A kunitz-type polypeptide from a coral, its encoding gene and use in neuroprotection

Abstract

The present application provides a kind of Kunitz polypeptide of coral origin and its encoding gene and application in neuroprotection, belong to polypeptide technical field.The Kunitz polypeptide of coral origin, amino acid sequence is as shown in SEQ ID NO:1.The Kunitz polypeptide has the property of producing specific binding with voltage-gated potassium ion channel Kv1.3, and can effectively repair 6-OHDA induced zebra fish larva movement behavior disorder and neuron damage and improve the increase of neutrophil aggregation and inflammatory factor in zebra fish brain caused by lipopolysaccharide.Therefore, the Kunitz polypeptide can be used as active ingredient in the preparation of drug for preventing and / or treating Parkinson's disease or anti-neuroinflammation.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide technology, specifically relating to a coral-derived Kunitz polypeptide, its encoding gene, and its application in neuroprotection. Background Technology

[0002] Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of dopamine neurons in the substantia nigra. This neuronal death leads to a decrease in dopamine levels, causing motor and other related symptoms. Currently, treatment for Parkinson's disease primarily aims to alleviate symptoms by increasing dopamine levels in the brain or mimicking the effects of dopamine. Commonly used drug treatments include dopamine prodrugs (such as levodopa), dopamine agonists (such as praridone), and deep brain stimulation (DBS). These treatments can help improve motor symptoms such as tremor, muscle rigidity, and bradykinesia. However, there are currently no effective treatment strategies to slow the progressive damage of PD. Therefore, there is an urgent need to explore new alternative treatment methods.

[0003] Voltage-gated potassium (Kv) channels are widely distributed in the nervous system and play a crucial role in regulating neurotransmitter release, neuronal excitability, and cell volume. Parkinson's disease is considered an ion channel disorder or neuronal channel disorder because it is closely related to potassium channel dysfunction. Kv channel blockers exert neuroprotective effects by inhibiting potassium efflux, thereby preventing neuronal death and apoptosis. Among the voltage-gated potassium channel family, Kv1.3 channels are expressed in dopamine neurons and participate in regulating their electrophysiological activity. Aberrant activation of Kv1.3 channels may lead to damage and death of dopamine neurons, thus promoting the development of Parkinson's disease. Therefore, Kv1.3 inhibitors have been investigated as a potential treatment strategy for Parkinson's disease. By inhibiting the activity of Kv1.3 channels, the risk of damage and apoptosis of dopamine neurons can be reduced, thereby protecting the nervous system. Furthermore, drug therapy using Kv1.3 channels has shown efficacy in alleviating motor and non-motor symptoms of Parkinson's disease. Therefore, molecules that regulate the Kv1.3 potassium channel subtype hold promise as novel drugs for treating Parkinson's disease.

[0004] Marine venomous animals (such as sea anemones, corals, and cone snails) possess abundant neurotoxic peptides, which can affect the physiological functions of other animals by modulating different subtypes of ion channels. Due to the high affinity and activity of these marine animal neurotoxic peptides for their targets, they have unique advantages in the development of drugs for treating nervous system diseases. For example, the Kunitz peptide SHPI-1 from the sunflower sea anemone (Stichodactyla helianthus) can achieve an IC50 concentration at nanomolar levels. 50Blocking Kv1.1, Kv1.2, and Kv1.6 channels can alleviate 6-OHDA-induced cytotoxicity in N2a cells. Another Kv channel blocker, ShK, from the sunflower anemone, exhibits picomolar affinity for Kv1.1, Kv1.3, Kv1.4, and Kv1.6 channels. Although both peptides are potent Kv blockers, their lack of target selectivity makes them unsuitable as therapeutic agents. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a coral-derived Kunitz polypeptide that specifically targets the potassium ion channel Kv1.3 and has anti-neuroinflammatory and therapeutic effects on Parkinson's disease.

[0006] This invention provides a coral-derived Kunitz polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] The present invention provides a gene encoding the Kunitz polypeptide derived from coral, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0008] The present invention provides a precursor peptide of the coral-derived Kunitz polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3.

[0009] This invention provides a neuroprotective drug, the active ingredient of which includes the coral-derived Kunitz polypeptide, the coding gene or the Kunitz polypeptide prepared by genetic engineering technology from biological materials containing the coding gene, or the mature peptide formed by processing the precursor peptide.

[0010] This invention provides a voltage-gated potassium ion channel Kv1.3 inhibitor, the active ingredient of which includes the coral-derived Kunitz polypeptide, the coding gene or Kunitz polypeptide prepared by genetic engineering technology from biomaterials containing the coding gene, or a mature peptide formed by processing the precursor peptide.

[0011] This invention provides the application of the coral-derived Kunitz polypeptide, the coding gene or biological material containing the coding gene prepared by genetic engineering, or the mature peptide formed by processing the precursor peptide in the preparation of neuroprotective drugs.

[0012] This invention provides the use of the coral-derived Kunitz polypeptide, the Kunitz polypeptide prepared by genetic engineering using the coding gene or biological material containing the coding gene, or the mature peptide formed by processing the precursor peptide in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0013] This invention provides the application of the coral-derived Kunitz polypeptide, the Kunitz polypeptide prepared by genetic engineering using the coding gene or biological material containing the coding gene, or the mature peptide formed by processing the precursor peptide in the preparation of anti-neuroinflammatory drugs.

[0014] Preferably, the biological material containing the coding gene includes at least one of the following biological materials: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene.

[0015] Preferably, the drug comprises injectable powder and / or injectable solution.

[0016] This invention provides a coral-derived Kunitz polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1. This invention screened a polypeptide with a typical Kunitz domain from coral transcripts and compared it with Kunitz polypeptide sequences from different species, confirming that this polypeptide is a novel Kunitz polypeptide. This invention also demonstrated, through molecular docking and patch-clamp techniques, that the polypeptide PlKuz1 can specifically bind to the voltage-gated potassium ion channel Kv1.3, with a half-repression constant of IC50. 50 The concentration was 3 μM. Simultaneously, zebrafish experiments confirmed that the Kunitz peptide reduced 6-OHDA-induced motor dysfunction and neuronal damage in juvenile zebrafish in a concentration-dependent manner; it also effectively alleviated LPS (lipopolysaccharide)-induced neutrophil aggregation and inflammatory factor increase in the zebrafish brain. In summary, the Kunitz peptide described in this invention has good neuroprotective effects and has significant application value in the treatment of Parkinson's disease and / or in the fight against neuroinflammation. Attached Figure Description

[0017] Figure 1 Figure 1 shows the sequence, base sequence, and multiple sequence alignment results of the PlKuz1 peptide. In the figure, A represents the full-length cDNA and amino acid sequence of PlKuz1, the yellow background represents the signal peptide sequence, the green background represents the precursor peptide sequence, and the red background represents the mature peptide sequence; B represents the multiple sequence alignment of the PlKuz1 peptide with Kunitz-type peptides from different species; C represents the disulfide bond pairing mode of the mature PlKuz1 peptide; and D represents the spatial structure simulation of PlKuz1.

[0018] Figure 2PlKuz1 can block voltage-gated potassium channels Kv1.3; A, B, and C represent the molecular docking analysis of PlKuz1 and rKv 1.3 and the interaction sites of the peptide-protein complex. PlKuz1 (red) and rKv 1.3 (green) are shown in cartoon form; D shows the patch-clamp experiment results of PlKuz1 on rKv 1.1 (E), rKv 1.2 (F), rKv 1.3 (G), and rKv 2.1.

[0019] Figure 3 To demonstrate that PlKuz1 can alleviate motor deficits and dopaminergic neuron loss in a 6-OHDA-induced zebrafish Parkinson's disease model, the study included: A. Mortality rates of zebrafish juveniles exposed to different concentrations of PlKuz1; B. Statistical analysis of total dynamic distance in zebrafish juveniles from different treatment groups; C. Representative morphological images of dopaminergic neurons in the zebrafish brain stained with TH; and TH staining in the thalamic region. + Neurons are indicated by yellow brackets; D. TH in each group + Quantitative analysis of neuronal area; ####p<0.0001 compared with the control group, **p<0.01, ***p<0.001, ****p<0.0001 compared with the 6-OHDA treatment group; Data represent the mean ± standard error of three independent experiments (n>=3); **p<0.001, ***p<0.001 compared with the control group;

[0020] Figure 4 Figure 1 shows the effect of PlKuz1 on reducing the expression of pro-inflammatory cytokines and neutrophil migration in LPS-induced zebrafish brain tissue; A. After LPS intraventricular injection, PlKuz1 reduced the recruitment of neutrophils in the brain of Tg(mpo:EGFP) zebrafish; Neutrophils (area within red circles) entered the head of larvae after LPS intraventricular injection; B. The number of neutrophils in the head of zebrafish after LPS intraventricular injection; C. Result of gene expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the head of zebrafish by qPCR; ####p<0.0001 compared with the control group, **p<0.01, ****p<0.0001 compared with the LPS-treated group. Detailed Implementation

[0021] This invention provides a coral-derived Kunitz polypeptide with the amino acid sequence shown in SEQ ID NO:1 (VCLLPAPVRGPCKGSFRRYFFHAISGQCNEFSYGGCRGNQNNFLTKLSCEQ KC).

[0022] In this invention, the Kunitz polypeptide is specifically derived from a common reef-building coral in China: *Porites lutea*, with a molecular weight of 5927.76 Daltons. The Kunitz polypeptide contains three pairs of disulfide bonds. The Kunitz polypeptide can be obtained through chemical synthesis or recombinant protein expression via genetic engineering.

[0023] The present invention provides a gene encoding the Kunitz polypeptide derived from coral, the nucleotide sequence of which is shown in SEQ ID NO:2(5'-GTTTGTCTTCTTCCCGCTCCGGTACGTGGC CCATGCAAAGGGTCTTTTCGACGATATTTCTTTCATGCAATAAGTGGACAGT GCAATGAATTCAGCTATGGGGGTTGCCGTGGAAACCAAAACAACTTTTTAA CCAAGTTAAGCTGCGAGCAAAAATGT-3').

[0024] This invention provides a precursor peptide of the coral-derived Kunitz polypeptide, the amino acid sequence of which is shown in SEQ ID NO:3 (MAFSLWLTMVLLLAAIAGLEGVEDSSLPPRVCLLPAPV RGPCKGSFRRYFFHAISGQCNEFSYGGCRGNQNNFLTKLSCEQKC), and the corresponding coding gene is shown in SEQ ID NO:4 (5'-ATGGCGTTTTCTCTTTGGCTTACAATGGTT CTTCTGTTAGCAGCTATTGCTGGGCTGGAAGGGGTGGAGGATTCTAGCTTGCCACCCAGGGTTTGTCTTCTTCCCGCTCCGGTACGTGGCCCATGCAAAGGGTCTTTTCGACGATATTTCTTTCATGCAATAAGTGGACAGTGCAATGAATTCAGCTATGGGGTTGCCGTGGAAACCAAAACAACTTTTTAACCAAGTTAAGCTGCGAGCAAAAATGT-3').

[0025] In this embodiment of the invention, molecular docking and patch-clamp techniques were used to confirm that the Kunitz peptide can specifically bind to the voltage-gated potassium channel Kv1.3, with a half-repression constant of IC1. 50 It is 3μM.

[0026] In this embodiment of the invention, a 6-OHDA-induced Parkinson's disease model in zebrafish was used as the experimental subject to analyze the efficacy of the Kunitz peptide. The results showed that the Kunitz peptide reduced 6-OHDA-induced motor dysfunction and neuronal damage in juvenile zebrafish in a concentration-dependent manner; simultaneously, it alleviated lipopolysaccharide (LPS)-induced neutrophil aggregation and inflammatory factor increase in the zebrafish brain. In summary, the peptide PlKuz1 described in this invention has potential application value in the treatment of Parkinson's disease and neuroinflammation.

[0027] Based on the excellent nerve cell repair function of the Kunitz polypeptide, the present invention provides a neuroprotective drug, the active ingredient of which includes the coral-derived Kunitz polypeptide, the coding gene or the Kunitz polypeptide prepared by genetic engineering technology from biological materials containing the coding gene, or the mature peptide formed by processing the precursor peptide.

[0028] In this invention, the biological material containing the coding gene preferably includes at least one of the following biological materials: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene. There are no particular limitations on the type of backbone vector in the recombinant vector containing the coding gene; common backbone vectors known in the art can be used, such as prokaryotic expression vectors, eukaryotic expression vectors, viral vectors, and mammalian cell expression vectors. The recombinant vector containing the coding gene is constructed by selecting a suitable backbone vector according to the type of host, and subsequently enters the host through transformation or transfection methods to obtain recombinant bacterial strains containing the coding gene, recombinant viral strains containing the coding gene, and recombinant cell lines containing the coding gene. The host preferably includes a prokaryotic expression system, a eukaryotic expression system, a virus, or mammalian cells. The prokaryotic expression system preferably includes *Escherichia coli*, lactic acid bacteria, etc. The eukaryotic expression system preferably includes yeast and / or insect cells. The virus is preferably adeno-associated virus and / or lentivirus. This invention does not impose any special limitation on the type of mammalian cell used; any mammalian cell well-known in the art can be used, such as Chinese hamster ovary cells (CHO cells). This invention also does not impose any special limitation on the preparation method of the biological material containing the encoding gene; any genetic engineering method well-known in the art can be used.

[0029] In this invention, the mature peptide formed after processing the precursor peptide is the coral-derived Kunitz polypeptide, which is converted into an active precursor peptide by enzymatic hydrolysis of the precursor peptide under the action of a specific protease. The hydrolysis sites are after LPPR and before VCLL.

[0030] In this invention, the drug further includes excipients. The excipients include at least one of the following: solvents, solubilizers and co-solvents, pH adjusters, antioxidants, osmotic pressure regulators, emulsifiers, antibacterial preservatives, complexing agents, antioxidants and reducing agents, buffers, fillers, and protectants. The dosage form of the drug preferably includes injectable powder and / or injectable solution. The effective concentration of the active ingredient in the drug is preferably 0.5–3 mg / g, and can be 1 mg / g or 2 mg / g.

[0031] Based on the characteristic of the Kunitz polypeptide to transfer and bind to the Kv1.3 channel, the present invention provides a voltage-gated potassium ion channel Kv1.3 inhibitor, the active ingredient of which includes the coral-derived Kunitz polypeptide, the coding gene or biological material containing the coding gene prepared by genetic engineering technology, or the mature peptide formed by processing the precursor peptide.

[0032] The present invention does not impose any special restrictions on the effective content of the active ingredient in the inhibitor; the effective concentration of ion channel inhibitors known in the art can be used.

[0033] This invention provides the application of the coral-derived Kunitz polypeptide, the coding gene or biological material containing the coding gene prepared by genetic engineering, or the mature peptide formed by processing the precursor peptide in the preparation of neuroprotective drugs.

[0034] This invention provides the use of the coral-derived Kunitz polypeptide, the Kunitz polypeptide prepared by genetic engineering using the coding gene or biological material containing the coding gene, or the mature peptide formed by processing the precursor peptide in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0035] In this invention, the treatment of Parkinson's disease is manifested in the Kunitz peptide specifically binding to the voltage-gated potassium ion channel Kv1.3, inhibiting the outflow of potassium ions from cells, thereby exerting a neuroprotective effect; the Kunitz peptide can also improve motor behavior defects and dopamine neuron loss in a 6-OHDA-induced zebrafish juvenile Parkinson's disease model, playing a role in restoring motor behavior and repairing dopamine neuron damage.

[0036] This invention provides the application of the coral-derived Kunitz polypeptide, the Kunitz polypeptide prepared by genetic engineering using the coding gene or biological material containing the coding gene, or the mature peptide formed by processing the precursor peptide in the preparation of anti-neuroinflammatory drugs.

[0037] In this invention, using a lipopolysaccharide-induced zebrafish inflammation model, the anti-neuroinflammatory effect of the Kunitz peptide was analyzed. The results showed that the Kunitz peptide reduced the expression levels of inflammatory factors such as TNF-α, IL-6 and IL-1β in zebrafish brain tissue stimulated by LPS, and could inhibit LPS-induced aggregation of neutrophils in the brain.

[0038] The following detailed description, in conjunction with embodiments, illustrates a coral-derived Kunitz polypeptide, its encoding gene, and its application in neuroprotection provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Screening of GcKuz1 peptide and its encoding gene

[0041] Blast annotation and Pfam analysis identified a transcript fragment (ID: >TRINITY_DN74292_c0_g2_i2::g.228717) with a typical Kunitz domain from the transcript sequence of Porites lutea. Multiple sequence alignment analysis with Kunitz peptide homologs from different species confirmed that PlKuz1 is a novel Kunitz peptide from corals.

[0042] The results are as follows Figure 1 As shown, PlKuz1 is a novel coral-derived Kunitz polypeptide with the mature peptide sequence shown in SEQ ID NO: 1 (VCLLPAPVRGPCKGSFRRYFFHAISGQCNEFSYGGCRGNQNNFLTK LSCEQKC), and the corresponding coding gene sequence is shown in SEQ ID NO: 2. The amino acid sequence of the PlKuz1 precursor peptide is shown in SEQ ID NO: 3 (MAFSLWLTMVLLLAAIAGL EGVEDSSLPPRVCLLPAPVRGPCKGSFRRYFFHAISGQCNEFSYGGCRGNQNNFLTKLSCEQKC).

[0043] Example 2

[0044] Determination of the target of PlKuz1 peptide

[0045] 1. Molecular docking

[0046] The 3D structure of PlKuz1 was predicted using the AlphaFold2 modeling system, and its structure diagram was drawn using the relaxed_ranked_001 structure. Human voltage-gated potassium channel Kv 1.3 (PDB: 7EJ1) was used as the target protein, and protein docking was performed using ClusPro software. The docking conformation with the lowest energy binding energy was selected. After docking, the interface residues of the peptide-protein complex were analyzed using Pymol software.

[0047] 2. Cell transfection and patch-clamp electrophysiological recording

[0048] Four recombinant potassium ion channel vectors were constructed based on the plasmid maps of rat Kv1.1 (accession number: Kv1.1 / RBG4), Kv1.2 (accession number: Kv1.2 / RBG4), Kv1.3 (accession number: Kv1.3 / RBG4), and Kv2.1 (accession number: Kv2.1 / RBG4) reported on the AddGene website (www.addgene.org).

[0049] HEK293T cells were cultured in Dulbecco medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated at 37°C with 5% CO2. After successful passage, the cells were transplanted into 35 mm diameter culture dishes. After 4–5 h, using Lipofectamine 3000 and P3000 (Life Technologies, USA) transfection reagents, following the manufacturer's instructions, 5 μg of the constructed potassium channel recombinant vector and 0.5 μg of green fluorescent protein particles (purchased from Sangon Biotech (Shanghai) Co., Ltd.) were co-transfected into HEK293T cells. 24–48 h post-transfection, cells were detached using 0.25% trypsin and prepared for patch-clamp recording. Once the cells attached to glass slides, cells exhibiting EGFP fluorescence were selected for whole-cell recording.

[0050] Electrophysiological experiments were performed at 25 ± 3 °C using an EPC-10 amplifier controlled by Patch-Master software (HEKA Elektronik, Lambrecht, Germany). For whole-cell recordings, all possible voltage errors were minimized by applying 80% series resistance compensation. Capacitance artifacts were eliminated via computer-controlled circuitry of the patch-clamp amplifier. The current sampling rate was 10 kHz, filtered at 2.9 kHz. Recording patches were made from high-purity borosilicate glass and flame-polished to achieve a resistance of 3–6 MΩ. For voltage-current recordings, the peak value of each trace was determined in the presence of either extracorporeal fluid or the test peptide. These potassium channel current values ​​were normalized to the maximum peak current in extracorporeal fluid application. After perfusion of cells with extracorporeal fluid for 30 s to generate a stable current, PlKuz1 peptide was added to a final concentration of 3 μM. Conductivity-voltage experiments were resolved by voltage steps starting from deep polarization voltage. To record the currents of different Kv channels, the extracellular fluid contained: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES (pH 7.3, adjusted with NaOH solution). The intracellular fluid contained: 140 mM KCl, 3 mM Na2ATP, 1 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH 7.3, adjusted with KOH solution).

[0051] The results are as follows Figure 2 As shown, PlKuz1 can specifically block the Kv1.3 channel (IC). 50 =3μM), without affecting other Kv channels (Kv1.1, Kv1.2 and Kv2.1).

[0052] Example 3

[0053] PlKuz1 can alleviate motor deficits and dopamine neuron loss in a 6-OHDA-induced Parkinson's disease model in juvenile zebrafish.

[0054] 1. Zebrafish kinetic behavior test

[0055] One day after fertilization, AB strain zebrafish larvae were treated with 25 μM 6-OHDA for two days. Subsequently, starting from day 3, the zebrafish were treated with 250 μM 6-OHDA for four days, followed by daily pretreatment with different concentrations of PlKuz1 (0.3 μM, 1 μM, 3 μM) for 2 hours. Finally, seven-day-old zebrafish were transferred to 48-well plates (one fish per well). The 48-well plates were placed in a Zebrabox device, and the swimming behavior of the zebrafish was monitored using an automated video tracking system (Viewpoint, ZebraLab, LifeSciences, Lyon, France). Before data acquisition, the zebrafish larvae were allowed to acclimatize to the Zebrabox environment. The swimming activity of each fish was recorded in six 10-minute intervals. The total distance traveled by the zebrafish larvae within each 10-minute interval was measured.

[0056] 2. Whole immunostaining against tyrosine hydroxylase (TH)

[0057] Zebrafish embryos, one day post-fertilization, were treated with PlKuz1 or without PlKuz1 for two consecutive days in the presence of 250 μM 6-hydroxydopamine (6-OHDA). The zebrafish were then fixed with 4% paraformaldehyde in PBS for 30 minutes, rinsed, and stored in absolute methanol at -20°C. Tyrosine hydroxylase-positive cells were semi-quantitatively assessed using ImageJ software, and the results are expressed as a percentage of the area of ​​tyrosine hydroxylase-positive cells in the control group.

[0058] The results are as follows Figure 3 As shown, in motor behavior tests, 6-OHDA significantly damaged dopaminergic (DA) neurons and altered swimming behavior in zebrafish. Pretreatment with 0.3–3 μM PlKuz1 alleviated 6-OHDA-induced dopaminergic neuronal loss and abnormal swimming behavior in zebrafish. These findings suggest that PlKuz1 has a protective effect against 6-OHDA-induced dopaminergic neuronal death and can alleviate motor deficits in juvenile zebrafish.

[0059] Example 4

[0060] PlKuz1 alleviated LPS-induced inflammatory cytokine production and neutrophil migration in juvenile zebrafish.

[0061] 1. Zebrafish brain neutrophil migration imaging

[0062] Following intracerebroventricular injection of 1 nL of 2 mg / mL LPS into the zebrafish brain, juvenile zebrafish were anesthetized with 0.1 mg / mL trichloroacetic acid and placed head-up on glass slides embedded with 1.5% low-melting-point agarose to observe neutrophil migration in the brain. Neutrophil aggregation in the Tg(mpo:EGFP) zebrafish brain neuronal regions was observed and photographed using a fluorescence stereomicroscope. The number of neutrophils in the Tg(mpo:EGFP) zebrafish brain regions was manually counted.

[0063] 2. Analysis of the expression levels of inflammatory factors in the zebrafish brain

[0064] Following intracerebroventricular injection of 1 μL of 2 mg / mL LPS into adult zebrafish, brain tissue was isolated from individual adult zebrafish for PCR analysis. TRIzol was used according to the manufacturer's instructions. TM Total RNA was extracted from zebrafish brain tissue. The obtained RNA was reverse transcribed into cDNA, and the reverse transcription was performed using an Evo M-MLV RT Premix for qPCR Kit (Accurate Biology, Shandong, China). qPCR analysis was performed using a SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Shandong, China) and a QuantStudio 7Flex real-time PCR system (ThermoFisher, USA). The cycling conditions were as follows: 50℃ for 2 min, 95℃ for 10 min, followed by 40 cycles, each consisting of 95℃ for 15 s and 60℃ for 30 s. The relative mRNA expression levels of inflammatory cytokines TNF-α, IL-6, and IL-1β were calculated by normalizing the internal reference gene elfα.

[0065] Table 1 Primer sequences used in real-time quantitative PCR

[0066] Primer name Primer sequence IL-1β forward (zebrafish) 5'-CATTTGCAGGCCGTCACA-3'(SEQ ID NO:5) IL-1β reverse (zebrafish) 5'-GGACATGCTGAAGCGCACTT-3'(SEQ ID NO:6) IL-6 forward (zebrafish) 5'-TCAACTTCTCCAGCGTGATG-3'(SEQ ID NO:7) IL-6 reverse (zebrafish) 5'-TCTTTCCCTCTTTTCCTCCTG-3'(SEQ ID NO:8) TNF-α forward (zebrafish) 5'-GCTGGATCTTCAAAGTCGGGTGTA-3'(SEQ ID NO:9) TNF-α reverse (zebrafish) 5'-TGTGAGTCTCAGCACACTTCCATC-3'(SEQ ID NO:10) elfαforward(zebrafish) 5'-GCTCAAACATGGGCTGGTTC-3'(SEQ ID NO:11) elfαreverse(zebrafish) 5'-AGGGCATCAAGAAGAGTAGTACCG-3'(SEQ ID NO:12)

[0067] The results are as follows Figure 4 As shown, PlKuz1 reduced the expression levels of inflammatory factors such as TNF-α, IL-6 and IL-1β in zebrafish brain tissue stimulated by LPS, and inhibited LPS-induced aggregation of neutrophils in the brain.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A neuroprotective drug, characterized in that, The active ingredient includes a coral-derived Kunitz polypeptide or a Kunitz polypeptide prepared by genetic engineering technology from a coral-derived Kunitz polypeptide encoding gene or a biomaterial containing the encoding gene; the amino acid sequence of the Kunitz polypeptide is shown in SEQ ID NO:1; the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

2. A voltage-gated potassium ion channel Kv1.3 inhibitor, characterized in that, The active ingredient includes a coral-derived Kunitz polypeptide or a Kunitz polypeptide prepared by genetic engineering technology from a coral-derived Kunitz polypeptide encoding gene or a biomaterial containing the encoding gene; the amino acid sequence of the Kunitz polypeptide is shown in SEQ ID NO:1; the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. The use of a coral-derived Kunitz polypeptide or a Kunitz polypeptide prepared by genetic engineering technology from the coding gene of the coral-derived Kunitz polypeptide or biological material containing the coding gene in the preparation of a drug for the prevention and / or treatment of Parkinson's disease, wherein the amino acid sequence of the coral-derived Kunitz polypeptide is shown in SEQ ID NO:1; and the nucleotide sequence of the coding gene is shown in SEQ ID NO:

2.

4. The use of coral-derived Kunitz polypeptide or Kunitz polypeptide prepared by genetic engineering technology from the coding gene of said coral-derived Kunitz polypeptide or biological material containing said coding gene in the preparation of anti-neuroinflammatory drugs, wherein the amino acid sequence of said coral-derived Kunitz polypeptide is shown in SEQ ID NO:1; and the nucleotide sequence of said coding gene is shown in SEQ ID NO:

2.

5. The application according to claim 3 or 4, characterized in that, Biological materials containing the coding gene include at least one of the following: a recombinant vector containing the coding gene, a recombinant bacterial strain containing the coding gene, a recombinant viral strain containing the coding gene, and a recombinant cell line containing the coding gene.

6. The application according to claim 3 or 4, characterized in that, The drug includes injectable powder and / or injectable solution.

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