Polypeptide and application thereof in neurodegenerative diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide and application thereof in neurodegenerative diseases. The amino acid sequence of the polypeptide provided by the invention is as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. Meanwhile, the invention provides a coding gene and related application of the polypeptide. The fusion polypeptide comprises a TAT cell penetrating peptide, an intermediate connecting peptide, a channel protein specific inhibition peptide and PEG modification, has a specific inhibition effect on Cx43, can block nerve injury caused by Cx43 and repair nerve injury, and can be used for treating Parkinson's disease and Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide and its application in neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases are a class of diseases that seriously affect human health, including Parkinson's Disease (PD), Alzheimer's Disease (AD), etc. These diseases are characterized by the gradual loss of specific types of neurons in the central nervous system, leading to cognitive and motor dysfunction. Studies in recent years have shown that inflammatory response plays a key role in the pathogenesis of these diseases. At present, the clinical treatments for these diseases mainly include drug therapy, surgical treatment and rehabilitation therapy, but these methods have limitations in delaying disease progression and improving symptoms.

[0003] 1. Medication (1) Parkinson's disease: Commonly used drugs include levodopa, dopamine receptor agonists (such as pramipexole and ropinirole), monoamine oxidase B inhibitors (such as selegiline) and catechol-O-methyltransferase inhibitors (such as entacapone). These drugs mainly relieve symptoms by supplementing dopamine or prolonging its duration of action, but they cannot prevent the progression of the disease. Long-term use may also lead to side effects and reduced efficacy.

[0004] (2) Alzheimer's disease: Commonly used drugs include cholinesterase inhibitors (such as donepezil, galantamine, and huperzine A) and NMDA receptor antagonists (such as memantine). These drugs can improve cognitive function but cannot prevent or reverse neuronal death.

[0005] 2. Surgery (1) Parkinson's disease: Deep brain stimulation (DBS) is a commonly used surgical method that regulates abnormal neural activity by implanting electrodes in specific brain areas, thereby improving motor symptoms. DBS is suitable for patients who have poor responses to drug treatment, but the surgical risk is high and the long-term effect needs further verification. At the same time, surgical stimulation is only used to relieve symptoms and cannot prevent the death of dopamine neurons.

[0006] Connexin43 (Cx43) is an important component of intercellular communication, and its dysfunction is associated with a variety of neurological diseases. Studies have shown that LPS (lipopolysaccharide) can induce a decrease in Cx43 gap junction communication and an increase in hemichannel function, thereby directly or indirectly causing neurotoxicity through excessive release of ATP and glutamate. Overactivation of connexin Cx43 is believed to be involved in the pathogenesis of Parkinson's disease or Alzheimer's disease, among which Cx43 inhibitory peptide GAP27 is believed to have neuroprotective effects and the potential to treat Parkinson's disease, but there is a lack of systematic research. Therefore, there is still a lack of effective drugs to delay disease progression and prevent or reverse neuronal death. Summary of the invention

[0007] The object of the present invention is to provide a polypeptide.

[0008] Another object of the present invention is to provide a gene encoding the above polypeptide.

[0009] Another object of the present invention is to provide a recombinant expression vector containing the above encoding gene.

[0010] Another object of the present invention is to provide a recombinant strain containing the above encoding gene.

[0011] Another object of the present invention is to provide a method for preparing the above polypeptide.

[0012] Another object of the present invention is to provide applications of the above polypeptide.

[0013] The amino acid sequence of the polypeptide ZT002-02 of the present invention is shown in SEQ ID No. 1: Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Pro-Phe-Ile-Ile-Ile.

[0014] The above-mentioned polypeptide ZT002-02 is a channel protein-specific inhibitory peptide. Compared with the commonly used CX43 inhibitory analog peptide Ser-Arg-Pro-Thr-Glu-Lys-Thr-Ile-Phe-Ile-Ile, the polypeptide sequence of the present invention has undergone multiple changes, among which, the second position Arg is changed to His, which reduces the positive charge of the polypeptide and thus enhances the transmembrane properties of the polypeptide; the eighth position Ile is changed to Lys, which increases the hydrophilicity of the polypeptide and balances the charge distribution, thereby increasing the transmembrane properties of the polypeptide; the ninth position Pro is added to increase the rigidity of the polypeptide, improve the stability of the polypeptide, and prolong the half-life of the polypeptide; the last position Ile is added to form the final Phe-Ile-Ile-Ile sequence, which enhances the hydrophobic tail of the polypeptide, facilitates fusion with the cell membrane, and enhances the binding ability with the channel protein, so as to enhance the inhibitory effect on Cx43.

[0015] The present invention also provides an optimized fusion polypeptide ZT002, whose amino acid sequence is shown in SEQ ID No. 2: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-(Gly)3-Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Pro-Phe-Ile-Ile-Ile.

[0016] In order to increase the ability of the ZT002-02 polypeptide to cross the blood-brain barrier, the fusion polypeptide ZT002 of the present invention selects to add the TAT cell-penetrating peptide sequence of the human immunodeficiency virus Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg at the front end of the ZT002-02 polypeptide. At the same time, a repeating sequence (Gly) 3 is added between the two polypeptide sequences to improve the overall flexibility of the fusion polypeptide and avoid the formation of steric hindrance.

[0017] The present invention also provides a PEG-modified polypeptide molecule PEG-ZT002, whose amino acid sequence is shown in SEQ ID No. 3: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-(Gly)3-Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Pro-Phe-Ile-Ile-Ile-(CH2CH2O)3-CH2OH.

[0018] The present invention further enhances the stability and half-life of the polypeptide molecule and increases the in vivo action time of the polypeptide molecule by chemically modifying the C-terminus of the fusion polypeptide ZT002 of SEQ ID NO. 2 with (PEG) 4.

[0019] The present invention provides a coding gene after codon optimization of the above fusion polypeptide, and its nucleotide sequence is shown in SEQ ID NO.4: TAT GGC CGT AAA AAA CGT CGT CAG CGC CGT CGT GGT GGT GGT TCT CAT CCGACC GAA AAG ACC AAG CCG TTC ATT ATC The present invention also provides a recombinant vector comprising a gene encoding the above polypeptide, preferably PET21a-ZT002. The polypeptide gene of the present invention is inserted between appropriate restriction enzyme sites of the expression vector, so that its nucleotide sequence is operably connected to the expression regulatory sequence. As a most preferred embodiment of the present invention, the fusion polypeptide ZT002 gene sequence of the present invention is inserted between restriction enzyme sites on the plasmid PET21a, so that the nucleotide sequence is located downstream of the promoter and is regulated by it, to obtain the recombinant expression plasmid PET21a-ZT002.

[0020] The present invention also provides a recombinant strain comprising the above polypeptide gene, preferably a recombinant Escherichia coli strain BL21 (DE3)-ZT002.

[0021] The present invention also provides the use of the above polypeptide, especially as a Cx43 inhibitor. The polypeptide molecule of the present invention has a specific inhibitory effect on Cx43 and can be used to prepare drugs for treating neurodegenerative diseases, such as drugs for Parkinson's disease or Alzheimer's disease.

[0022] The PEG-modified polypeptide molecule PEG-ZT002 of the present invention comprises a TAT cell penetrating peptide, an intermediate connecting peptide, a channel protein specific inhibitory peptide and a PEG modification, and has a plasma half-life of up to 80 hours and a half-life in the brain of up to 120 hours; it has a specific inhibitory effect on Cx43; it can be used to treat Parkinson's disease and Alzheimer's disease; it can inhibit neuroimmune reactions and block the resulting nerve damage; it can promote the production of nerve growth factor, repair nerve damage, increase the level of neurotransmitters, and reduce the nerve damage caused by the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The mass spectrum of the fusion polypeptide ZT002 is shown.

[0025] Figure 2 Shows the inhibitory effect of polypeptide molecules on Cx43.

[0026] Figure 3 It shows that PEG-ZT002 can reverse dopamine neuron damage in mice caused by neuroimmunity.

[0027] Figure 4It showed that PEG-ZT002 could reverse the decrease of dopamine neurotransmitter in the striatum of mice caused by neuroimmunity.

[0028] Figure 5 Shows the changes in the concentrations of PEG-ZT002 and ZT002-02 peptides in the blood.

[0029] Figure 6 Shows the changes in the concentrations of PEG-ZT002 and ZT002-02 peptides in brain tissue. DETAILED DESCRIPTION

[0030] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0031] Example 1 (1) Construction of expression plasmid: Design and synthesize the target gene sequence and add the start codon and stop codon.

[0032] Its nucleotide sequence is as follows: ATG TAT GGC CGT AAA AAA CGT CGT CAG CGC CGT CGT GGT GGT GGT TCT CATCCG ACC GAA AAG ACC AAG CCG TTC ATT ATC ATT The above sequence was inserted into the PET21a plasmid to construct the expression plasmid PET21a-ZT002.

[0033] (2) Fermentation expression The above expression plasmid was transferred into Escherichia coli BL21 (DE3) to prepare the production strain BL21 (DE3)-ZT002, and the strain was fermented and expressed to obtain the target polypeptide, which was purified by chromatography to obtain a high-purity polypeptide.

[0034] The bacterial strain fermentation expression adopts the segmented Feed-Batch fermentation process, and the specific process is as follows: 1) Resuscitation of bacterial strains: inoculate 100 µL of glycerol solution from the working seed batch directly into a 100 mL bottle of LB medium and culture at 30±2℃ until OD600 is 0.5±0.2; 2) Fermentation: The production bacteria prepared after bacterial resuscitation were inoculated into the fermentation tank, the culture medium volume was 2±1L, and the fermentation temperature was 37°C; when the OD600 was about 0.6±0.1, 0.6 mM IPTG was added to activate the expression of the target polypeptide. The fermentation was segmented, and the Fed-batch Phase feeding fermentation mode was carried out when the OD600 value was about 10, and the feed was fed at a flow rate of 40 mL / h.

[0035] 3) Bacteria collection: After fermentation for a total of about 18±3 hours (OD600 value can reach up to 200), end the fermentation; cool to 25℃ or below for 30 minutes. Centrifuge the bacterial solution at 6000±1000 rpm at 4℃ for 20±10 minutes to collect the bacteria, transfer the bacteria directly to liquid nitrogen for quick freezing, weigh them, and then divide them into cryopreservation boxes and store them below -60℃.

[0036] 4) Peptide purification: The peptide ZT002 was expressed in the form of inclusion bodies. After the bacterial cells were permeated with a buffer containing 0.5% TritonX-100, the inclusion bodies were obtained by centrifugation at 10000±1000 rpm for 15 minutes. The inclusion bodies were washed twice with the permeation buffer and then subjected to a denaturation step to obtain the renatured polypeptide molecules, which were then purified by ultrafiltration and molecular sieve chromatography. The purity of the polypeptide can reach more than 98%.

[0037] (3) Peptide identification The molecular weight of the peptides was confirmed by mass spectrometry (MS).

[0038] The results are as follows Figure 1 As shown, mass spectrometry showed that the molecular weight of the purified polypeptide was 3223.8, which was consistent with the theoretical molecular weight of ZT002, 3223.79.

[0039] (4) PEG modification The purified polypeptide backbone ZT002 was modified at its C-terminus with PEG to obtain PEG-ZT002. The specific steps are as follows: 1) Dissolve PEG chain (0.5 mmol) in DMSO to a concentration of 10 mM; dissolve N-hydroxysuccinimide (NHS) (0.5 mmol) and N,N'-dicyclohexylcarbodiimide (DCC) (0.5 mmol) in appropriate amounts of DMF to a concentration of 10 mM. Add the PEG chain in the ice bath to the mixed solution of NHS and DCC and stir evenly; react at room temperature for 30 minutes with constant stirring to form activated PEG4-NHS ester.

[0040] 2) Dissolve the peptide backbone ZT002 in an appropriate amount of PBS buffer (pH 7.4) to a concentration of 10 mg / mL. Slowly add the activated PEG4-NHS ester solution to the peptide backbone solution, react overnight (12-16 hours) at room temperature, and purify the reaction mixture by HPLC.

[0041] Finally, the modified polypeptide molecule PEG-ZT002 is obtained: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-(Gly)3-Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Pro-Phe-Ile-Ile-Ile-(CH2CH2O)3-CH2OH.

[0042] Example 2 Investigating the inhibitory effect of polypeptide on Cx43 The inhibitory effect of Cx43 was carried out by fluorescence transfer assay: 1) Human astrocytes HA1800, a cell line with high expression of CX43, were cultured in 6-well plates until 90% confluence, and each well was divided into 8 treatment groups: blank control (Control), Cx43 mimetic peptide (GAP27, commercially available), and 6 inhibitory peptides ZT002-01 to ZT002-06 with different amino acids were designed and chemically synthesized by solid phase synthesis method.

[0043] ZT002-01:Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Phe-Ile-Ile-Ile; ZT002-02: Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Pro-Phe-Ile-Ile-Ile; ZT002-03: Ser-His-Pro-Thr-Glu-Lys-Thr-Ile-Pro-Phe-Ile-Ile-Ile; ZT002-04:Ser-His-Pro-Thr-Glu-Lys-Thr-Ile-Trp-Phe-Ile-Ile-Ile; ZT002-05:Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Trp-Phe-Ile-Ile-Ile; ZT002-06: Ser-His-Pro-Thr-Glu-Lys-Thr-Lys-Tyr-Phe-Ile-Ile-Ile.

[0044] After the cells were washed once with PBS, 2 mL of serum-free medium containing 100 nM of each group of drugs was added to each well and cultured at 37°C for another 30 min; 2) Add 0.5 mL of 2.5 mM Lucifer Yellow to each well and continue incubating at 37°C for 30 min; wash three times with ice-cold PBS, and then read the fluorescence intensity using a fluorescence spectrophotometer (430 nm excitation, 530 nm emission). The fluorescence intensity of the control group was calculated as 100%, and the percentages of other wells were calculated based on the readings. The lower the value, the stronger the Cx43 channel inhibition effect.

[0045] The results are as follows Figure 2 As shown, ZT002-02 polypeptide has the best inhibitory effect on Cx43.

[0046] The present invention optimizes the membrane penetration and long-term effect of the ZT002-02 polypeptide to obtain the polypeptide molecule PEG-ZT002, which has no effect on the inhibitory effect on Cx43.

[0047] Example 3 Establishment of animal model: 8-week-old SPF male C57BL / 6 mice were used to establish the LPS injury model. The dopamine neuron injury model was established by injecting 2μl LPS (5μg) or 6-OHDA (2.5μg) into the substantia nigra region of the midbrain of mice using a brain positioning injection apparatus. Brain pathology performed 2 weeks later showed significant dopamine neuron damage in the substantia nigra region of the brain after LPS injection, and a decrease in dopamine levels in the striatum.

[0048] Drug treatment: 24 hours before LPS injection, mice were subcutaneously injected with saline or PEG-ZT002 (100 μg). After LPS injection, the mice were fed for 2 weeks and then sacrificed for brain pathology analysis.

[0049] Analysis of dopamine neuron damage in substantia nigra: The mouse brain tissue was removed and fixed in 4% paraformaldehyde solution for 24 hours, then dehydrated with 30% sucrose for 24 hours and frozen sections were made. Each section was 35 μm thick and stored at 4°C. Part of the substantia nigra and striatum brain slices were stained with TH, a dopamine neuron marker, and then developed with DAB (diaminobenzidine) and photographed under a microscope.

[0050] like Figure 3 As shown in A and B, PEG-ZT002 administration can significantly reduce the damage of TH-positive dopamine neurons in the substantia nigra of mice (A) and increase the level of dopamine in the striatum (B). The number of dopamine neurons and the intensity of TH-positive staining area were manually calculated using Image J and marked on the right.

[0051] Analysis of striatal neurotransmitter levels: Striatal tissue was homogenized in 0.1 M perchloric acid and centrifuged at 12000 rpm for 30 minutes. The supernatant was filtered through a 0.2 μm nylon filter and then the levels of dopamine (DA) and its metabolites DOPAC and HVA were detected by HPLC.

[0052] like Figure 4 As shown, PEG-ZT002 can significantly restore the decreased levels of striatal dopamine (DA) and its metabolites (DOPAC, HVA) caused by LPS.

[0053] Example 5 Investigation of the pharmacokinetics of polypeptides 1. Experimental Design 1.1 Animal experiments Twenty-four 2-month-old male C57BL / J mice were selected and injected with 100 μl of 50 nmol of peptide ZT002-02 or PEG-modified fusion peptide molecule PEG-ZT002 through the tail vein to evaluate the serum and brain pharmacokinetics of the peptide.

[0054] 1.2 Blood sample collection 0.1 mL of blood was collected before administration (0 hour) and at different time points after administration (0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours). The collected blood samples were immediately centrifuged (3000 rpm, 10 minutes) to separate the serum, which was divided into cryopreservation tubes and stored in a -80°C refrigerator for testing.

[0055] 1.3 Tissue sample collection At different time points after administration (such as 1, 4, 8, 24, 48, 72, 96, and 120 hours), 3 animals were anesthetized and killed each time, and brain tissue was obtained after ice-cold PBS perfusion. Brain tissue samples were added to an appropriate amount of ice-cold PBS and homogenized using a tissue homogenizer in an ice bath; the homogenized samples were centrifuged (3000 rpm, 10 minutes) and the supernatant was collected; the supernatant was divided into cryopreservation tubes and stored in a -80°C refrigerator for testing.

[0056] 2. Determination of drug concentration 2.1 Sample preparation Take 100 μl of sample (PBS supplement if serum is insufficient) and add 100 μl of 5% TCA solution to pretreat the sample, incubate on ice for 30 minutes, centrifuge at high speed (13,000 rpm, 10 minutes), take the supernatant, filter it through a 0.2 μm filter and prepare for sample testing.

[0057] 2.2 High performance liquid chromatography (HPLC) determination 20 μl of the treated sample was loaded onto a C18 HPLC reverse phase chromatography column to determine the drug concentration.

[0058] The mobile phase A was PBS and B was 100% acetonitrile (ACN). Gradient elution was performed from 95% A solution and 5% B solution to 5% A solution and 95% B solution for 30 min. The abundance of the peptide sample ZT002 was detected at 214 nm, and the corresponding concentration was calculated using the standard curve.

[0059] Standard curve: 20 μl of ZT002 standards with concentrations of 0, 5nM, 10nM, 20nM, 50nM, 100nM, and 200nM were loaded for testing, and the peak area and corresponding concentration of ZT002 were determined to fit the linear standard curve, and the linear standard equation was obtained: y=11.6x, where: y is the peak area (AU), and x is the peptide concentration (nM). The concentration of the sample peptide ZT002 was calculated based on the standard curve.

[0060] The results are as follows Figure 5 , 6 As shown in the figure, after PEG-ZT002 injection, the half-life of the peptide in plasma can reach 80 hours, and the half-life in the brain can reach 120 hours. However, the half-life of the unmodified and optimized peptide ZT002-02 in serum is only 1.4 hours, and it is difficult to enter the brain and accumulate.

[0061] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

2. The polypeptide encoding gene according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant expression vector comprising the coding gene according to claim 2.

4. A recombinant strain comprising the encoding gene according to claim 2.

5. A method for preparing the polypeptide according to claim 1, characterized in that: The method comprises the following steps: (1) transforming a host cell with a recombinant expression vector containing a gene encoding a polypeptide to obtain a recombinant strain; (2) culturing the recombinant strain to induce expression of the polypeptide; (3) Purifying the polypeptide.

6. The method according to claim 5, characterized in that The C-terminus of the purified polypeptide is chemically modified with PEG to obtain a PEG-modified polypeptide molecule.

7. Use of the polypeptide according to claim 1.

8. Use of the polypeptide according to claim 1 in the preparation of a Cx43 inhibitor.

9. Use of the polypeptide according to claim 1 in the preparation of a drug for treating neurodegenerative diseases.

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