 The retinal cell ultrastructure of the positive drug control group in Example 4 of the present application is shown.

[0052] Figure 13 The retinal cell ultrastructure of the drug group in Example 4 of the present application is shown.

[0053] Figure 14 The HE staining results in Example 4 of the present application are shown.

[0054] Figure 15 The retinal ganglion cell number results after HE staining in Example 4 of the present application are shown. Compared with the model group: p < 0.01; compared with the model group: ## p < 0.01; compared with the model group p < 0.05. DETAILED DESCRIPTION

[0055] The present application will be further described in detail below in conjunction with specific embodiments, and the given examples are only for illustrating the present application, but not for limiting the scope of the present application.

[0056] Example 1: Preparation and confirmation of GCNM01 protein

[0057] 1. Construction of expression plasmid

[0058] The construction of the expression plasmid was completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0059] Specifically, Shengong Bioengineering (Shanghai) Co., Ltd. synthesized the optimized gene sequence (SEQ ID NO. 15) encoding the GCNM01 protein, and constructed the pcDNA3.1-GCNM01 recombinant plasmid.

[0060] The amino acid sequence of the GCNM01 protein encoded by SEQ ID NO. 15 is shown in SEQ ID NO. 16: KLATMEPRLLLALLLGSTPAVGSVQLTISPKTGVVREKIGADIVLVNDKLGEGGFGIVYLCNPRSESNTPPTEAAAKRRKTYFRSDSLPDLQKVVEKLRGQNLLVHECLFEVGEAYLVMELVKGGDLFDAITSTSLTKTERRIMDDFEACLRLHDSLHPGPGPLHRIMDDFEACFYKLDTLLKSVRSLPTFQKLLHQLFHGHRMSLIDLGLAFKKGGGGSAPCRKSCFRAVRAPPPYDMQDGDMRPEIWIAQELRRIGDEFNGSTPPTMSERPTEDEELDTSKALRQRQIRGFSDSSCLLLHHHHHH GS The basic information of the plasmid is as follows: (1) Host cell: E. coli TOP10; (2) Enzyme cutting site: HindIII / BamHI; (3) Resistance: ampicillin (Ampicillin); (4) Culture temperature: 37°C; (5) Length: 930; (6) Vector: pcDNA3.1 (+).

[0061]

[0062] The detection results show that the recombinant plasmid pcDNA3.1-GCNM01 is successfully constructed, and the map thereof is shown in FIG. 1. Figure 2

[0063] 2. Expression and purification of GCNM01 protein in CHO-S cells

[0064] (1) Expression: The pcDNA3.1-GCNM01 plasmid was transfected into CHO-S suspension cells by using electroporation method. Batch fed culture was carried out at 37°C and 5% CO2 for 7 days. The cell culture supernatant was collected by centrifugation.

[0065] (2) Affinity chromatography: After filtering the supernatant, it was loaded into a pre-equilibrated Ni Sepharose Excel affinity chromatography column. The target protein was eluted with a buffer containing 250 mM imidazole after washing with a buffer containing 20 mM imidazole.

[0066] ​(3) Ion exchange chromatography: The affinity chromatography eluate was replaced with 20 mM Tris-HCl, pH 8.0 buffer solution by ultrafiltration tube. It was loaded into Q Sepharose FF anion exchange column and eluted with NaCl linear gradient, and the main peak was collected.

[0067] (4) Molecular sieve chromatography: The sample after ion exchange purification was concentrated and loaded into HiLoad 16 / 600 Superdex75 pg chromatography column, and eluted with PBS buffer, and the GCNM01 monomer peak was collected.

[0068] (5) QC detection.

[0069] The protein concentration was determined to be 0.2 mg / mL using BCA method (BCA protein quantification kit, Thermo, item number 23225).

[0070] The results of SDS-PAGE detection are shown in Figure 3 , which shows that the results are a single band and the molecular weight is correct.

[0071] The endotoxin concentration was detected to be <0.1 EU / μg using chromogenic method (endotoxin detection kit, Biyun Tian).

[0072] Example 2: In vitro evaluation of the prevention and treatment efficacy of GCNM01 protein on oxidative stress damage

[0073] SHSY5Y cells (human neuroblastoma cell line) damaged by hydrogen peroxide (H2O2) are the most classic and commonly used in vitro oxidative stress damage model in neuroscience research. This embodiment evaluates the prevention and treatment efficacy of GCNM01 protein on oxidative stress damage in vitro with the aid of this cell model.

[0074] (I) Experimental materials

[0075] (II) Experimental scheme and results

[0076] The experimental process of this embodiment is as follows: 1. Cell plating For SHSY5Y adherent cells, wash the cells with PBS for 2 times, add 0.25% trypsin, digest at 37°C for 1-2 min, and when the cells are about to fall off, add DMEM / F12 basic medium to terminate digestion, and then transfer the cells to a 15 ml or 50 ml centrifuge tube after blowing several times, and centrifuge at 1000 rpm at room temperature for 3 min.

[0077] Discard the supernatant, resuspend the cell pellet with an appropriate amount of DMEM / F12 basic medium, mix well, and count.

[0078] According to the counting result, dilute the cells with PBS to adjust the cell density to 1×10 5 cells / ml, and inoculate the cells in a 96-well plate at a density of 1×10 4 cells / well, i.e., 100 μl / well.

[0079] 2. Cell modeling and drug administration

[0080] After the cells are inoculated in the plate for 24 h, three groups are set up: a normal group, a model group, and a drug group. The model group and the drug group are damaged by 250 μM H2O2 for 30 min, and then the supernatant is discarded. The drug group is administered with GCNM01 protein at two concentrations of 100 ng / ml and 10 ng / ml, 4 replicates for each concentration, 100 μl per well. The model group and the normal group are treated with DMEM / F12 basic medium, and continue to be cultured for 72 h. The cell viability is detected by CellTiter-glo luminescence method.

[0081] Note: 250 μM H2O2 needs to be prepared on site, which is diluted from a mother liquor of 30% H2O2 (about 10 M) by DMEM / F12 basic medium.

[0082] 3. Cell viability detection

[0083] The cell viability is detected by the Biyun Tian CellTiter-Lumi™ II cell activity detection kit by chemiluminescence method at a wavelength of 590 nm. The results are shown in Table 2 and Figure 4 .

[0084]

[0085] The results of the cell experiment show that the recombinant protein of the application can significantly reverse the decrease in cell viability caused by oxidative stress, and exhibits a strong protective or therapeutic effect.

[0086] Example 3: Efficacy evaluation of GCNM01 protein on retinitis pigmentosa (RP)

[0087] RD mice (i.e., retinal degeneration mice) are mice carrying a mutation in the Pde6b gene rd1, which is a classic, spontaneous, and rapidly degenerative animal model for studying retinitis pigmentosa (RP). In this embodiment, the prevention and treatment effects of GCNM01 protein on RP, as well as the delivery efficiency and bioavailability of the protein, are studied by intraperitoneal injection of GCNM01 protein into RD mice and visual electrophysiology and HE staining.

[0088] (I) Experimental scheme

[0089] 1. Preventive intervention

[0090] The 14-day-old RD10 mice were randomly divided into a model group and a prevention group, 10 mice in each group. Among them, the protein solution prepared by PBS and GCNM01 protein was injected into the abdominal cavity of the mice in the prevention group at 18 μg GCNM01 protein / g mouse / day. In addition, 14-day-old C57BL / 6J mice (10 mice) were used as wild type control group, and 0.1 mL / day of PBS was injected into the abdominal cavity of the mice in the model group and the wild type control group. When the mice in each group were 28 days old, the administration was ended, and HE staining was performed. The specific process is shown in Figure 5 .

[0091] 2. Therapeutic intervention

[0092] The 1-month-old RD10 mice were randomly divided into a model group and a treatment group, 10 mice in each group. Among them, the protein solution prepared by PBS and GCNM01 protein was injected into the abdominal cavity of the mice in the treatment group at 18 μg GCNM01 protein each time. C57BL / 6J mice (10 mice) were used as wild type control group, and 0.15 mL / time of PBS was injected into the abdominal cavity of the mice in the wild type control group and the model group, 2 times a week, for a total of 4 weeks. When the mice in each group were 60 days old, the administration was ended, and HE staining was performed.

[0093] 3. Visual electrophysiological detection

[0094] Another batch of RD10 mice and wild type mice were administered according to the scheme in “1. Preventive intervention”, and when the mice in each group were 28 days old, the administration was ended, and visual electrophysiological detection was performed.

[0095] (II) Index detection

[0096] (1) Visual electrophysiology

[0097] The equipment used was Phoenix Micron IV Ganzfeld ERG (Phoenix Research Labs, USA, model Phoenix Micron IV). After the administration was ended, the mice were moved to a dark room for dark adaptation for more than 8 hours, and then the visual function of the mice was detected under weak red light in the dark room. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.1 ml / 10g), and the pupil was fully dilated using compound tropicamide eye drops after satisfactory anesthesia. The mouse was placed on a constant temperature mouse table, and the right eyeball was fully exposed to the light source. A drop of normal saline was dropped on the cornea of the mouse, a metal electrode (black) was attached to the center of the cornea of the mouse, a reference electrode (red) was attached to the subcutaneous position between the two ears of the mouse, and a ground electrode (green) was attached to the tail. After starting the software, the dark response 1×10 -2 (cd·s) / m2 Intensity of light stimulus, scotopic response 1×100 (cd·s) / m 2 Intensity of light stimulation and scotoma should be 1×10 3 (cd·s) / m 2 Intensity of light stimulation and light adaptation 1×10 3 (cd·s) / m 2 Electroretinogram waveforms under high intensity stimulation light, with each intensity interval of 10s, comparing the magnitudes of wave a and wave b.

[0098] (2) HE staining

[0099] Under anesthesia, both eyeballs of mice were gouged out. After gouging out the left eyeball, it was placed in an EP tube containing ocular fixative. After fixation for 24 hours, the specimen was removed from the fixative and subjected to gradient dehydration by sequentially placing it in 75%, 85%, and 95% alcohol solutions, each lasting 15 minutes. Subsequently, the specimen was immersed in xylene I and xylene II solutions for 15 minutes each. Finally, the specimen was placed in paraffin I and paraffin II at 56°C for 1 hour each. Then, it was embedded and sectioned. The section thickness was approximately 5 μm. After dewaxing, the paraffin sections were stained in hematoxylin solution, then differentiated with differentiation solution, and then blued with lithium carbonate solution. After dehydration, it was stained in an eosin staining tank for 2 minutes. The sections were mounted with neutral resin. The samples were examined under a microscope, and images were acquired.

[0100] (III) Test Results

[0101] 1. Preventive intervention

[0102] Results of HE staining of the retina in each group are as follows Figure 6 As shown, the thickness of the retinal photoreceptor cell layer is as follows: Figure 7 As shown in the figure. Compared with the control group, the thickness of the retinal photoreceptor cell layer in the model group was significantly reduced, and the difference was statistically significant (P<0.01). Compared with the model group, the thickness of the retinal photoreceptor cell layer in the drug group (i.e., the prevention group in the above experimental protocol) was significantly increased, and the difference was statistically significant (P<0.05).

[0103] Depend on Figure 6 It can be seen that the control group has intact and tightly arranged retinal layers; the model group has significantly thinned retinal photoreceptor cell layer and sparse cell nuclei; and the drug group has significantly restored retinal photoreceptor cell layer thickness and its structure tends to be normal. Figure 6 The results show that the recombinant protein of the present invention can effectively inhibit the loss of photoreceptor cells caused by photodamage and protect the morphology of the retina.

[0104] Depend on Figure 7It can be seen that the thickness of the retinal photoreceptor layer in the model group was significantly lower than that in the control group (P<0.001); the thickness of the retinal photoreceptor layer in the drug group was significantly increased (P<0.05). Figure 7 The results show that the recombinant protein of the present invention can effectively increase the thickness of the retinal photoreceptor cell layer, suggesting that the recombinant protein of the present invention has therapeutic and protective effects on retinal damage.

[0105] 2. Electrophysiological Function Verification

[0106] Electroretinogram (ERG) is a series of complex potential changes generated by retinal cells under light stimulation. The basic principle is as follows: Stimulating light passes through the refractive system, aqueous humor, and outer retinal cells to reach the receptors. The receptors perform photoelectric conversion, generating the a-wave of the ERG. The signal generated by the receptors reaches the bipolar cells connected to the receptors and is amplified into the b-wave of the ERG. Müller cells participate in the related rectification and conduction process. Therefore, the a-wave is mainly generated by photoreceptor cells, while the b-wave is mainly influenced by Müller cells and bipolar cells. These potential changes reflect retinal function to a certain extent. ERG test results are shown below. Figure 8 As shown.

[0107] The results of the a-wave detection are shown in Table 3. Compared with the control group, the model group mice showed better performance in dark adaptation at 1×10⁻⁶ Hz. -2 (cd·s) / m 2 and 1×10 0 (cd·s) / m 2 Under intense light stimulation, there was no statistically significant difference in the amplitude of the a-wave in the mouse retina. P >0.05); the model group mice were in dark adaptation at 1×10 3 (cd·s) / m 2 And Ming adapts 1×10 3 (cd·s) / m 2 Under intense light stimulation, the amplitude of the a-wave in the mouse retina decreased significantly, and the difference was statistically significant. P <0.01 or P <0.05). Compared with the model group, the drug group mice showed better performance in dark adaptation at 1×10⁻⁵. -2 (cd·s) / m 2 and 1×10 0 (cd·s) / m 2 Under intense light stimulation, the amplitude of the a-wave in the mouse retina increased, but the difference was not statistically significant. P >0.05); the drug-treated mice were in the dark adaptation 1×10 3 (cd·s) / m 2 And Ming adapts 1×10 3 (cd·s) / m2 Under intense light stimulation, the amplitude of the a-wave in the mouse retina was significantly increased, and the difference was statistically significant. P <0.01 or P <0.05).

[0108]

[0109] The data in Table 3 show that, in dark-adapted high flash intensity (DA10) 3 ) and light adaptation (LA10) 3 Under the above conditions, the amplitude of α-waves in the retina of mice in the model group was significantly lower than that in the control group (P < 0.05 or P < 0.01); however, after intervention with the recombinant protein of the present invention, the amplitude under the above conditions significantly recovered and was better than that in the model group (P < 0.05 or P < 0.01), suggesting that the recombinant protein of the present invention significantly improves the retinal photoreceptor function of model mice.

[0110] The results of b-wave detection are shown in Table 4. Compared with the control group, the model group mice showed better performance in dark adaptation at 1×10⁻⁶ Hz. -2 (cd·s) / m 2 Under intense light stimulation, the amplitude of the b-wave in the mouse retina decreased, but the difference was not statistically significant. P >0.05); the model group mice were in dark adaptation at 1×10 0 (cd·s) / m 2 1×10 3 (cd·s) / m 2 And Ming adapts 1×10 3 (cd·s) / m 2 Under intense light stimulation, the amplitude of the b-wave in the mouse retina decreased significantly, and the difference was statistically significant. P <0.01 or P <0.05). Compared with the model group, the drug group mice showed better performance in dark adaptation at 1×10⁻⁵. -2 (cd·s) / m 2 Under intense light stimulation, the amplitude of the b-wave in the mouse retina increased, but the difference was not statistically significant. P >0.05); the drug-treated mice were in the dark adaptation 1×10 0 (cd·s) / m 2 1×10 3 (cd·s) / m 2 And Ming adapts 1×10 3 (cd·s) / m 2 Under intense light stimulation, the amplitude of the b-wave in the mouse retina significantly increased ( P <0.05 or P <0.01).

[0111]

[0112] The data in Table 4 show that in dark adaptation (DA10) 0 DA10 3 ) and LA10 3 Under these conditions, the amplitude of the b-wave in the retina of the model group mice was significantly lower than that of the control group ( P < 0.05 P < 0.01 indicates that light damage significantly impairs the function of retinal bipolar cells. After intervention with the recombinant protein of the present invention, the amplitude of the b-wave under the above conditions significantly increased, and under high light intensity dark adaptation and light adaptation conditions, it not only recovered to the control level, but also significantly exceeded the model group (#P < 0.05, ##P < 0.01), indicating that the recombinant protein of the present invention can effectively reverse and enhance the retinal bipolar cell response of model mice.

[0113] Example 4: Efficacy evaluation of GCNM01 protein in glaucoma

[0114] (a) Experimental materials

[0115] 1. Animal models: SPF-grade 40-week-old DBA / 2J glaucoma model mice (n = 20) were selected, whose intraocular pressure could be stably maintained at 25-30 mmHg; the control group was set as age-matched SPF-grade DBA / 2J normal mice (n = 5), whose intraocular pressure fluctuated in the range of 10-15 mmHg.

[0116]

[0117] 3. Test kit: BCA protein quantification kit (purchased from Thermo, catalog number 23225); HE staining kit (purchased from Solarbio, catalog number G1120). Electron microscopy fixative: prepared from 2.5% glutaraldehyde and 1% osmium tetroxide.

[0118] 3. Instruments and Equipment: Hitachi H-7650 transmission electron microscope, Bio-Rad ChemiDoc XRS + gel imaging system, Olympus BX53 optical microscope.

[0119] (II) Experimental Methods

[0120] 1. Grouping and Dosing Regimen

[0121] Twenty glaucoma model mice were randomly divided into three groups, and five normal mice were set as a blank group. The specific grouping and drug administration regimens are shown in Table 5.

[0122]

[0123] 2. Index detection

[0124] 2.1 WB detection of Brn3a protein expression

[0125] Protein extraction: At the end of the administration period, the experimental mice were euthanized. Bilateral retinal tissue was immediately isolated and placed in RIPA lysis buffer with protease inhibitors and ground thoroughly in an ice bath. The samples were then centrifuged at 4°C for 30 minutes at 12,000 rpm, and the supernatant was collected for subsequent experimental analysis.

[0126] Protein quantification analysis: The BCA (Bicinchoninic Acid) method was used to determine the concentration of the extracted protein solution. Based on the determination results, the amount of protein loaded in each group was accurately adjusted to 30 μg to ensure the consistency and comparability of the experimental samples.

[0127] Electrophoresis separation and membrane transfer operation: 10% SDS-PAGE gel electrophoresis was carried out, with a constant voltage of 80V for 30 minutes in the concentrated gel stage, and a constant voltage of 120V for 90 minutes in the separation gel stage. After electrophoresis, the proteins in the gel were transferred to the PVDF membrane by wet transfer method, and the transfer process was carried out at a constant current of 300 mA for 90 minutes, so as to realize efficient transfer of proteins.

[0128] Immunodetection and data analysis: The PVDF membrane was placed in 5% skim milk blocking solution and blocked at room temperature for 1 hour to reduce non-specific binding. Then Brn3a specific primary antibody was added and incubated overnight at 4°C. The next day, the corresponding secondary antibody was incubated at room temperature for 50 minutes, and color development was detected by ECL chemiluminescence method. Finally, the gray scale ratio of the target protein Brn3a and the internal reference protein Actin was quantitatively analyzed by ImageJ image analysis software.

[0129] 2.2 Transmission electron microscopy observation of cell ultrastructure

[0130] Sample preparation: The right eyeball of the experimental mouse was taken, and after the retinal tissue was separated, it was cut into 1 mm 3 cuboid tissue blocks. The tissue blocks were fixed with 2.5% glutaraldehyde solution at 4°C for 24 hours, and then post-fixed with 1% osmium acid solution for 2 hours. Dehydration was carried out using 30%-100% gradient ethanol solution, and finally embedding was completed using epoxy resin.

[0131] Section preparation and observation: Ultra-thin sections with a thickness of 50-70 nm were prepared, and uranyl acetate-citrate lead double staining method was used for staining. Hitachi H-7650 transmission electron microscope was used for observation and image collection. Three random fields were selected for each section, and the observation magnification was set to x8000-x15000.

[0132] 2.3 HE staining to detect retinal thickness

[0133] Sample processing: The left eyeballs of experimental mice were fixed with 4% paraformaldehyde solution for 24 hours, then dehydrated with gradient ethanol, and then the samples were paraffin-embedded and prepared into continuous tissue sections with a thickness of 5 pm.

[0134] Staining and quantitative analysis: After deparaffinization and hydration, the sections were stained with hematoxylin-eosin (HE), with hematoxylin staining for 5 minutes and eosin staining for 30 seconds. After staining, neutral balsam was used for mounting. The samples were observed using an Olympus BX53 optical microscope, and the retinal whole layer thickness was measured using Image-Pro Plus 6.0 image analysis software. Five random fields were selected for each eyeball, and the average value was calculated as the final measurement value of the retinal thickness of the sample.

[0135] 2.4 Statistical methods

[0136] IBM SPSS Statistics 26.0 statistical analysis software was used for data processing. For normally distributed and homoscedastic measurement data, statistical description was performed in the form of mean ± standard deviation (x ± s). Single factor analysis of variance (One-Way ANOVA) was used to test the significance of differences between groups to evaluate whether there were overall differences between multiple treatment groups. Further pairwise comparison between groups used the least significant difference method (Least Significant Difference, LSD) to determine the statistical differences between specific groups. P < 0.05 was considered as the standard for judging whether the difference between groups had statistical significance.

[0137] (Three) Experimental results

[0138] 1. Molecular level: Brn3a protein expression

[0139] The WB detection results are shown in Table 6 and Figure 9 .

[0140]

[0141] Table 6 and Figure 9The results show that, compared with the model group and the positive drug control group, the expression level of Brn3a protein in the drug group shows a statistically significant upward trend P <0.01 and P <0.05). The results fully demonstrate that the recombinant protein of the application can significantly promote the expression of retinal ganglion cell (RGC) specific markers by regulating related signal pathways, and effectively inhibit the programmed cell death process of RGC, thereby playing a protective role on RGC.

[0142] In view of the main pathological feature of glaucoma is the progressive death of retinal ganglion cells (RGC) leading to irreversible loss of vision, and the recombinant protein of the application can not only promote the expression of RGC specific markers to maintain cell characteristics and functions, but also effectively inhibit the key mechanism of RGC loss, i.e. programmed cell death, it can be seen that the recombinant protein of the application can block the disease progression of glaucoma from the upstream, and play a clear neuroprotective effect, thereby slowing down or preventing visual function damage, thus having important value for the treatment of glaucoma.

[0143] 2. Retinal cell ultrastructure

[0144] The transmission electron microscopy images of the retinal cell ultrastructure of each group are shown in Figure 10 to Figure 13 .

[0145] Figure 10 The cells of the model group show irregular shape, slightly deep cytoplasm density, and moderate number of organelles; the nucleus (N) is pyknotic, slightly sunken, chromatin is condensed, nuclear membrane is visible, and perinuclear space is widened; mitochondria (M) are obviously swollen, membrane structure is blurred and damaged, matrix and cristae are largely dissolved; rough endoplasmic reticulum (RER) is expanded, and surface ribosomes are shed; individual autophagic bodies (AP) are visible.

[0146] Figure 11 The cells of the blank control group show oval shape, uniform cytoplasm distribution, and moderate number of organelles; the nucleus (N) is approximately irregular oval, slightly sunken, chromatin is sparse, nuclear membrane is blurred, and perinuclear space is not widened; mitochondria (M) are mostly small in shape, with intact membrane, uniform matrix, and clear cristae; rough endoplasmic reticulum (RER) is expanded, and membrane-adjacent ribosomes are sparse; Golgi body (Go) cisternae are expanded; no typical autophagic structure is visible.

[0147] Figure 12The cells of the positive drug control group showed irregular shape, cytoplasm density deepened, and the number of organelles was moderate; the nucleus (N) was irregular, with multiple indentations, the chromatin was uniform, the nuclear membrane was blurred, and the perinuclear space was not widened; the mitochondria (M) were swollen to different degrees, most of which had complete membranes, and the matrix and crista were dense, part of which was obviously swollen, the membrane was damaged, and the matrix and crista were exuded; the rough endoplasmic reticulum (RER) was expanded, and the ribosomes on it were shed; the Golgi body (Go) was flat and expanded; and no typical autophagic structure was observed.

[0148] Figure 13 The cells of the drug group showed approximately oval shape, uniform cytoplasm density, and moderate number of organelles; the nucleus (N) was oval, slightly indented, the chromatin was uniformly distributed, the nuclear membrane was clear, and the perinuclear space was not widened; the mitochondria (M) were obviously swollen, the membrane structure was complete, the matrix was partially dissolved, and the crista was partially broken and disappeared; the rough endoplasmic reticulum (RER) was expanded, and the ribosomes on it were sparse; the Golgi body (Go) was partially expanded; and no typical autophagic structure was observed.

[0149] From Figure 10 to Figure 13 It can be seen that the cells of the model group showed a serious overall state of damage and stress, and these typical damage models indicated that the modeling successfully induced cell damage centered on the mitochondrial-mediated apoptosis pathway and endoplasmic reticulum stress. The cells of the blank control group were basically normal, with slight aging and resting characteristics, indicating that the cells were in a basic metabolic state. The cells of the positive drug control group were still damaged, indicating that the cells were only partially protected, which indicated that the positive drug blocked the nuclear apoptosis process to some extent and had a protective effect on part of the mitochondria, but failed to completely reverse the damage to the organelles, and the protection effect was incomplete. The structure of the cells of the drug group was basically maintained, and there was no apoptosis, which indicated that the recombinant protein of the application exhibited a clear neuroprotective effect, and its core function may be to stabilize the mitochondrial membrane and block the apoptosis signal transduction, thereby maintaining the basic functional structure at the organelle level, and the protection effect is better than or different from that of the positive drug.

[0150] Therefore, from the ultrastructure level, the recombinant protein of the application has a significant protective effect on the cell damage induced by the model, and its effect mainly lies in effectively maintaining the normal morphology of the nucleus and stabilizing the mitochondrial membrane structure, thereby completely preventing the cell from going to apoptosis. The key link of its protective effect may be at the mitochondrial level, and under the experimental conditions, its comprehensive effect is better than that of the positive control drug.

[0151] 3. Tissue level

[0152] The HE staining results are shown in Figure 14 From Figure 14As can be seen, the retinal layers in the blank control group (especially the ganglion cell layer (GCL), nuclear layer (INL), and outer nuclear layer (ONL)) are structurally intact and clearly layered, with densely packed and abundant cells. The thickness of the entire retinal layer is uniform and within the normal physiological range, indicating that the retina is undamaged and represents a normal tissue morphology baseline. Compared with the blank control group, the model group shows a significantly thinner overall retina, especially with a reduced number and looser cell arrangement in the ganglion cell layer (GCL) and outer nuclear layer (ONL). In some areas, cell layers even show breakage or increased gaps, indicating successful model construction and significant pathological damage to the retina (consistent with the characteristics of neuronal loss in retinal diseases such as glaucoma). The retinal layers in the drug group have a structure similar to the blank control group, but are significantly thicker than the model group. The cells in the ganglion cell layer and outer nuclear layer are relatively densely packed and clearly layered, only slightly less than the blank control group. This indicates that the recombinant protein of this invention can effectively reduce retinal damage, protect the cell quantity and structural integrity of each retinal layer, and slow down the thinning process of the retina. The retinal thickness in the positive control group was better than that in the model group, but slightly thinner than that in the drug group. The cell layer arrangement was also slightly less dense than that in the drug group, indicating that although the positive drug has a certain protective effect on the retina, its effect is weaker than that in the drug group.

[0153] Overall, Figure 14 The results show that the recombinant protein of the present invention can effectively protect the morphological structure of the retina, reduce thinning and cell loss, and has a better protective effect than positive control drugs.

[0154] The number of retinal ganglion cells after HE staining was calculated based on the HE staining results, as shown below. Figure 15 As shown. Figure 15 The results showed that the number of retinal ganglion cells in the model group was significantly lower than that in the blank control group (p < 0.01), indicating that the retinal ganglion cells of mice were severely damaged. The number of retinal ganglion cells in the drug group was significantly higher than that in the model group (p < 0.01), which shows that the recombinant protein of the present invention can effectively increase the number of retinal ganglion cells in model mice and has an effective protective effect on the retina.

[0155] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. A method for preparing a recombinant protein for improving retinal oxidative stress, comprising the following steps: Step 1, connecting a polynucleotide encoding the recombinant protein to an expression vector to construct a recombinant expression vector; Step 2, transforming the recombinant expression vector into an expression cell, inducing expression and isolating and purifying to obtain the recombinant protein; wherein the recombinant protein comprises an immunoglobulin Fc region and a functional domain, the amino terminus of the functional domain is connected to the carboxyl terminus of the immunoglobulin Fc region; the amino acid sequence of the functional domain is selected from: (i) the amino acid sequence shown in SEQ ID NO. 1; (ii) an amino acid sequence having substitution, deletion or addition of one or more amino acids compared with the amino acid sequence shown in SEQ ID NO. 1; (iii) an amino acid sequence chemically modified from the amino acid sequence shown in SEQ ID NO. 1; or (iv) an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the immunoglobulin Fc region is selected from: (i) the amino acid sequence shown in SEQ ID NO. 2; (ii) an amino acid sequence having substitution, deletion or addition of one or more amino acids compared with the amino acid sequence shown in SEQ ID NO. 2; (iii) an amino acid sequence chemically modified from the amino acid sequence shown in SEQ ID NO. 2; or (iv) an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2; the amino terminus of the functional domain is connected to the carboxyl terminus of the immunoglobulin Fc region through a linker; preferably, the amino acid sequence of the linker consists of 1 to 3 amino acid sequences shown in SEQ ID NO. 3; more preferably, the amino acid sequence of the linker is shown in SEQ ID NO. 3; optionally, the carboxyl terminus of the functional domain is connected to a sIgA mimetic tail; preferably, the amino acid sequence of the sIgA mimetic tail comprises SEQ ID NO. 6 or is shown in SEQ ID NO. 6; the amino terminus of the immunoglobulin Fc region is further connected to a hinge region and a signal peptide in sequence; preferably, the amino acid sequence of the hinge region comprises SEQ ID NO. 4 or is shown in SEQ ID NO. 4; preferably, the amino acid sequence of the signal peptide comprises SEQ ID NO. 5 or is shown in SEQ ID NO. 5; the amino acid sequence of the recombinant protein is selected from: (i) the amino acid sequence shown in SEQ ID NO. 7; (ii) an amino acid sequence having substitution, deletion or addition of one or more amino acids compared with the amino acid sequence shown in SEQ ID NO. 7; (iii) an amino acid sequence chemically modified from the amino acid sequence shown in SEQ ID NO. 7; or (iv) an amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.

7. ​ ​ ​ ​ ​ ​ ​ 2. The production method according to claim 1, wherein, ​ ​ ​ ​ ​ 3. The production method according to claim 1 or 2, wherein ​ ​ 4. The production method according to any one of claims 1 to 3, wherein, ​ ​ ​ 5. The production method according to any one of claims 1 to 4, wherein, ​ ​ ​ ​ ​ 6. The production method according to any one of claims 1 to 5, wherein, In step 1, the polynucleotide comprises a HindIII restriction site and a BamHI restriction site at two ends respectively; Preferably, the step 1 further comprises the following steps: Step 101, using HindIII endonuclease and BamHI endonuclease to respectively digest the polynucleotide and the expression vector; Step 102, connecting the digested polynucleotide and the expression vector to obtain a connection product; Step 103, transforming the connection product into a host cell, and then culturing on a culture medium containing ampicillin; Step 104, screening positive clones to obtain a recombinant expression vector.

7. The production method according to any one of claims 1 to 6, wherein The nucleotide sequence of the polynucleotide comprises SEQ ID NO: 8; Preferably, the nucleotide sequence of the polynucleotide comprises SEQ ID NO: 15 or as shown in SEQ ID NO:

15.

8. The production method according to claim 6 or 7, wherein The expression vector is a eukaryotic expression vector, preferably, the expression vector is a pcDNA3.1 vector; Optionally, the host cell is a prokaryotic host cell, preferably, the host cell is Escherichia coli, for example, Escherichia coli TOP10.

9. The production method according to any one of claims 1 to 8, wherein, In step 2, the expression cell is a mammalian cell line, preferably, the expression cell is a Chinese hamster ovary cell (CHO).

10. The production method according to any one of claims 1 to 9, wherein, In step 2, the separation and purification comprises: ultrasonic crushing the expression cell; removing cell debris by centrifugation, and collecting the supernatant; chromatography on the supernatant; Preferably, the chromatography comprises affinity chromatography, ion exchange chromatography and molecular sieve chromatography in sequence. In step 2, the separation and purification comprises: ultrasonic crushing the expression cell; removing cell debris by centrifugation, and collecting the supernatant; chromatography on the supernatant; Preferably, the chromatography comprises affinity chromatography, ion exchange chromatography and molecular sieve chromatography in sequence.

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