Peptides targeting neurofilament light chain protein and their applications

Abstract

The present invention provides a polypeptide targeting neurofilament light chain protein (NFL) and its application. The polypeptide probe has high detection sensitivity, strong specificity, high affinity with neurofilament light chain protein, can effectively screen the serum of AD patients and normal people through NFL in serum, has the advantages of short detection time, low detection cost, and automatic operation. It provides a new liquid biopsy method and idea for diagnosing and monitoring neurodegenerative diseases such as AD.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to a polypeptide targeting neurofilament light chain protein and application thereof. Background Art

[0002] Neurofilaments (NF) are important components of the neuronal cytoskeleton. They maintain the normal morphology of neurons and the elasticity of nerve fibers to prevent breakage. Neurofilament light (NFL) is considered to be the most important part of neurofilaments. Neurofilaments are a neuron skeleton protein that is highly expressed in large-diameter myelinated axons. When central nervous system axonal damage is caused by inflammation, neurodegeneration, trauma or cerebrovascular disease, neurofilaments will be released into the extracellular fluid and cerebrospinal fluid, and released into the peripheral blood at a lower concentration. The amount of release depends on the severity of axonal damage. In recent years, NFL has been used as a candidate biomarker for axonal injury and is receiving more and more attention. With the rapid development of science and technology, the technology for detecting NFL has become more accurate. Clinical studies have found that NFL is closely related to various diseases, and it has important guiding significance in disease diagnosis, guiding disease prognosis, biomarkers, estimating disease severity, testing drug effectiveness, and predicting prognosis. Studies have shown that NFL, as a marker of axonal damage, plays an important guiding role in the diagnosis, medication efficacy, and prognosis evaluation of different neurological diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), traumatic brain injury (TBI), Huntington's disease (HD), and acute ischemic stroke (AIS).

[0003] Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. AD patients usually show changes in memory, judgment, behavior and emotions, which eventually interfere with the patient's control over the body. Due to the aging of the population and the improvement of the quality of life, the number of AD patients will increase several times in the next few decades, from about 50 million people worldwide at present to more than 100 million people in 2050. Alzheimer's disease not only makes patients disabled and suffer great pain, but also causes a large financial burden. In addition, the incidence of Alzheimer's disease gradually increases with age. According to epidemiological data, the incidence rate is about 5% for people over 65 years old, rising to 10% for those over 70 years old, and 30% for those over 80 years old. So far, there is no effective drug for the treatment of AD. The existing drugs can improve the symptoms of some patients, but they are expensive and have limited efficacy, and they cannot prevent or reverse the progression of the disease. With the aging of the population, while waiting for the much-needed drug breakthrough, people have turned their attention to secondary prevention such as early identification and treatment of AD. There is an urgent need for an ideal AD diagnostic technology that has high accuracy and reliability, is non-invasive or minimally invasive, and cost-effective and can be used for large-scale screening to improve pathological diagnosis, achieve early diagnosis, and increase the possibility of treatment.

[0004] By detecting the changes of relevant biomarkers through biochemical methods, the progression of the disease can be reflected in a timely manner to a certain extent. According to the detection method, it can be divided into imaging biomarkers, cerebrospinal fluid biomarkers and blood biomarkers. AD-related imaging biomarkers mainly include MRI and PET diagnosis, which reveal brain atrophy and the aggregation of Aβ, tau and other proteins in the brain respectively. However, due to the complex operation and high cost of these technologies, their application at the primary care level is limited. Cerebrospinal fluid (CSF) is the most reliable and accurate biofluid for AD biomarker assessment, because CSF directly interacts with the extracellular space in the brain, thereby reflecting relevant biochemical / pathological changes, and has been included in the AD diagnostic criteria. However, the disadvantages of CSF extraction are complex and invasive, which limits the application of these methods in the detection of Alzheimer's patients and mild cognitive impairment. Due to the routine and easy popularization of blood testing, samples can be collected and processed using existing systems, and blood-based AD biomarkers can meet the requirements of primary care institutions for extensive screening of large populations. Confirming the cause through blood testing has the three advantages of cost-effectiveness, non-invasiveness and ease of implementation, which is expected to make large-scale dementia diagnosis a new reality. However, blood-based biomarkers have picogram concentrations and require ultrasensitive sensors and analytical techniques to detect.

[0005] NFL levels in cerebrospinal fluid and serum have been investigated from many aspects for their potential as AD biomarkers. Several studies have shown that neurofilament light chain levels in cerebrospinal fluid and blood increase with the progression of AD disease and have good predictive value for future cognitive decline. According to the results of a dominantly inherited Alzheimer's disease network study, a significant increase in the annual rate of change of serum NFL in AD pathogenic mutation carriers can be observed 16.2 years before clinical onset. Moreover, data show that plasma neurofilament light chain levels in AD patients are 1 / 30–1 / 40 of cerebrospinal fluid levels, and the levels of the two are closely correlated, so it is reliable to use neurofilament light chain protein as a blood biomarker. In a study of elderly people without cognitive impairment, it was observed that the cerebrospinal fluid neurofilament light chain levels were elevated in people who experienced cognitive decline during the follow-up period, so it is expected to be used for preventive screening in the preclinical stage of AD. Histopathological examination of NFL-positive nerves in APP / PS1 mice revealed abnormalities, which is consistent with the increase of NFL in AD, indicating potential axonal damage. The study also showed that early elevated serum NFL concentrations were closely associated with the progression of AD-like pathology, and serum neurofilament light chain concentrations decreased in response to anti-Aβ immunotherapy, suggesting that serum neurofilament light chain may be a biomarker of treatment response.

[0006] As a biomarker of axonal injury, accurate measurement of NFL concentration can help understand the development of various diseases. Due to axonal injury in the central nervous system caused by various disease states, NFL is released into the extracellular fluid, cerebrospinal fluid and peripheral blood. The NFL in peripheral blood is lower than that in extracellular fluid and cerebrospinal fluid. In clinical practice, cerebrospinal fluid sampling is a traumatic examination and is not easy to obtain compared to peripheral blood sampling. In clinical practice, it is more desirable to use an accurate determination method to detect NFL in peripheral blood to guide clinical work.

[0007] Therefore, the measurement of NFL concentration in body fluids, especially in blood, which is easily accessible, is expected to be used for preventive screening in the preclinical stage of AD. However, compared with cerebrospinal fluid, blood components are more complex and the content of biomarkers is lower, so it is necessary to establish a blood detection method for AD markers with higher sensitivity and specificity.

[0008] Serum neurofilament light chain protein is a blood marker that can reflect the degree of damage to nerve axons. It can assist in diagnosis, assess the severity of the disease and determine the prognosis in many neurological diseases. At present, there are relatively few studies on probes targeting NfL, clinical data is not sufficient, and specificity and sensitivity analysis of various diseases is still insufficient. This will be a direction and goal of future research.

[0009] Compared with macromolecules such as antibodies, peptides as diagnostic probes have the advantages of being easy to synthesize, easy to chemically modify, good stability, and strong penetration. Developing new peptide probes with high sensitivity and high specificity for NFL, and assembling them into nanostructures to achieve highly sensitive AD serum detection, is expected to provide a new method for the early diagnosis of AD and other neurodegenerative diseases. Summary of the invention

[0010] The purpose of the present invention is to provide a polypeptide targeting neurofilament light chain protein and its application.

[0011] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a polypeptide targeting neurofilament light chain protein, and the general structural formula of the polypeptide is as follows:

[0012] X9X8X7X6X5X4X3X2X1C;

[0013] wherein C is cysteine; X1 is lysine, asparagine or isoleucine; X2 is valine, proline or glutamic acid; X3 is aspartic acid, serine or tryptophan; X4 is glutamic acid, valine, glycine, leucine or threonine; X5 is histidine, tyrosine, arginine or aspartic acid; X6 is aspartic acid, phenylalanine, serine or glutamine; X7 is tryptophan, aspartic acid, isoleucine or tyrosine; X8 is glutamine, aspartic acid, glutamic acid or arginine; and X9 is alanine, leucine, histidine or phenylalanine.

[0014] Furthermore, the polypeptide is Pep12, Pep15, Pep18 Pep30 or Pep39, and the amino acid sequences are (SEQ ID NO: 1-5):

[0015] Pep12: FQWFYENVKC;

[0016] Pep15: FNDFHVWPNC;

[0017] Pep18:FEWSRVWVKC;

[0018] Pep30: HKIDYENVKC;

[0019] Pep39:AEIFYLSVKC.

[0020] Preferably, the polypeptide is Pep12.

[0021] The amino acid residues contained in the polypeptide of the present invention are L-type and / or D-type, as well as sequence variants of amino acid residues in reverse order or cyclic peptide structures.

[0022] In a second aspect, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the polypeptide.

[0023] In a third aspect, the present invention provides a biological material containing the polynucleotide, wherein the biological material includes but is not limited to recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a transgenic cell line.

[0024] In a fourth aspect, the present invention provides a bivalent or multivalent body formed by self-assembly of the polypeptide, wherein the bivalent or multivalent body is formed by covalently linking a linking molecule, or by mixing with a polymer and non-covalently linking.

[0025] The linking molecule can be fluorescein thiocyanate, 6-tert-butyloxycarbonylhydrazinonicotinic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or N-hydroxysuccinimide.

[0026] The polymer may be at least one of polyethylene glycol, polyvinyl alcohol, cyclodextrin, polyamide-amine dendrimer, polylactic acid, polylactic acid-ethanolamine, and the like.

[0027] The bivalent or multivalent form of the present invention has the property of targeting neurofilament light chain protein.

[0028] In a fifth aspect, the present invention provides use of the polypeptide, the polynucleotide, the biomaterial, or the bivalent or multivalent in the preparation of a drug, a diagnostic reagent, a diagnostic kit or a developer for the prevention or treatment of NFL-related diseases.

[0029] The disease includes, but is not limited to, neurodegenerative diseases, inflammation, traumatic or vascular diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, multiple sclerosis, traumatic brain injury, Huntington's disease or acute ischemic stroke.

[0030] In a sixth aspect, the present invention provides a detection reagent, which comprises the polypeptide or the bivalent or multivalent body, and a pharmaceutically acceptable excipient.

[0031] The auxiliary material may be at least one of an excipient, a diluent, a carrier, a flavoring agent, a binder or a filler.

[0032] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0033] (I) The polypeptide of the present invention has a strong binding ability with NFL. The equilibrium dissociation constant KD of the binding kinetic constant between the polypeptide and NFL obtained by surface plasmon resonance technology is 10 -9 Mole / liter order of magnitude.

[0034] (ii) Surface plasmon resonance technology or ELISA combined with NFL peptide can be used to detect the blood signal intensity comparison between AD patients and normal people. This peptide can be used to clearly distinguish patients from normal people.

[0035] (III) The AD diagnostic technology based on this peptide can be used for detection in the early stages of the disease without causing trauma to the patient, and has high detection accuracy and good specificity.

[0036] (iv) The polypeptide of the present invention is easy to synthesize with high efficiency and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the mass spectrometry identification results of the polypeptide molecules Pep12, Pep15, Pep18, Pep30, and Pep39 in the preferred embodiments of the present invention.

[0038] Figure 2 The results of surface plasmon resonance detection of the binding of polypeptide molecules Pep12, Pep15, Pep18 and Pep39 to NFL at concentrations of 7.72nM, 15.44nM, 30.88nM, 61.75nM and 123.5nM in the preferred embodiment of the present invention are shown in Figure 1. Among them, a represents the SPRi binding curve of the positive peptide screened from the high-throughput library to NFL. b represents the binding affinity fitting curve of Pep12, Pep15, Pep18 and Pep39 to NFL.

[0039] Figure 3 This is a graph showing the test results of the specific binding of the polypeptide Pep12 to NFL in a preferred embodiment of the present invention.

[0040] Figure 4 This is the immunofluorescence staining imaging of 5×FAD mouse brain slices with polypeptide Pep12 labeled FITC in a preferred embodiment of the present invention. Among them, ac is the staining of 5×FAD mouse brain tissue slices with FITC-Pep12 probe, a is the polypeptide FITC channel, b is the DAPI nuclear staining channel, and c is the superimposed channel. df is the staining of C57BL / 6 mouse brain tissue slices with FITC-Pep12 probe, d is the polypeptide FITC channel, e is the DAPI nuclear staining channel, and f is the superimposed channel.

[0041] Figure 5 TEM, AFM and DLS characterization of the self-assembly of the polypeptide Pep12 to form a nanostructure in a preferred embodiment of the present invention. Among them, a represents the transmission electron microscopy image of the self-assembly of Pep12 to form a sheet, b represents the atomic force microscopy image of the self-assembly of Pep12 to form a sheet, and c represents the DLS image obtained by assembling Pep12 in water for 0, 4 and 24 hours.

[0042] Figure 6The figure shows the affinity comparison between the assembly, monomer and NFL formed by the polypeptide Pep12 in the preferred embodiment of the present invention.

[0043] Figure 7 The peptide Pep12 assembly combined with SPRi in the preferred embodiment of the present invention detects serum samples of AD patients and healthy people and ROC curve analysis. Wherein, a represents the peptide self-assembly probe detecting serum samples of AD patients (n=31) and healthy control group (n=31). b represents the receiver operating characteristic curve analysis of the test results.

[0044] Figure 8 This is a graph showing the results of an experimental test for evaluating the detection limit of a polypeptide in a preferred embodiment of the present invention in detecting diluted serum spiked with different concentrations of NFL in an AD diagnostic system.

[0045] Fig. 9 This is a graph showing the sensitivity test results of the polypeptide in detecting serum in the AD diagnostic system in a preferred embodiment of the present invention.

[0046] Fig.10 The polypeptide Pep12 of the present invention uses polypeptide self-assembly, monomer and NFL antibody as probes combined with SPRi detection to distinguish serum samples of AD patients and normal people.

[0047] Fig.11 The polypeptide Pep12 assembly formed in the preferred embodiment of the present invention is combined with the PLISA method to detect serum samples of AD patients and healthy people and the ROC curve analysis. Wherein, a represents the polypeptide self-assembly probe based on the PLISA method to detect serum samples of AD patients, MCI and healthy controls. b represents the receiver operating characteristic curve analysis of the test results. DETAILED DESCRIPTION

[0048] The present invention provides a polypeptide targeting neurofilament light chain protein and its application in the diagnosis of Alzheimer's disease. The polypeptide probe has high detection sensitivity, strong specificity, high affinity with neurofilament light chain protein (NFL), can effectively screen the serum of AD patients and normal people through NFL in the serum, has the advantages of short detection time, low detection cost, and automatic operation. It provides a new liquid biopsy method and idea for diagnosing and monitoring neurodegenerative diseases such as AD.

[0049] The polypeptide provided by the present invention can detect the NFL content in the blood in a simpler, more sensitive, lower-cost and non-invasive method to diagnose and monitor Alzheimer's disease, and can effectively distinguish and diagnose patients and normal people through the NFL in body fluids such as serum.

[0050] The present invention adopts the following technical solution:

[0051] In the first aspect, a polypeptide targeting neurofilament light chain protein (NFL) is provided, wherein the polypeptide can specifically bind to the NFL protein, and the general formula of the polypeptide is:

[0052] X9X8X7X6X5X4X3X2X1C;

[0053] wherein X1 is lysine, asparagine or isoleucine; X2 is valine, proline or glutamic acid; X3 is aspartic acid, serine or tryptophan; X4 is glutamic acid, valine, glycine, leucine or threonine; X5 is histidine, tyrosine, arginine or aspartic acid; X6 is aspartic acid, phenylalanine, serine or glutamine; X7 is tryptophan, aspartic acid, isoleucine or tyrosine; X8 is glutamine, aspartic acid, glutamic acid or arginine; X9 is alanine, leucine, histidine or phenylalanine; and C is cysteine.

[0054] The amino acid residues that make up a polypeptide can be L-type, D-type, or a mixture of L-type and D-type.

[0055] Furthermore, the polypeptide provided by the present invention is as follows:.

[0056] Pep12: FQWFYENVKC;

[0057] Pep15: FNDFHVWPNC;

[0058] Pep18:FEWSRVWVKC;

[0059] Pep30: HKIDYENVKC;

[0060] Pep39:AEIFYLSVKC.

[0061] The small molecule affinity peptide of the present invention specifically binds to NFL. Further, the small peptide of the present invention can be used to detect NFL.

[0062] In a second aspect, the present invention provides a DNA fragment comprising a nucleotide sequence encoding the polypeptide described in the first aspect of the present invention.

[0063] In a third aspect, the present invention provides an expression vector comprising at least one copy of the DNA fragment.

[0064] In a fourth aspect, the present invention provides a prokaryotic or eukaryotic host cell, wherein the host cell contains the expression vector described in the third aspect of the present invention.

[0065] In a fifth aspect, the present invention provides a bivalent or multivalent body assembled from the polypeptide described in the first aspect of the present invention.

[0066] The bivalent or multivalent form of the present invention has the property of targeting neurofilament light chain protein.

[0067] As a preferred technical solution, the divalent or multivalent body of the present invention is formed by covalently linking linking molecules; or is formed by non-covalently linking through self-assembly; or is formed by mixing with polymers and non-covalently linking.

[0068] Preferably, the linking molecule is fluorescein isothiocyanate (FITC), 6-tert-butyloxycarbonylhydrazinonicotinic acid (HYNIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS); the linking molecule is a new type of non-toxic cross-linking agent with good biocompatibility.

[0069] The polymer of the present invention can be selected according to specific needs, for example, it can be any one of polyethylene glycol (PEG), polyvinyl alcohol (PVA), cyclodextrin, polyamide-amine dendrimer (PAMAM), polylactic acid (PLA), polylactic acid-ethanolamine (PLGA) or a mixture of at least two thereof.

[0070] In a sixth aspect, the present invention provides an NFL detection agent, wherein the NFL detection agent comprises the polypeptide as described above.

[0071] The polypeptide of the present invention can be used as a detection agent for detecting NFL, or as a component of a detection agent for detecting NFL.

[0072] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the polypeptide as described above.

[0073] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0074] Preferably, the pharmaceutically acceptable excipient is any one of an excipient, a diluent, a carrier, a flavoring agent, a binder or a filler, or a combination of at least two thereof.

[0075] In an eighth aspect, the present invention provides the use of the above-mentioned polypeptide, bivalent or multivalent body or pharmaceutical composition in the preparation of a drug for detecting, diagnosing and / or monitoring a disease associated with NFL. The details are as follows:

[0076] (1) Use in the preparation of drugs, diagnostic reagents, diagnostic kits or imaging preparations for the prevention or treatment of NFL-related diseases;

[0077] (2) Use in the diagnosis, prevention or treatment of NFL-related diseases;

[0078] (3) Application in the preparation of diagnostic reagents for detecting the expression level of NFL in biological samples such as human tissues, cells, serum, plasma, urine, and cerebrospinal fluid;

[0079] (4) Application in detecting the expression level of NFL in body fluids or peripheral blood;

[0080] The disease includes neurodegenerative lesions, inflammation, traumatic or vascular lesions, etc. Preferably, the disease includes any one of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, traumatic brain injury, Huntington's disease, acute ischemic stroke, etc.

[0081] Preferably, the application means comprises using the polypeptide, bivalent or multivalent body or pharmaceutical composition that specifically recognizes NFL as described above as a probe molecule, combined with surface plasmon resonance imaging technology, ELISA, PLISA and biochip to detect the content of NFL in serum.

[0082] Furthermore, the application method also includes using the polypeptide, bivalent or multivalent body labeled with the fluorescent dye that specifically recognizes NFL as described above as a probe molecule, and using a fluorescence spectrometer or an enzyme marker to detect the NFL content in the serum adsorbed on the carrier.

[0083] Preferably, the fluorescent dye is fluorescein isothiocyanate (FITC), Cy3, Cy5, etc.; the carrier is any one of an immunoassay plate, a 96-well plate, an epoxy-modified glass slide, and a plasma gold chip.

[0084] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0085] The SPRi instrument used in the following examples is Plexera Kx5V2, Plexera Bioscience LLC, which is mainly equipped with a 660nm LED light source, a CCD image collector and a sensor chip with a microfluidic channel. The instrument displays the change of the reflected light intensity at each monitoring point over time and records it as an SPR curve.

[0086] Unless otherwise specified, the "AD patient" in the present invention refers to a patient with Alzheimer's disease.

[0087] Unless otherwise specified, "μM" in the present invention means "μmol / L" and "mM" means "mmol / L".

[0088] Unless otherwise specified, the solvents of the aqueous solutions used in the following examples are all sterile ultrapure aqueous solutions.

[0089] Unless otherwise specified, all reagents used in the following examples were analytically pure.

[0090] Unless otherwise specified, the PBS solutions used in the following examples are all 1×PBS solutions.

[0091] Example 1 Construction and screening of NFL targeting peptide library

[0092] This example provides a method for constructing and screening an NFL targeting polypeptide library, comprising the following steps:

[0093] 1. Experimental instruments and materials

[0094] N-Methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), ethanedithiol (EDT), N,N-dimethylformamide (DMF), anhydrous ether, resin, methanol, various Fmoc-protected amino acids, streptavidin magnetic beads (MB-Streptavidin), biotin labeling kit, peptide synthesis tube, shaker, vacuum water pump, rotary evaporator, laser confocal microscope (ZEISS LSM 710), the above reagents and materials were obtained from commercial sources.

[0095] 2. Solvent preparation

[0096] Deprotection solvent - piperidine: N,N-dimethylformamide = 1:4 (volume ratio).

[0097] Reaction solution: N-methylmorpholine: N,N-dimethylformamide = 1:24 (volume ratio).

[0098] Lysis solution: trifluoroacetic acid (92.5% by volume), triisopropylsilane (2.5% by volume), ethanedithiol (2.5% by volume) and ultrapure water (2.5% by volume).

[0099] Ninhydrin test solution - Ninhydrin: Vitamin C: Phenol = 1:1:1 (mass ratio)

[0100] 3. Synthesis of NFL "one bead one substance" peptide library

[0101] The peptide library was synthesized by the Fmoc solid phase synthesis strategy using the "mixed splitting" method with TentaGel resin (loading capacity 0.26mmol / g) as the carrier. During the OBOC library synthesis process, the resin beads were equally separated in each cycle, and different Fmoc-amino acids and HBTU dissolved in DMF containing 0.4mol / L NMM were added, and each coupling time was 1h. The Fmoc group was then removed in DMF containing 20% ​​(v / v) piperidine, and each deprotection time was 10min. The ninhydrin experiment was used to test whether the Fmoc protecting group was completely removed and whether each terminal amino group on the resin was coupled to a new amino acid. Finally, a cleavage reagent mixture (95% TFA: 5% water, v / v) was used to remove the side chain protecting groups of each amino acid for subsequent screening.

[0102] The specific steps are as follows:

[0103] 1) Resin swelling: Weigh 600 mg of resin and add DMF to swell for 1 h.

[0104] 2) First, 700 mg of methionine and an equal amount of peptide coupling reagent HBTU were added to react. After deprotection and washing, 700 mg of glycine and an equal amount of peptide coupling reagent HBTU were added in the second step.

[0105] 3) Divide into 3 tubes, add 250 mg of lysine and an equal amount of HBTU, asparagine and an equal amount of HBTU, isoleucine and an equal amount of HBTU respectively for coupling, and react for 1-2 hours. When the reagent does not change color, it means that the coupling is complete. Add hexahydropyridine solution to each tube to remove the amino protecting group at the N terminal. When the reagent changes color significantly, it means that the reaction is complete. Then wash the resin with DMF and DCM and merge the three tubes of resin into one tube.

[0106] 4) The mixture was then divided into 3 tubes, and valine and an equal amount of HBTU, proline and an equal amount of HBTU, and glutamic acid and an equal amount of HBTU were added to react respectively. After the reaction, piperidine solution was added to remove the Fmoc protecting group, and DMF and DCM were used for washing three times alternately, and then mixed into one tube.

[0107] 5) Divide the mixture into three parts, add aspartic acid and an equal amount of peptide coupling agent, serine and an equal amount of HBTU, tryptophan and an equal amount of HBTU respectively for coupling, deprotection, washing with DMF and DCM three times each and then mixing.

[0108] 6) Divide the mixture into 5 equal portions, add glutamic acid, valine, glycine, leucine, threonine and an equal amount of HBTU solution respectively to start the reaction, and after the reaction is completed, add piperidine solution for deprotection, wash with DMF and DCM three times each and then mix again.

[0109] 7) Divide the mixture into 4 equal parts, add histidine, tyrosine, arginine, aspartic acid and an equal amount of peptide coupling agent to each tube for coupling. After the reaction is completed, deprotect the mixture in each tube, wash it with DMF and DCM three times each, and then mix them again.

[0110] 8) Divide the mixture into 4 equal parts, add aspartic acid, phenylalanine, serine, glutamine and an equal amount of HBTU to each tube for coupling. After the reaction, deprotect the mixture in each tube, wash the mixture three times and then mix the mixture.

[0111] 9) Divide the mixture into 4 equal parts, add tryptophan, aspartic acid, isoleucine, tyrosine and an equal amount of HBTU to each tube for coupling, and after the reaction is completed, deprotect the mixture in each tube, wash it three times and then mix it.

[0112] 10) Divide the mixture into 4 equal parts, add glutamine, aspartic acid, glutamic acid, arginine and an equal amount of peptide coupling agent to each tube for coupling, and after the reaction is completed, deprotect the mixture in each tube, wash it three times and then mix it.

[0113] 11) Divide the mixture into 4 equal parts, add alanine, leucine, histidine, phenylalanine and an equal amount of HBTU to each tube for coupling, and after the reaction is completed, deprotect the mixture in each tube, wash the mixture three times and then mix the mixture.

[0114] 12) Add 700 mg of cysteine ​​and an equal amount of HBTU to react. After the reaction is completed, deprotect each tube, wash three times and then mix.

[0115] 13) Add cleavage solution to the resin after the reaction is completed, react at room temperature for 2 hours, remove the protecting group, wash and condense with anhydrous methanol, drain and store in a -20°C refrigerator.

[0116] 4. Screening of NFL-positive peptides

[0117] (1) The dried peptide library was swollen with 1× PBS overnight, then washed three times with 1× PBS, 5% skim milk was added to block the surface of the peptide beads on a vortexer at 37°C for 2 h, and then washed three times with 1× PBS;

[0118] (2) Label the NFL protein using the biotin labeling kit, mix the biotin-labeled NFL protein with the peptide library, incubate at 37°C for 2 h, and then wash three times with 1× PBS;

[0119] (3) Then take 100 μL MB-Streptavidin and add it to the peptide library. Incubate it in a vortexer at 37°C in the dark for 2 h. Place the EP tube containing the peptide library after incubation on a magnetic rack. The positive peptides are adsorbed to the side wall of the EP tube by the magnetic force, while the negative peptides settle to the bottom of the EP tube due to gravity.

[0120] When the positive peptide beads are incubated with the biotin-labeled receptor protein, the positive peptide beads specifically recognize the protein, and the magnetic ball labeled with streptavidin recognizes the positive peptide beads by recognizing biotin. The surface of the positive peptide beads will be coated with a layer of magnetic beads with magnetism, so that they can be captured by the magnetic field. In order to crack by cyanogen bromide, secondary mass spectrometry identification is performed, and then the corresponding sequence information is solved using the Mascot database. The positive peptide part is resynthesized according to the sequence and marked with fluorescence, MALDI-TOF identification and HPLC purification are used for subsequent experiments. The polypeptide of the present invention is obtained by chemical synthesis ( Figure 1 ).

[0121] Example 2 Testing the binding ability between polypeptide and NFL using surface plasmon resonance imaging technology

[0122] The binding ability between peptide and NFL was tested by surface plasmon resonance imaging technology. The specific method is as follows:

[0123] 1. Dissolve the polypeptide in ddH2O to a concentration of 1-1000 μM. Spot the polypeptide sample prepared by the method of Example 1 on the surface of a 3D chip, repeat 3 spots for each sample, incubate at 4°C for 12 hours, and then wash with 10×PBS, 1×PBS, and ultrapure water. Then seal the chip with 5% (w / v) skim milk at 4°C for 12 hours, then wash with 10×PBS, 1×PBS, and ddH2O for 10 minutes each, and finally blow dry with clean nitrogen.

[0124] 2. Install the chip on the SPRi instrument, measure the SPRi angle and adjust it to the optimal optical position, select relevant detection points in the detection area, including sample points and blank points, and set the experimental flow rate to 2 μL / s.

[0125] 3. Select PBST as the buffer and pass it into the circulation pool until the baseline is stable. Then use NFL with concentrations of 7.72nM, 15.44nM, 30.88nM, 61.75nM and 123.5nM for detection in sequence. The binding time is 300 seconds, and the dissociation time is 300 seconds. Phosphoric acid is passed between each concentration for regeneration.

[0126] Test results see Figure 2 (AU is a unit used to reflect the strength of binding signals in surface plasmon resonance imaging and is a dimensionless unit) as shown, after fitting, the kinetic constants and binding dissociation constants are calculated, so as to select the sequence with the strongest interaction.

[0127] Table 1

[0128] serial number Amino acid sequence of the peptide <![CDATA[K D (mol / L)]]> Pep12 FQWFYENVK <![CDATA[1.39×10 –9 ]]> Pep15 FNDFHVWPNC <![CDATA[7.51×10 –9 ]]> Pep18 FEWSRVWVKC <![CDATA[5.74×10 –7 ]]> Pep30 HKIDYENVKC <![CDATA[9.78×10 –6 ]]> Pep39 AEIFYLSVKC <![CDATA[4.81×10 –7 ]]>

[0129] The peptide sequences and corresponding KD values ​​are shown in Table 1. The affinity range of the five positive peptides is 10 -6 Up to 10 -9 mol / L. Among them, the dissociation constant value of Pep12 and NFL is 1.39×10 -9 mol / L, indicating that the peptide had a fairly high affinity level for NFL, so Pep12 was selected for subsequent research.

[0130] Example 3 Investigation of the specificity of peptide binding to NFL

[0131] In this example, surface plasmon resonance imaging technology was used to test the specificity of the peptide binding to NFL. The specific method is as follows:

[0132] 1. As in Example 2, the same SPRi chip was manufactured and installed on the SPRi instrument. The SPRi angle was measured and adjusted to the optimal optical position. Relevant detection points were selected in the detection area, including sample points and blank points. The experimental flow rate was set to 2 μL / s.

[0133] 2. PBST was selected as the buffer and passed into the circulation pool until the baseline was stable. Then, NFL, bovine serum albumin (BSA), immunoglobulin M, β-amyloid protein, and Tau protein with a concentration of 123.5 nM were tested in turn. The binding time was 300 seconds and the dissociation time was 300 seconds. Phosphoric acid was passed between each concentration for regeneration.

[0134] At the same concentration, the ΔAU values ​​of Pep12 binding signals to BSA, Aβ, Tau and other proteins were all below 0.4, while the ΔAU value of Pep12 binding signals to NFL was around 1.8, much higher than other proteins, indicating that it has good specificity ( Figure 3 ), the binding of the Pep12 polypeptide of the present invention to NFL has high specificity.

[0135] Example 4 Detection of NFL in Mouse Brain Tissue by Peptides

[0136] Mouse brain slice staining: Frozen sections were prepared from brain tissues of 5×FAD transgenic AD model mice carrying five familial gene mutations (AD model mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and C57BL / 6 mice. The brain slices were rinsed in TBST three times, 10 minutes each time. Then they were blocked with 5% BSA overnight and washed three times with TBST. Then, 1mg / mL FITC-labeled peptide solution was prepared with 5% BSA, and 50uL of peptide solution was added to the surface of the slices, and incubated at room temperature for 2h in the dark. Nuclear staining was performed with 1:200 Hoechst 33342 dye solution, and incubated at room temperature in the dark for ten minutes. Wash three times with TBST and image under a single-photon confocal microscope.

[0137] Using 5×FAD transgenic mice as a model, FITC-labeled peptides were used for fluorescent staining. First, FITC-Pep12 peptides were incubated with frozen sections of brain tissues of 5×FAD mice and C57BL / 6 mice, and the cell nuclei were stained with Hoechst dye. Among them, FITC was displayed by the green channel and Hoechst was displayed by the blue channel. The results showed that FITC-Pep12 had a stronger binding effect on brain tissue cells of 5×FAD mice ( Figure 4 ), but had almost no specific binding effect on the brain slices of C57BL / 6 mice.

[0138] Example 5 Preparation and characterization of polypeptide self-assembly

[0139] With the help of 0.1% DMSO, the Pep12 peptide was dissolved in ddH2O to make a 1mM solution, placed in a constant temperature shaker at 37°C, and incubated for 0, 4 and 24 hours respectively to obtain the assembly. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) characterization of surface morphology: The assembled peptide solution was diluted ten times with water. For AFM, 20μL of peptide solution was placed on mica and incubated for 15 minutes, carefully washed twice with deionized water, and blown dry with an ear bulb. The test was performed in an air environment using Multimode 8 in smart mode, and the obtained image was flattened using Nanoscale software. For TEM characterization, 10μL of solution was dropped on a copper grid and incubated for 10 minutes. After being sucked away, it was stained with uranyl acetate and the excess dye was washed off with ddH2O. After the sample was dried, it was imaged by HT-7700TEM.

[0140] TEM and AFM results show that Pep12 initially formed a nanostructure, which evolved into a larger size within 24 hours. It can be seen that the nanostructure was enlarged 5 times to a size of about 2 μm after 4 hours and continued to grow larger after 24 hours ( Figure 5 ). DLS spectroscopy further confirmed the time dependence of the size changes of the peptides in water, with the size of the nanostructures increasing over time.

[0141] Example 6 Detection of AD patients

[0142] Pep12 polypeptide self-assembly (prepared in Example 5), Pep12 polypeptide monomer and anti-NFL antibody (purchased from Abcam) were used as probes to detect 30 AD serum samples and 30 healthy control serum samples provided by the Union Hospital Affiliated to Fujian Medical University. It was found that there was a significant difference between the healthy control group and the AD group in the detection of Pep12 polypeptide self-assembly as a probe, and the signal of the AD group was higher than that of the normal group (P<0.001). The detection effect of Pep12 polypeptide monomer (P<0.01) was inferior to that of polypeptide self-assembly ( Figure 6). When anti-NFL antibody was used as a probe, the overall signal was weak and the discrimination ability was the weakest (P<0.05). The detection results of the peptide self-assembly were subjected to receiver operating characteristic curve (ROC) analysis, and the area under the curve (AUC) was calculated. Figure 7 As shown, the AUC value of Pep12 polypeptide self-assembly for the healthy control group and AD group was 0.7791.

[0143] Example 7 Evaluation of polypeptide detection performance

[0144] Serum sample detection and peptide performance evaluation: With 0.1% DMSO as a solubilizer, the peptide was dissolved in ddH2O to make a 1mM solution, and placed in a 37°C constant temperature shaker for incubation for 24 hours to obtain a peptide self-assembly solution; the peptide powder was dissolved in 1% ammonia water to obtain a 1mg / mL monomer solution. The assembled and monomeric peptide solution and antibody solution were dropped on the designated area of ​​the thiol-modified bare gold chip, with three replicates for each sample point. Among them, the serum sample was diluted with buffer at 1:2000, and the regeneration solution was replaced with 5mM NaOH solution. For the test of serum sensitivity, the sample was diluted twice from 1:2000 to 1:64000 with PBST. In the detection limit evaluation experiment, NFL protein was added to the diluted serum, so that the final concentration of the added protein ranged from 1.2pM to 12000pM as the mobile phase. In the antibody detection experiment, diluted protein and NFL antibody were used as mobile phases, and the detection program was set: PBS was passed at a flow rate of 2 μL / s for 120 s to generate a baseline; the protein solution was passed at a flow rate of 2 μL / s for 300 s; the PBST solution was passed at a flow rate of 2 μL / s for 300 s for desorption; the NFL antibody was passed at a flow rate of 2 μL / s for the detection process; and 5 mM NaOH was passed at a flow rate of 2 μL / s for 300 s for the regeneration process.

[0145] Since serum components are complex and contain a wide variety of proteins, the ability of peptide self-assemblies to detect NFL in the complex system of serum needs to be further tested. Figure 8 As shown in the figure, different concentrations of NFL protein were added to the serum sample and then used as the mobile phase to combine with the peptide self-assembly. It was found that in the range of 1.2pM to 12000pM, there was a good linear relationship between the spiked NFL concentration and the detection signal, R 2 It is 0.9784.

[0146] Example 8 Testing the Sensitivity of AD Diagnostic System Using Peptides

[0147] This example uses surface plasmon resonance imaging technology to test the detection limit of the AD diagnostic system for serum. The specific method is as follows:

[0148] 1. As in Example 2, the same SPRi chip was manufactured and installed on the SPRi instrument. The SPRi angle was measured and adjusted to the optimal optical position. Relevant detection points were selected in the detection area, including sample points and blank points. The experimental flow rate was set to 2 μL / s.

[0149] 2. PBST was selected as the buffer and passed into the circulation pool until the baseline was stable. Then, serum dilutions of different patients and normal subjects were passed into the circulation pool. The dilution concentrations were 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000, respectively. The binding time was 300 seconds, and the dissociation time was 300 seconds. 5 mM NaOH was passed between each sample for regeneration.

[0150] In order to evaluate the sensitivity of the detection method, serum was gradiently diluted to different concentrations as the mobile phase. Fig. 9 As shown, the results showed that when the serum sample was diluted at 1:2000, the Pep12 polypeptide self-assembly and AD blood samples produced a signal of about 1400ΔRU, while the blood samples of healthy controls of similar age only produced about 1000ΔRU. The results showed that this dilution factor can significantly distinguish the AD group from the NC group, and is suitable for serum detection under this system. As the dilution factor gradually increased, it can be seen that the ΔRU values ​​of the two groups of test results continued to decrease; when the dilution ratio was 1:4000, it still had the ability to distinguish AD from normal controls. When the dilution factor was increased to 1:8000, the binding signal with the patient's serum showed the extremely high sensitivity of the system for the diagnosis of AD blood samples. Subsequently, the dilution factor was further increased, but the serum of AD patients and healthy controls was almost indistinguishable. The test results are shown in Fig. 9 As shown, when the serum dilution ratio is less than or equal to 1:16000, AD patients can be clearly distinguished from normal people, showing extremely high sensitivity.

[0151] Example 9 Detection of serum signals using polypeptides

[0152] This example uses surface plasmon resonance imaging technology to test peptides to detect serum from AD patients and normal human serum. The specific method is as follows:

[0153] 1. As in Example 2, the same SPRi chip was manufactured and installed on the SPRi instrument. The SPRi angle was measured and adjusted to the optimal optical position. Relevant detection points were selected in the detection area, including sample points and blank points. The experimental flow rate was set to 2 μL / s.

[0154] 2. PBST was selected as the buffer and passed into the circulation pool until the baseline was stable. Then, serum dilutions of different patients and normal subjects (1:2000) were passed into the circulation pool respectively. The dissociation time was 300 seconds. 5 mM NaOH was passed between each sample for regeneration.

[0155] Test results such as Fig.10 As shown, Pep12 monomers and assemblies can clearly distinguish AD patients from normal people. The result of peptide self-assembly is the best, followed by peptide monomers. When anti-NFL antibody is used as a probe, it is found that the overall signal is weak and the ability to distinguish is the weakest.

[0156] Example 10 Detection of serum samples of AD patients by ELISA based on polypeptide Pep12

[0157] PLISA method:

[0158] 1. After washing the 96-well plate with PBS, add 3 μL of serum to the plate, dilute to 100 μL with PBS, and incubate at 37°C for 2 h.

[0159] 2. After discarding the liquid, block the plate with 5% BSA and incubate at 37°C for 1 hour;

[0160] 3. Wash the plate twice with wash buffer;

[0161] 4. Add 10 μg / mL FITC-labeled peptide solution and incubate at 37°C for 1 h;

[0162] 5. After discarding the liquid, wash the plate twice with washing buffer;

[0163] 6. Dissolve the polypeptide molecules on the well plate with DMSO and mix well by pipetting;

[0164] 7. Read the plate using an ELISA reader.

[0165] Test results such as Fig.11 As shown, Pep12 assembly can clearly distinguish AD patients, MCI and normal people. The detection sensitivity of PLISA method using peptide instead of antibody is similar to that of SPRi detection method, indicating that Pep12 peptide has good specificity and sensitivity and is suitable for a variety of detection methods.

[0166] In summary, it can be concluded from Experimental Examples 1-10 that the affinity NFL polypeptide of the present invention, especially Pep12, is a polypeptide that binds to NFL in serum with high sensitivity and specificity, and provides a new liquid biopsy method and idea for diagnosing and monitoring AD.

[0167] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A polypeptide targeting neurofilament light chain protein, characterized in that: The polypeptide is Pep12, and the amino acid sequence is: Pep12: FQWFYENVKC; The amino acid residues contained in the polypeptide are in L-form.

2. A bivalent or multivalent body formed by self-assembly of the polypeptide according to claim 1, characterized in that: The divalent or multivalent body is formed by non-covalent linkage.

3. Use of the polypeptide according to claim 1, or the bivalent or multivalent polypeptide according to claim 2 in the preparation of a diagnostic reagent or a diagnostic kit for Alzheimer's disease.

4. A detection reagent, characterized in that: The detection reagent comprises the polypeptide of claim 1 or the bivalent or multivalent of claim 2, and pharmaceutically acceptable excipients; Wherein, the auxiliary material is at least one of an excipient, a diluent, a carrier, a binder or a filler.

Images