WO2016078546A1 - Alzheimer's disease (ad) preventive drug - Google Patents
Alzheimer's disease (ad) preventive drug Download PDFInfo
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- WO2016078546A1 WO2016078546A1 PCT/CN2015/094568 CN2015094568W WO2016078546A1 WO 2016078546 A1 WO2016078546 A1 WO 2016078546A1 CN 2015094568 W CN2015094568 W CN 2015094568W WO 2016078546 A1 WO2016078546 A1 WO 2016078546A1
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
Definitions
- the invention relates to the field of drug research and development, and particularly relates to a polypeptide and application thereof in preparing a medicament for treating or preventing senile dementia.
- AD Alzheimer Disease
- AD is a common neurodegenerative disease. Its main symptoms are memory loss, low cognitive ability and slow thinking, and progressive aggravation. Finally, life cannot take care of itself and die. ⁇ 10 years. With the acceleration of the aging process of society, the number of patients with AD has risen sharply, and it has become a serious disease that threatens the physical and mental health of the elderly, especially the elderly, and brings serious social, economic and family problems.
- NFTs neurofibrillary tangles
- a ⁇ ⁇ -amyloid
- NFTs Since the discovery that the main component of NFTs is a double helix formed by the aggregation of abnormal, hyperphosphorylated Tau protein (Morris M, Maeda S, Vossel K, Mucke L. The many faces of tau. Neuron. 2011 May 12; 70(3) : 410-26), domestic and foreign scholars generally agree that the abnormality and hyperphosphorylation of the skeleton protein tau is a key step in the formation of NFTs. The accumulation of hyperphosphorylated tau protein is not only present in entangled form, but it is also a major component of dystrophic processes in neuropilic fibrils and senile plaques.
- Alzheimer's disease There are currently no specific treatments and methods for the pathological features of Alzheimer's disease.
- Commonly used drugs to alleviate Alzheimer's disease are: (1) Improve choline neurotransmitter: One of the main causes of Alzheimer's disease is memory loss due to choline deficiency.
- acetylcholinesterase (AchE) inhibitors play an important role in the treatment of senile dementia by enhancing cholinergic effects;
- drugs that improve cerebral blood circulation and brain cell metabolism these drugs can dilate cerebral blood vessels to improve senile dementia Patient's sugar, protein, nucleic acid, Metabolic disorders such as lipids;
- calcium antagonists these drugs easily pass the blood-brain barrier, selectively dilate the cerebral blood vessels, thereby improving memory and cognitive function;
- hormonal drugs mainly relieve the symptoms of female patients.
- these drugs only slightly improve the symptoms of dementia in patients with advanced senile dementia or delay the progression of dementia. Therefore, the development of new drugs for the pathological features of Alzheimer's disease has become a global expectation.
- TAT is a kind of cell penetrating peptides, which is an efficient transport carrier discovered in recent years. TAT can penetrate the cell membrane, nuclear membrane, carrying peptides, proteins and DNA molecules to enter the cytoplasm and nucleus through receptor transport to exert corresponding biological effects (Kilic E, Kilic U, Hermann DM.TAT fusion proteins against ischemic stroke: Current status and future perspectives. Front Biosci. 2006 May 1; 11:1716-21). Current research shows that HIV-TAT is able to cross all tissue cells without significant toxic side effects. TAT can bring the polypeptides connected to it into the cells within a few minutes, and can enter the neurons across the blood-brain barrier. The polypeptides brought into the cells retain their original biological activities and thus play a biological role.
- PTPN1 also known as PTP1B, Tonks NK.Protein tyrosine
- Phosphases--from housekeeping enzymes to masterregulators of signal transduction FEBS J. 2013 Jan; 280(2): 346-78.
- Alzheimer's disease transgenic animal model By applying to the in vivo Alzheimer's disease transgenic animal model, it effectively exerts its biological function of blocking the binding of miR-124 to PTPN1 3'-UTR, promotes PTPN1 expression, and improves microtubule-associated protein in senile dementia model animals.
- Tau hyperphosphorylation, amyloid deposition, and animal learning and memory disorders provide molecular targets for further development of clinical treatments for Alzheimer's disease.
- polypeptide selected from the group consisting of:
- polypeptide comprising a substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1.
- the polypeptide of the invention is TAT-siP-PTPN1, the sequence of which is set forth in SEQ ID NO: 1. That is, the TAT transmembrane peptide (GRKKRRQRRRPRQ) was ligated with siP-PTPN1 (a polypeptide that blocks the binding site of miR-124 and PTPN1, we named silencing peptide PTPN1, abbreviated as siP-PTPN1), of which siP-PTPN1 The sequence is PYGCRVIQRIGNYVIQHVASNNVEKIGGYVIRHVGGYVIRHVGNYVIQHVGNYVIQHVGCRVIQRIL.
- the polypeptide of the present invention utilizes the transmembrane function of TAT to transport the siP-PTPN1 polypeptide into the bloodstream through the blood-brain barrier and is taken up by the brain nerve cells to exert its biological function.
- a tag polypeptide such as HA (YPYDVPDYA)
- HA YPYDVPDYA
- a preferred polypeptide sequence of the invention is the sequence in which HA (YPYDVPDYA) is added at the end of SEQ ID NO: 1.
- a preferred polypeptide of the invention is TAT-siP-PTPN1-HA (SEQ ID NO: 4).
- Another object of the invention is also to provide a nucleotide sequence encoding a polypeptide of the invention.
- the nucleotide sequence of the present invention is a nucleotide sequence encoding the small molecule polypeptide TAT-siP-PTPN1, preferably shown by SEQ ID NO: 2, but is not limited thereto. Due to the degeneracy of the codons, all nucleotide sequences encoding TAT-siP-PTPN1 polypeptides are within the scope of the invention.
- the nucleotide sequence encoding the TAT-siP-PTPN1-HA polypeptide is SEQ ID NO:3.
- the invention still further provides a nucleic acid construct comprising a nucleotide sequence of the invention operably linked to one or more controls that direct production of the polypeptide in a suitable host sequence.
- the invention still further provides a recombinant expression vector comprising the nucleic acid construct.
- Another object of the present invention is to provide a use of the polypeptide of the present invention for the preparation of a medicament for the treatment or prevention of Alzheimer's disease.
- the present invention provides the use of a polypeptide TAT-siP-PTPN1 for the preparation of a medicament for treating or preventing Alzheimer's disease.
- the polypeptide of the present invention is made into an intravenous injection, and after intravenous injection, it is found to be effective for improving hyperphosphorylation, amyloid deposition, animal learning and/or memory of the microtubule-associated protein tau exhibited by the senile dementia model animal. Obstacles provide molecular targets for further development of drugs for clinical treatment of Alzheimer's disease.
- Another object of the present invention is to provide a medicament comprising the small molecule polypeptide of the present invention. Composition.
- the technical measures adopted by the present invention are: Applicants have found that the 3'-UTR of m iR-124 and PTPN1 in the dementia model animal bind to each other and mediate the hyperphosphorylation of the downstream microtubule-associated protein tau, Amyloid deposition, as well as animal learning and memory disorders. Blocking the 3'-UTR of miR-124 and PTPN1 to each other can effectively improve the above symptoms.
- the polypeptide enters the bloodstream and crosses the blood-brain barrier and is taken up by the brain's nerve cells to exert its biological function.
- TAT-scramble-HA SEQ ID NO: 5
- TAT-siP-PTPN1-HA SEQ ID NO: 4
- This control sequence was obtained by artificially scrambling the siP-PTPN1 sequence.
- the TAT-siP-PTPN1-HA polypeptide and the control TAT-scramble-HA were obtained by the applicant by prokaryotic expression, collection, and purification.
- the invention proves that TAT-siP-PTPN1-HA has an exact therapeutic effect on the Alzheimer's model by applying TAT-siP-PTPN1-HA in an animal model of Alzheimer's disease.
- the present invention has the following characteristics: (1) The small molecule polypeptide of the present invention is easy to induce expression purity, is completely soluble, is suitable for intravenous injection, and has no toxic side effects. (2) The polypeptide disclosed by the present invention is taken up by neurons through the blood-brain barrier, and can be transformed into a nervous system disease such as Alzheimer's disease, so that it has practical feasibility.
- Coding sequence means a polynucleotide that directly specifies the amino acid sequence of a polypeptide.
- the boundaries of the coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA).
- the coding sequence can be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
- control sequence means a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide of the invention.
- Each control sequence may be native (ie, from the same gene) or foreign (ie, from a different gene) to the polynucleotide encoding the polypeptide, or be native or foreign to each other.
- control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At least, the control sequence includes a promoter, And transcription and translation termination signals.
- These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linkage of these control sequences to the coding region of a polynucleotide encoding a polypeptide.
- expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
- Expression vector means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence providing for its expression.
- host cell means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
- host cell encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication.
- nucleic acid construct means a single or double stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner that is not inherent in nature. A segment containing a nucleic acid, or a synthetic nucleic acid molecule comprising one or more control sequences.
- operably linked means a construct in which the control sequence is placed in position relative to the coding sequence of the polynucleotide such that the control sequence directs expression of the coding sequence.
- Sequence identity The degree of association between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
- EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.) 16:276-277
- Needleman-Wunsch algorithm implemented in the Needle program (Needleman and Wunsch) (1), J. Mol. Biol. 48: 443-453) to determine sequence identity between two amino acid sequences.
- These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
- the output of the "longest identity" of the Nieder annotation (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows: (consistent residue ⁇ 100) / (alignment length - total number of gaps in the alignment)
- Niedel as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al, 2000, supra) (preferably version 5.0.0 or later)
- the Nederman-Wengsch algorithm (Nedleman and Wunsch, 1970, supra) implemented in the program was used to determine sequence identity between two deoxyribonucleotide sequences. These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
- the output of the "longest identity" of the Niedel label (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows: (consistent deoxyribonucleotide ⁇ 100) / (alignment length - ratio The total number of vacancies in the pair)
- the present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
- the polypeptide of the present invention has the polypeptide of the amino acid sequence of SEQ ID NO: 1.
- the present invention also relates to a polypeptide comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:1, which polypeptides are capable of For the treatment or prevention of senile dementia.
- the invention relates to a polypeptide comprising a substitution, deletion and/or insertion of one or more (several) amino acid residues in the amino acid sequence of SEQ ID NO: 1.
- the polypeptide of the invention differs from the mature polypeptide of SEQ ID NO: 1 by no more than 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
- amino acid changes can have subtle properties, ie, conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein.
- conservative substitutions are within the scope of the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and day) Asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine) , threonine and methionine).
- Amino acid substitutions that do not generally alter specific activity are known in the art and are for example, by H. Neurat and RL Hill, 1979, The Proteins, Academic Press (Academic Press) ), described in New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg , Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
- amino acid changes have the property of altering the physicochemical properties of the polypeptide.
- amino acid changes can increase the thermal stability of the polypeptide, alter substrate specificity, change the pH optimum, and the like.
- Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known methods of mutagenesis, recombination and/or shuffling, followed by relevant screening procedures, such as by Reidhaar (Reidhaar) -Olson) and Sauer, 1988, Science 241: 53-57; Bowie and Saar, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625.
- Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and regional targeting Change (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
- the mutagenesis/shuffling method can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896).
- Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.
- Variants of these polypeptides may be based on a polynucleotide presented in the form of a mature polypeptide coding sequence (eg, a subsequence thereof), and/or by introduction of an amino acid sequence that does not alter the polypeptide, but corresponding to a host intended to produce the enzyme
- the nucleotide substitution of the codon used by the organism, or by the introduction of nucleotide substitutions that may result in different amino acid sequences For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.
- Mutations in the putative contact site amino acid can also be combined, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling. Physical analysis of the structure to determine the active site of the enzyme or other biological interactions. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899 - 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. It is also possible to infer the identification of essential amino acids from the alignment with related polypeptides.
- a tagged amino acid sequence such as HA, AviTag, Calmodulin-tag, Strep, Flag-tag, His, myc, Xpress, V5, TC, VSV, etc.
- the detection does not affect the biological function of the peptide/protein.
- a start codon such as ATG, is added, so a methionine (M) is added to the beginning of the translated polypeptide, which is required for expression of the polypeptide without affecting the polypeptide/protein.
- M methionine
- the invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences which, under conditions compatible with the control sequences, direct the coding sequence at the appropriate Expression in host cells.
- Polynucleotides can be manipulated in a variety of ways to provide expression of the polypeptide. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.
- the control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell to express a polynucleotide encoding a polypeptide of the present invention.
- the promoter comprises transcriptional control sequences that mediate expression of the polypeptide.
- the promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and heterozygous promoters, and may be extracellularly encoded by a homologous or heterologous source encoding the host cell. Or the gene acquisition of the intracellular polypeptide.
- the invention also relates to recombinant expression vectors comprising the polynucleotides, promoters, and transcriptional and translational termination signals of the invention.
- Different nucleotide and control sequences can be joined together to create a recombinant expression vector which can include one or more convenient restriction sites. Polynucleotides encoding the variant are allowed to be inserted or substituted at these sites.
- the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
- the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequence for expression.
- the invention also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences which direct the production of a polypeptide of the invention .
- the construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier.
- the host cell can be any cell useful for recombinant production of a polypeptide of the invention, such as a prokaryotic cell (bacterial cell) or a eukaryotic cell (e.g., a mammalian, insect, plant, fungal cell).
- Figure 1 is a Coomassie blue staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
- Figure 2 is a diagram of immunoblot staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
- Figure 3 is a statistical diagram of the water maze results of TAT-siP-PTPN1-HA in improving learning and memory in Tg2576 mice.
- Figure 4 is a graph showing the results of TAT-siP-PTPN1-HA improving the phosphorylation of tau protein in Tg2576 mice.
- Figure 5 is a graph showing the improvement of A ⁇ deposition results in Tg2576 mice by TAT-siP-PTPN1-HA.
- Figure 6 is a statistical diagram showing the results of Golgi staining of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
- Figure 7 is a graph showing the results of long-term potentiation of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
- the present invention can improve the senile plaque, tau hyperphosphorylation and senile senescence of senile dementia model animals by injecting TAT-siP-PTPN1-HA polypeptide into 12-month-old Tg2576 mice (purchased from The Jackson Laboratory). Learning and / or memory impairment.
- polypeptides TAT-scramble-HA and TAT-siP-PTPN1-HA were directly dissolved in physiological saline to a final concentration of 10 mg/kg.
- mice Male Tg2576 mice, SPF grade, weighing 25-32 grams, 20, were housed in a conventional environment.
- Experimental group 1 control group (wild type control mouse Tg2576 mice); 2 model group (Tg2576 mice injected with TAT-scramble-HA); 3 treatment group (Tg2576 injected with TAT-siP-PTPN1-HA small) Rat); 10 per group.
- Tg2576 mice 12-month-old Tg2576 mice were given a TAT-scramble-HA polypeptide by a single injection in the tail vein at a dose of 10 mg/kg.
- Tg2576 mice 12-month-old Tg2576 mice were given TAT-siP-PTPN1-HA polypeptide by single injection in the tail vein at a dose of 10 mg/kg.
- the nucleotide sequence of the peptide was synthesized by Wuhan Qingke Company, and it was constructed into the prokaryotic expression vector pGEX-5X-1 (purchased from GE Healthcare China) by molecular biology technology, and was competent in BL21 (purchased from Beijing full version). Transformation and amplification in the gold company).
- pGEX-5X-1 purchased from GE Healthcare China
- BL21 purchased from Beijing full version
- Transformation and amplification in the gold company When the bacterial solution OD600 reached 0.6, 2 mM IPTG was added to induce 0.5 hour, 1 hour, and 2 hours, respectively, and finally, protein expression was observed by SDS-PAGE electrophoresis and Coomassie blue staining.
- polypeptide After the polypeptide was induced to express, it was purified, and subjected to shearing using Factor Xa enzyme (purchased from NEB) to isolate the small molecule polypeptide of interest (about 8 kDa). Protein samples were purified and assayed by Western blotting using HA-specific antibodies (purchased from Abcam). Expression of TAT-siP-PTPN1-HA was detected by staining for specific antibodies recognizing HA, and the results confirmed the expression was successful.
- Factor Xa enzyme purchased from NEB
- mice's ability to learn and remember Morris water maze training and testing (see Figure 3)
- the Morris water maze test system was used in the experiment, including a circular pool with a diameter of 120 cm and a height of 60 cm.
- the cylindrical plexiglass platform has a diameter of 10 cm and a height of 40 cm.
- the water level in the pool is about 45cm high, and the room temperature and water temperature are kept at 26 ⁇ 2°C.
- the platform is placed at the center of a certain quadrant and is not about 2cm below the water surface.
- the mice were placed in the water from the head of the 1/2 radians in either quadrant to the pool wall, and the incubation period (ie, the time from the entry point of the mouse to the platform) was measured.
- the path was used to measure the learning and memory of the mice.
- the indicator of test scores was used in the experiment, including a circular pool with a diameter of 120 cm and a height of 60 cm.
- the cylindrical plexiglass platform has a diameter of 10 cm and a height of 40 cm.
- the water level in the pool is about 45cm
- the swimming time limit was 60 seconds. That is, the platform was not automatically found within 60 seconds.
- the incubation period was recorded as 60 seconds. The tester guided him to the stage and rested. The next training will take place after 30 seconds.
- the first training time was 6 days. On the 7th day, the time required for the mice to find the plateau was recorded, and the latency required for the mice to find the platform was recorded. Take a day off, go to the platform, calculate the dwell time of the mouse in the target quadrant within 1 minute, and analyze the number of crossings at the position of the platform to evaluate its memory ability.
- mice in the treatment group had simple trajectory recovery and shorter latency; the mice in the model group had the shortest time and distance through the platform, and the number of times was the least, indicating that the mice in the model group Memory ability is impaired, the position of the platform is not remembered; the mice in the treatment group recovered to the time and distance of crossing the platform compared to the model group. Normally consistent, the number of crossing platforms increased accordingly, and its memory ability was significantly restored after TAT-siP-PTPN1-HA administration.
- mice were sacrificed by decapitation of the cervical vertebrae, and the brain tissue was quickly removed and placed in a 0-4 ° C, 0.05 M Tris buffered saline solution (TB, pH 7.0) to rapidly separate the bilateral hippocampus.
- Tris buffered saline solution TB, pH 7.0
- the % protein homogenate was centrifuged at 1000 rpm for 5 minutes, the supernatant was taken, and the protein content was determined and used.
- Sample preparation was performed with immunoblotting, and the antibody was washed with A ⁇ 42 antibody (purchased from A ⁇ eta GmBH, Germany), and the sample was added to develop color with tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB). And read at a wavelength of 450 nm on a spectrophotometer.
- a ⁇ 42 has a high toxic effect.
- the results showed that the release of A ⁇ 42 fragment in the model group was significantly increased.
- TAT-siP-PTPN1-HA After treatment with TAT-siP-PTPN1-HA, the A ⁇ 42 in the treatment group returned to the normal group, further demonstrating that TAT-siP-PTPN1-HA can improve Alzheimer's disease.
- mice were anesthetized and fixed on a stereotaxic instrument, and local craniotomy was performed.
- the stimulation electrodes and recording electrodes were placed in the hippocampal CA3 and CA1 regions, respectively. Stimulate the field potential induced by the CA3 region. Select the appropriate stimulus and record electrode position. I/O curves were drawn, and 40% of the maximum amplitude of the induced fEPSP was selected as the stimulation intensity.
- a series of high frequency stimulation 100 Hz, 1 second
- Long-term time-course enhancement (LTP) recorded for 120 minutes.
- the indicator of tau protein was related to Alzheimer's disease.
- the A ⁇ content was also significantly reduced compared with the model group, indicating that TAT-siP-PTPN1-HA can alleviate the abnormal hyperphosphorylation of tau in the hippocampus of Tg2576 mice, reduce amyloid A ⁇ deposition and improve animal learning and/or memory impairment.
- the polypeptide of the present invention can be used for the prevention and treatment of senile dementia.
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Abstract
Disclosed are a low molecular weight polypeptide, nucleotide sequence encoding the polypeptide, and use of the polypeptide in preparing a drug for treating and preventing Alzheimer's disease (AD).
Description
本发明涉及药物研发领域，具体涉及一种多肽及其在制备治疗或预防老年痴呆药物中的应用。The invention relates to the field of drug research and development, and particularly relates to a polypeptide and application thereof in preparing a medicament for treating or preventing senile dementia.
老年性痴呆(Alzheimer Disease,AD)是一种常见的神经退行性疾病，其主要症状表现为记忆力减退，认知能力低下和思维迟钝，且进行性加重，最后生活不能自理而死亡，病程一般8～10年。随着社会老龄化进程的加快，AD的患病人数急剧上升，已成为威胁老龄特别是高龄人口身心健康的严重疾病，并带来了严重的社会、经济和家庭问题。Alzheimer Disease (AD) is a common neurodegenerative disease. Its main symptoms are memory loss, low cognitive ability and slow thinking, and progressive aggravation. Finally, life cannot take care of itself and die. ~10 years. With the acceleration of the aging process of society, the number of patients with AD has risen sharply, and it has become a serious disease that threatens the physical and mental health of the elderly, especially the elderly, and brings serious social, economic and family problems.
AD病人主要脑病理改变是形成大量神经原纤维缠结(neurofibrillary tangles，NFTs)和大量老年斑。其分子标志物分别是异常磷酸化的tau蛋白(一种微管结合蛋白)和β-淀粉样蛋白(Aβ)。国外学者已采用转基因技术，在小鼠成功地复制出β-淀粉样蛋白沉积形成的老年斑样病变，从而使得未来AD治疗将减少Aβ沉积作为药物干预的靶标成为可能。NFTs的数量与AD临床痴呆程度正相关。因此，干预NFTs对AD的防治尤为重要。自从发现NFTs的主要成分是异常、过度磷酸化的Tau蛋白聚集形成的双螺旋丝以来(Morris M,Maeda S,Vossel K,Mucke L.The many faces of tau.Neuron.2011May 12；70(3):410-26)，国内外学者普遍公认，骨架蛋白tau的异常、过度磷酸化是NFTs形成的关键步骤。过度磷酸化tau蛋白的聚集不仅以缠结的形式存在，它也是神经毡纤丝和老年斑中的营养不良突起的主要成分。The main brain pathological changes in AD patients are the formation of a large number of neurofibrillary tangles (NFTs) and a large number of age spots. Its molecular markers are abnormally phosphorylated tau (a microtubule-binding protein) and β-amyloid (Aβ). Foreign scholars have used transgenic technology to successfully replicate senile plaque-like lesions formed by β-amyloid deposition in mice, making it possible for future AD treatment to reduce Aβ deposition as a target for drug intervention. The number of NFTs is positively correlated with the degree of clinical dementia in AD. Therefore, intervention in NFTs is particularly important for the prevention and treatment of AD. Since the discovery that the main component of NFTs is a double helix formed by the aggregation of abnormal, hyperphosphorylated Tau protein (Morris M, Maeda S, Vossel K, Mucke L. The many faces of tau. Neuron. 2011 May 12; 70(3) : 410-26), domestic and foreign scholars generally agree that the abnormality and hyperphosphorylation of the skeleton protein tau is a key step in the formation of NFTs. The accumulation of hyperphosphorylated tau protein is not only present in entangled form, but it is also a major component of dystrophic processes in neuropilic fibrils and senile plaques.
目前尚没有针对老年性痴呆病理特征的特效治疗药物和方法，常用的减缓老年痴呆发作药物有：(1)改善胆碱神经传递药物：老年痴呆的一个主要原因是胆碱不足导致记忆衰退。因此乙酰胆碱酯酶(AchE)抑制剂通过增强胆碱能作用在老年痴呆治疗方面发挥了重要作用；(2)改善脑血液循环和脑细胞代谢的药物：此类药物可以扩张脑血管以改善老年痴呆患者的糖、蛋白、核酸、
脂质等代谢障碍；(3)钙拮抗剂：此类药物易于通过血脑屏障，选择性扩张脑血管，从而改善记忆和认知功能；(4)激素类药物：主要缓解女性患者的症状。然而，这些药物仅对中晚期老年性痴呆患者的痴呆症状有所轻度改善或延缓痴呆进展。因此研发新的针对老年性痴呆病理学特征的药物，成为全球的期待。There are currently no specific treatments and methods for the pathological features of Alzheimer's disease. Commonly used drugs to alleviate Alzheimer's disease are: (1) Improve choline neurotransmitter: One of the main causes of Alzheimer's disease is memory loss due to choline deficiency. Therefore, acetylcholinesterase (AchE) inhibitors play an important role in the treatment of senile dementia by enhancing cholinergic effects; (2) drugs that improve cerebral blood circulation and brain cell metabolism: these drugs can dilate cerebral blood vessels to improve senile dementia Patient's sugar, protein, nucleic acid,
Metabolic disorders such as lipids; (3) calcium antagonists: these drugs easily pass the blood-brain barrier, selectively dilate the cerebral blood vessels, thereby improving memory and cognitive function; (4) hormonal drugs: mainly relieve the symptoms of female patients. However, these drugs only slightly improve the symptoms of dementia in patients with advanced senile dementia or delay the progression of dementia. Therefore, the development of new drugs for the pathological features of Alzheimer's disease has become a global expectation.
TAT是一种细胞穿膜多肽(cell penetrating peptides)，是近年来发现的一种高效运输载体。TAT能够穿透细胞膜、细胞核膜，携带多肽、蛋白质以及DNA分子等通过受体转运方式进入胞质和胞核发挥相应的生物效应(Kilic E,Kilic U,Hermann DM.TAT fusion proteins against ischemic stroke：current status and future perspectives.Front Biosci.2006May 1；11:1716-21)。目前研究显示HIV-TAT，能够穿过所有组织细胞，且无明显的毒副作用。TAT能将与之相连的多肽在数分钟内带入细胞，而且可以跨越血脑屏障进入神经元内，被带入细胞的多肽则保留了它们原有的生物活性，从而发挥生物学作用。TAT is a kind of cell penetrating peptides, which is an efficient transport carrier discovered in recent years. TAT can penetrate the cell membrane, nuclear membrane, carrying peptides, proteins and DNA molecules to enter the cytoplasm and nucleus through receptor transport to exert corresponding biological effects (Kilic E, Kilic U, Hermann DM.TAT fusion proteins against ischemic stroke: Current status and future perspectives. Front Biosci. 2006 May 1; 11:1716-21). Current research shows that HIV-TAT is able to cross all tissue cells without significant toxic side effects. TAT can bring the polypeptides connected to it into the cells within a few minutes, and can enter the neurons across the blood-brain barrier. The polypeptides brought into the cells retain their original biological activities and thus play a biological role.
发明内容Summary of the invention
基于申请人最近的研究与TAT技术，申请人合成了一种小分子多肽，该多肽能与脑内高表达的小RNA分子miR-124竞争性结合到PTPN1(又名PTP1B，Tonks NK.Protein tyrosine phosphatases--from housekeeping enzymes to masterregulators of signal transduction.FEBS J.2013Jan；280(2):346-78.)的3′-UTR。通过运用到在体的老年痴呆转基因动物模型上，有效发挥其阻断miR-124与PTPN1的3′-UTR结合的生物学作用，促进PTPN1表达，改善老年痴呆模型动物表现出的微管相关蛋白tau的过度磷酸化、淀粉样蛋白沉积，以及动物学习、记忆障碍，为进一步开发临床治疗老年痴呆的药物提供了分子靶点。Based on the applicant's recent research and TAT technology, Applicants synthesized a small molecule peptide that competitively binds to PTPN1 (also known as PTP1B, Tonks NK.Protein tyrosine), a small RNA molecule miR-124 that is highly expressed in the brain. Phosphases--from housekeeping enzymes to masterregulators of signal transduction. FEBS J. 2013 Jan; 280(2): 346-78.) 3'-UTR. By applying to the in vivo Alzheimer's disease transgenic animal model, it effectively exerts its biological function of blocking the binding of miR-124 to PTPN1 3'-UTR, promotes PTPN1 expression, and improves microtubule-associated protein in senile dementia model animals. Tau hyperphosphorylation, amyloid deposition, and animal learning and memory disorders provide molecular targets for further development of clinical treatments for Alzheimer's disease.
本发明的目的在于提供一种多肽，该多肽选自下组：It is an object of the present invention to provide a polypeptide selected from the group consisting of:
(a)一种包括SEQ ID NO:1的氨基酸序列的多肽；(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(b)一种与SEQ ID NO:1的氨基酸序列的多肽具有至少80％序列同一性的氨基酸序列的多肽；或(b) a polypeptide having an amino acid sequence having at least 80% sequence identity to a polypeptide of the amino acid sequence of SEQ ID NO: 1;
(c)一种在SEQ ID NO:1的氨基酸序列上包括一个或多个的氨基酸残基的取代、缺失和/或插入的多肽。
(c) a polypeptide comprising a substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1.
在一个具体实施方式中，本发明的多肽是TAT-siP-PTPN1，其序列为SEQ ID NO:1所示。也就是将TAT穿膜肽(GRKKRRQRRRPRQ)，与siP-PTPN1(阻断miR-124与PTPN1结合位点的多肽，我们命名为silencing peptide for PTPN1,缩写为siP-PTPN1)连接，其中siP-PTPN1的序列为PYGCRVIQRIGNYVIQHVASNNVEKIGGYVIRHVGGYVIRHVGNYVIQHVGNYVIQHVGCRVIQRIL。In a specific embodiment, the polypeptide of the invention is TAT-siP-PTPN1, the sequence of which is set forth in SEQ ID NO: 1. That is, the TAT transmembrane peptide (GRKKRRQRRRPRQ) was ligated with siP-PTPN1 (a polypeptide that blocks the binding site of miR-124 and PTPN1, we named silencing peptide PTPN1, abbreviated as siP-PTPN1), of which siP-PTPN1 The sequence is PYGCRVIQRIGNYVIQHVASNNVEKIGGYVIRHVGGYVIRHVGNYVIQHVGNYVIQHVGCRVIQRIL.
本发明的多肽利用TAT的穿膜功能将siP-PTPN1多肽输送进入血液穿过血脑屏障，并被大脑神经细胞摄取从而发挥其生物学功能。The polypeptide of the present invention utilizes the transmembrane function of TAT to transport the siP-PTPN1 polypeptide into the bloodstream through the blood-brain barrier and is taken up by the brain nerve cells to exert its biological function.
优选地，为了后期对肽和表达蛋白的检测，申请人在本发明所述的多肽后面添加了标签多肽，例如HA(YPYDVPDYA)。本发明优选的多肽序列为SEQ ID NO:1末端添加HA(YPYDVPDYA)的序列。进一步，本发明优选的多肽为TAT-siP-PTPN1-HA(SEQ ID NO:4)。Preferably, for later detection of the peptide and the expressed protein, Applicants add a tag polypeptide, such as HA (YPYDVPDYA), following the polypeptide of the present invention. A preferred polypeptide sequence of the invention is the sequence in which HA (YPYDVPDYA) is added at the end of SEQ ID NO: 1. Further, a preferred polypeptide of the invention is TAT-siP-PTPN1-HA (SEQ ID NO: 4).
本发明的另一个目的还在于提供了一种编码本发明所述多肽的核苷酸序列。Another object of the invention is also to provide a nucleotide sequence encoding a polypeptide of the invention.
在一个具体实施方式中，本发明的核苷酸序列是编码小分子多肽TAT-siP-PTPN1的核苷酸序列，优选为SEQ ID NO：2所示，但不局限于该序列。由于密码子的简并性，所有编码TAT-siP-PTPN1多肽的核苷酸序列都为本发明的保护范围。In a specific embodiment, the nucleotide sequence of the present invention is a nucleotide sequence encoding the small molecule polypeptide TAT-siP-PTPN1, preferably shown by SEQ ID NO: 2, but is not limited thereto. Due to the degeneracy of the codons, all nucleotide sequences encoding TAT-siP-PTPN1 polypeptides are within the scope of the invention.
优选编码TAT-siP-PTPN1-HA多肽的核苷酸序列为SEQ ID NO：3。Preferably, the nucleotide sequence encoding the TAT-siP-PTPN1-HA polypeptide is SEQ ID NO:3.
本发明还进一步提供了一种核酸构建体，包含本发明所述的核苷酸序列，该核苷酸序列可操作地连接到一种或多种指导所述多肽在合适的宿主中生产的控制序列。The invention still further provides a nucleic acid construct comprising a nucleotide sequence of the invention operably linked to one or more controls that direct production of the polypeptide in a suitable host sequence.
本发明还进一步提供了一种包含所述的核酸构建体的重组表达载体。本发明的另一个目的在于提供了一种本发明所述的多肽在制备治疗或预防老年痴呆药物中的应用。优选地，本发明提供了一种多肽TAT-siP-PTPN1在制备治疗或预防老年痴呆药物中的应用。将本发明所述的多肽制成静脉注射药，静脉注射后，发现其可有效改善老年痴呆模型动物表现出的微管相关蛋白tau的过度磷酸化、淀粉样蛋白沉积，动物学习和/或记忆障碍，为进一步开发临床治疗老年痴呆的药物提供了分子靶点。The invention still further provides a recombinant expression vector comprising the nucleic acid construct. Another object of the present invention is to provide a use of the polypeptide of the present invention for the preparation of a medicament for the treatment or prevention of Alzheimer's disease. Preferably, the present invention provides the use of a polypeptide TAT-siP-PTPN1 for the preparation of a medicament for treating or preventing Alzheimer's disease. The polypeptide of the present invention is made into an intravenous injection, and after intravenous injection, it is found to be effective for improving hyperphosphorylation, amyloid deposition, animal learning and/or memory of the microtubule-associated protein tau exhibited by the senile dementia model animal. Obstacles provide molecular targets for further development of drugs for clinical treatment of Alzheimer's disease.
本发明的另一个目的在于提供了一种包含本发明所述的小分子多肽的药
物组合物。Another object of the present invention is to provide a medicament comprising the small molecule polypeptide of the present invention.
Composition.
为了实现上述目的，本发明采用的技术措施是：申请人发现，老年痴呆模型动物中m iR-124和PTPN1的3′-UTR相互结合，并介导下游微管相关蛋白tau的过度磷酸化、淀粉样蛋白沉积，以及动物学习、记忆障碍。阻断miR-124和PTPN1的3′-UTR相互结合，能有效改善上述症状。针对这一发现，申请人将TAT穿膜肽与siP-PTPN1-H A连接，得到具有生物活性的TAT-siP-PTPN1-HA多肽，通过在体静脉注射方式，使TAT-siP-PTPN1-HA多肽进入血液穿过血脑屏障并被大脑神经细胞摄取发挥其生物学功能。In order to achieve the above object, the technical measures adopted by the present invention are: Applicants have found that the 3'-UTR of m iR-124 and PTPN1 in the dementia model animal bind to each other and mediate the hyperphosphorylation of the downstream microtubule-associated protein tau, Amyloid deposition, as well as animal learning and memory disorders. Blocking the 3'-UTR of miR-124 and PTPN1 to each other can effectively improve the above symptoms. In response to this finding, Applicants linked the TAT transmembrane peptide to siP-PTPN1-H A to obtain a biologically active TAT-siP-PTPN1-HA polypeptide, which was TAT-siP-PTPN1-HA by intravenous injection. The polypeptide enters the bloodstream and crosses the blood-brain barrier and is taken up by the brain's nerve cells to exert its biological function.
申请人设计了TAT-siP-PTPN1-HA(SEQ ID NO：4)的对照序列TAT-scramble-HA(SEQ ID NO：5)。该对照序列是将siP-PTPN1序列人为打乱而得到的。TAT-siP-PTPN1-HA多肽和对照TAT-scramble-HA由申请人通过原核表达、收集、纯化得到。Applicants designed the control sequence TAT-scramble-HA (SEQ ID NO: 5) of TAT-siP-PTPN1-HA (SEQ ID NO: 4). This control sequence was obtained by artificially scrambling the siP-PTPN1 sequence. The TAT-siP-PTPN1-HA polypeptide and the control TAT-scramble-HA were obtained by the applicant by prokaryotic expression, collection, and purification.
本发明通过对TAT-siP-PTPN1-HA在老年痴呆动物模型上的应用，证明了TAT-siP-PTPN1-HA对老年痴呆模型具有确切的治疗作用。The invention proves that TAT-siP-PTPN1-HA has an exact therapeutic effect on the Alzheimer's model by applying TAT-siP-PTPN1-HA in an animal model of Alzheimer's disease.
与现有技术相比，本发明具有以下特点：(1)本发明所涉及的小分子多肽容易诱导表达纯度，完全可溶，适合静脉注射，无毒副作用。(2)本发明公开的多肽经血液穿过血脑屏障被神经元摄取，可以转化应用于神经系统疾病如老年痴呆，使其具有实际操作的可行性。Compared with the prior art, the present invention has the following characteristics: (1) The small molecule polypeptide of the present invention is easy to induce expression purity, is completely soluble, is suitable for intravenous injection, and has no toxic side effects. (2) The polypeptide disclosed by the present invention is taken up by neurons through the blood-brain barrier, and can be transformed into a nervous system disease such as Alzheimer's disease, so that it has practical feasibility.
定义：definition:
编码序列：术语“编码序列”意指直接指定一个多肽的氨基酸序列的多核苷酸。编码序列的边界一般由一个开放阅读框架决定，该开放阅读框架从一个起始密码子(如ATG、GTG或TTG)开始并且以一个终止密码子(如TAA、TAG或TGA)结束。编码序列可以是一种基因组DNA、cDNA、合成DNA或其组合。Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA). The coding sequence can be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
控制序列：术语“控制序列”意指对于表达编码本发明的成熟多肽的多核苷酸所必需的核酸序列。每个控制序列对于编码该多肽的多核苷酸来说可以是天然的(即，来自相同基因)或外源的(即，来自不同基因)，或相对于彼此是天然的或外源的。此类控制序列包括但不限于前导子、聚腺苷酸化序列、前肽序列、启动子、信号肽序列、以及转录终止子。至少，控制序列包括启动子，
以及转录和翻译终止信号。出于引入有利于将这些控制序列与编码一种多肽的多核苷酸的编码区连接的特异性限制酶切位点的目的，这些控制序列可以提供有多个接头。Control sequence: The term "control sequence" means a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide of the invention. Each control sequence may be native (ie, from the same gene) or foreign (ie, from a different gene) to the polynucleotide encoding the polypeptide, or be native or foreign to each other. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At least, the control sequence includes a promoter,
And transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linkage of these control sequences to the coding region of a polynucleotide encoding a polypeptide.
表达：术语“表达”包括涉及多肽产生的任何步骤，包括但不限于，转录、转录后修饰、翻译、翻译后修饰、以及分泌。Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
表达载体：术语“表达载体”意指线性或环状DNA分子，该分子包括编码多肽的多核苷酸并且该多核苷酸可操作地与提供用于其表达的控制序列相连接。Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence providing for its expression.
宿主细胞：术语“宿主细胞”意指任何细胞类型，该细胞类型对于用包含本发明的多核苷酸的核酸构建体或表达载体进行转化、转染、转导等是易感的。术语“宿主细胞”涵盖由于复制期间发生的突变而与亲本细胞不同的亲本细胞的任何后代。Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication.
核酸构建体：术语“核酸构建体”意指单链或双链的一种核酸分子，该核酸分子是从天然存在的基因中分离的，或者以一种本来不存在于自然界中的方式被修饰成含有核酸的区段，或者是合成的，该核酸分子包含一个或多个控制序列。Nucleic acid construct: The term "nucleic acid construct" means a single or double stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner that is not inherent in nature. A segment containing a nucleic acid, or a synthetic nucleic acid molecule comprising one or more control sequences.
可操作地连接：术语“可操作地连接”意指如下的构造，其中，控制序列相对于多核苷酸的编码序列安置在适当位置处，从而使得该控制序列指导该编码序列的表达。Operably linked: The term "operably linked" means a construct in which the control sequence is placed in position relative to the coding sequence of the polynucleotide such that the control sequence directs expression of the coding sequence.
序列同一性：两个氨基酸序列之间或者两个核苷酸序列之间的关联度通过参数“序列同一性”来描述。Sequence identity: The degree of association between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
出于本发明的目的，使用如在EMBOSS包(EMBOSS：欧洲分子生物学开放软件套件(The European Molecular Biology Open Software Suite)，赖斯(Rice)等人，2000，遗传学趋势(Trends Genet.)16:276-277)(优选5.0.0版或更新版本)的尼德尔(Needle)程序中所实施的尼德尔曼-翁施(Needleman-Wunsch)算法(Needleman(尼德尔曼)和Wunsch(翁施)，1970，分子生物学杂志(J.Mol.Biol.)48:443-453)来确定两个氨基酸序列之间的序列同一性。使用的这些参数是空位开放罚分10、空位延伸罚分0.5，以及EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。尼德尔标注的“最长的同一性””的输出(使用-非简化选项获得)被用作百分比同一性，并且如下计算：
(一致的残基×100)/(比对长度-比对中的空位总数)For the purposes of the present invention, use, for example, in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.) 16:276-277) (preferably version 5.0.0 or later) The Needleman-Wunsch algorithm implemented in the Needle program (Needleman and Wunsch) (1), J. Mol. Biol. 48: 443-453) to determine sequence identity between two amino acid sequences. These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest identity" of the Nieder annotation (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows:
(consistent residue × 100) / (alignment length - total number of gaps in the alignment)
出于本发明的目的，使用如在EMBOSS包(EMBOSS：欧洲分子生物学开放软件套件，赖斯(Rice)等人，2000，见上文)(优选5.0.0版或更新版本)的尼德尔程序中所实施的尼德尔曼-翁施算法(尼德尔曼(Needleman)和翁施(Wunsch)，1970，见上文)来确定两个脱氧核糖核苷酸序列之间的序列同一性。使用的这些参数是空位开放罚分10、空位延伸罚分0.5以及EDNAFULL(NCBI NUC4.4的EMBOSS版本)取代矩阵。尼德尔标注的“最长的同一性”的输出(使用-非简化选项获得)被用作百分比同一性，并且如下计算：(一致的脱氧核糖核苷酸×100)/(比对长度-比对中的空位总数)For the purposes of the present invention, use Niedel as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al, 2000, supra) (preferably version 5.0.0 or later) The Nederman-Wengsch algorithm (Nedleman and Wunsch, 1970, supra) implemented in the program was used to determine sequence identity between two deoxyribonucleotide sequences. These parameters used are gap open penalty of 10, gap extension penalty of 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the "longest identity" of the Niedel label (obtained using the -non-simplification option) is used as the percent identity and is calculated as follows: (consistent deoxyribonucleotide × 100) / (alignment length - ratio The total number of vacancies in the pair)
发明详细说明Detailed description of the invention
用于治疗或预防老年痴呆的多肽Polypeptide for treating or preventing Alzheimer's disease
本发明涉及一种包括SEQ ID NO:1的氨基酸序列的多肽。优选地，本发明的多肽具有SEQ ID NO:1所示的氨基酸序列的多肽。本发明还涉及包括与SEQ ID NO：1所示的氨基酸序列具有至少85％，90％，95％，96％，97％，98％，99％或100％的同一性的多肽，这些多肽能用于治疗或预防老年痴呆。The present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. Preferably, the polypeptide of the present invention has the polypeptide of the amino acid sequence of SEQ ID NO: 1. The present invention also relates to a polypeptide comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO:1, which polypeptides are capable of For the treatment or prevention of senile dementia.
另一个方面，本发明涉及一种在SEQ ID NO:1的氨基酸序列上包括一个或多(几)个的氨基酸残基的取代、缺失和/或插入的多肽。在这种情况下，本发明的多肽与SEQ ID NO:1的成熟多肽相差不超过10个氨基酸，例如1、2、3、4、5、6、7、8或9个氨基酸。In another aspect, the invention relates to a polypeptide comprising a substitution, deletion and/or insertion of one or more (several) amino acid residues in the amino acid sequence of SEQ ID NO: 1. In this case, the polypeptide of the invention differs from the mature polypeptide of SEQ ID NO: 1 by no more than 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.
这些氨基酸变化可以具有微小性质，即，不会显著地影响蛋白质的折叠和/或活性的保守氨基酸取代或插入。保守取代的实例是在下组的范围内：碱性氨基酸(精氨酸、赖氨酸及组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺和天冬酰胺)、疏水性氨基酸(亮氨酸、异亮氨酸及缬氨酸)、芳香族氨基酸(苯丙氨酸、色氨酸及酪氨酸)及小氨基酸(甘氨酸、丙氨酸、丝氨酸、苏氨酸及甲硫氨酸)。一般不会改变特异性活性的氨基酸取代是本领域已知的并且例如由H.诺伊拉特(Neurath)和R.L.希尔(Hill)，1979在蛋白质(The Proteins)，学术出版社(Academic Press)，纽约中描述。常见取代是Ala/Ser、Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、Ala/Glu、和Asp/Gly。
These amino acid changes can have subtle properties, ie, conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein. Examples of conservative substitutions are within the scope of the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and day) Asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine) , threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are for example, by H. Neurat and RL Hill, 1979, The Proteins, Academic Press (Academic Press) ), described in New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg , Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
可替代地，氨基酸改变具有这样一种性质:改变多肽的物理化学特性。例如，氨基酸改变可以提高多肽的热稳定性、改变底物特异性、改变最适pH，等等。Alternatively, amino acid changes have the property of altering the physicochemical properties of the polypeptide. For example, amino acid changes can increase the thermal stability of the polypeptide, alter substrate specificity, change the pH optimum, and the like.
可以做出单个或多个氨基酸取代、缺失和/或插入并且使用诱变、重组和/或改组的已知方法进行测试，随后进行相关筛选程序，如由里德哈尔-奥尔森(Reidhaar-Olson)和萨奥尔(Sauer)，1988，科学(Science)241:53-57；博维(Bowie)和萨奥尔，1989，美国科学院院刊(Proc.Natl.Acad.Sci.USA)86:2152-2156；WO 95/17413；或WO 95/22625所披露的那些。可以使用的其他方法包括易错PCR、噬菌体展示(例如，洛曼(Lowman)等人，1991，生物化学(Biochemistry)30:10832-10837；美国专利号5,223,409；WO 92/06204)以及区域定向诱变(德比什尔(Derbyshire)等人，1986，基因(Gene)46:145；内尔(Ner)等人，1988，DNA 7:127)。Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known methods of mutagenesis, recombination and/or shuffling, followed by relevant screening procedures, such as by Reidhaar (Reidhaar) -Olson) and Sauer, 1988, Science 241: 53-57; Bowie and Saar, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or those disclosed in WO 95/22625. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al, 1991, Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and regional targeting Change (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
可以结合诱变/改组方法与高通量自动化筛选方法来检测由宿主细胞表达的克隆的、诱变的多肽的活性(内斯(Ness)等人，1999，自然生物技术(Nature Biotechnology)17:893-896)。编码活性多肽的诱变的DNA分子可以回收自宿主细胞，并且使用本领域的标准方法对其进行迅速测序。这些方法允许迅速确定多肽中单个氨基酸残基的重要性。The mutagenesis/shuffling method can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.
这些多肽的变体可以基于以成熟多肽编码序列(例如其子序列)形式呈现的多核苷酸，和/或通过引入不会改变该多肽的氨基酸序列，但对应于预定用于产生该酶的宿主有机体的密码子使用的核苷酸取代，或通过引入可能产生不同氨基酸序列的核苷酸取代来构建。对于核苷酸取代的一般描述，参见例如福德(Ford)等人，1991，蛋白表达与纯化(Protein Expression and Purification)2:95-107。Variants of these polypeptides may be based on a polynucleotide presented in the form of a mature polypeptide coding sequence (eg, a subsequence thereof), and/or by introduction of an amino acid sequence that does not alter the polypeptide, but corresponding to a host intended to produce the enzyme The nucleotide substitution of the codon used by the organism, or by the introduction of nucleotide substitutions that may result in different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.
可以根据本领域中已知的程序，如定点诱变或丙氨酸扫描诱变(坎宁汉(Cunningham)和威尔斯(Wells)，1989，科学(Science)244:1081-1085)来鉴定多肽中的必需氨基酸。在后一项技术中，在该分子中的每个残基处引入单个丙氨酸突变，并且对所得突变体分子的α-淀粉酶活性进行测试以鉴定对于该分子的活性至关重要的氨基酸残基。还参见，希尔顿(Hilton)等人，1996，生物化学杂志(J.Biol.Chem.)271:4699-4708。也可结合假定接触位点氨基酸的突变，如通过以下技术例如核磁共振、结晶学、电子衍射、或光亲和标记进行确定
的对结构进行物理学分析，从而确定酶的活性位点或其他生物学相互作用。参见，例如，德弗斯(de Vos)等人，1992，科学(Science)255:306-312；史密斯(Smith)等人，1992，分子生物学杂志(J.Mol.Biol.)224:899-904；乌乐达维尔(Wlodaver)等人，1992，欧洲生化学会联合会快报(FEBS Lett.)309:59-64。还可以从与相关多肽的比对来推断鉴别必需氨基酸。It can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). An essential amino acid in a polypeptide. In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the alpha-amylase activity of the resulting mutant molecule is tested to identify amino acids that are critical to the activity of the molecule. Residues. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Mutations in the putative contact site amino acid can also be combined, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling.
Physical analysis of the structure to determine the active site of the enzyme or other biological interactions. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899 - 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. It is also possible to infer the identification of essential amino acids from the alignment with related polypeptides.
本领域技术人员知道，在多肽的两端添加一个或几个氨基酸常常并不影响多肽的功能。例如，在多肽的末端添加一段标签氨基酸序列，例如HA，AviTag,Calmodulin-tag,Strep,Flag-tag,His,myc,Xpress,V5,TC,VSV等，是为了用于后期对肽和表达蛋白的检测，并不会影响多肽/蛋白的生物学功能。另外，在生物学表达时，会加入起始密码子，如ATG，因此翻译的多肽的起始端就会增加一个蛋氨酸(M)，这些都是多肽表达的需要，而不会影响多肽/蛋白的生物学功能。Those skilled in the art will recognize that the addition of one or several amino acids at both ends of the polypeptide often does not affect the function of the polypeptide. For example, a tagged amino acid sequence, such as HA, AviTag, Calmodulin-tag, Strep, Flag-tag, His, myc, Xpress, V5, TC, VSV, etc., is added to the end of the polypeptide for use in late peptides and expressed proteins. The detection does not affect the biological function of the peptide/protein. In addition, in biological expression, a start codon, such as ATG, is added, so a methionine (M) is added to the beginning of the translated polypeptide, which is required for expression of the polypeptide without affecting the polypeptide/protein. biological functions.
核酸构建体Nucleic acid construct
本发明还涉及核酸构建体，这些核酸构建体包括可操作地连接至一个或多个控制序列的本发明的多核苷酸，在与控制序列相容的条件下，这些控制序列指导编码序列在合适的宿主细胞中的表达。可以按多种方式操纵多核苷酸，以提供多肽的表达。取决于表达载体，在其插入载体以前操纵多核苷酸可以是希望的或必需的。用于利用重组DNA方法修饰多核苷酸的技术是本领域熟知的。The invention also relates to nucleic acid constructs comprising a polynucleotide of the invention operably linked to one or more control sequences which, under conditions compatible with the control sequences, direct the coding sequence at the appropriate Expression in host cells. Polynucleotides can be manipulated in a variety of ways to provide expression of the polypeptide. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.
该控制序列可以是一个启动子，即，被宿主细胞识别以对编码本发明多肽的多核苷酸进行表达的一种多核苷酸。该启动子包含转录控制序列，这些序列介导了该多肽的表达。该启动子可以是在宿主细胞中显示出转录活性的任何多核苷酸，包括突变型、截短型及杂合型启动子，并且可以是由编码与该宿主细胞同源或异源的细胞外或细胞内多肽的基因获得。The control sequence may be a promoter, i.e., a polynucleotide recognized by a host cell to express a polynucleotide encoding a polypeptide of the present invention. The promoter comprises transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and heterozygous promoters, and may be extracellularly encoded by a homologous or heterologous source encoding the host cell. Or the gene acquisition of the intracellular polypeptide.
表达载体Expression vector
本发明还涉及包括本发明的多核苷酸、启动子、以及转录和翻译终止信号的重组表达载体。不同的核苷酸和控制序列可以连接在一起以产生一个重组表达载体，这一重组表达载体可以包括一个或多个便利的限制酶切位点以
允许在这些位点处插入或取代编码该变体的多核苷酸。可替代地，该多核苷酸可以通过将该多核苷酸或包括该多核苷酸的核酸构建体插入用于表达的适当载体中来表达。在产生该表达载体时，该编码序列是位于该载体中，这样使得该编码序列与该供表达的适当控制序列可操作地连接。The invention also relates to recombinant expression vectors comprising the polynucleotides, promoters, and transcriptional and translational termination signals of the invention. Different nucleotide and control sequences can be joined together to create a recombinant expression vector which can include one or more convenient restriction sites.
Polynucleotides encoding the variant are allowed to be inserted or substituted at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In generating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequence for expression.
宿主细胞Host cell
本发明还涉及重组宿主细胞，这些重组宿主细胞包括本发明的多核苷酸，该多核苷酸可操作地连接至一个或多个控制序列，该一个或多个控制序列指导本发明的多肽的产生。将包含多核苷酸的构建体或载体引入到宿主细胞中，这样使得该构建体或载体被维持作为染色体整合体或作为自主复制的染色体外载体，如早前所描述。该宿主细胞可以是有用于重组产生本发明的多肽的任何细胞，例如原核细胞(细菌细胞)或真核细胞(如哺乳动物、昆虫、植物、真菌细胞)。The invention also relates to recombinant host cells comprising a polynucleotide of the invention operably linked to one or more control sequences which direct the production of a polypeptide of the invention . The construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The host cell can be any cell useful for recombinant production of a polypeptide of the invention, such as a prokaryotic cell (bacterial cell) or a eukaryotic cell (e.g., a mammalian, insect, plant, fungal cell).
所有图中，*P<0.05,**P<0.01与正常组比较；#P<0.05,##P<0.01与模型组比较。正常组，不做任何处理；模型组，Tg2576小鼠给予TAT-scramble-HA；治疗组：Tg2576小鼠给予TAT-siP-PTPN1-HA。单次尾静脉注射，剂量为10mg/kg。In all the figures, *P<0.05, **P<0.01 compared with the normal group; #P<0.05, ##P<0.01 compared with the model group. In the normal group, no treatment was performed; model group, Tg2576 mice were given TAT-scramble-HA; treatment group: Tg2576 mice were given TAT-siP-PTPN1-HA. A single tail vein injection at a dose of 10 mg/kg.
图1为一种小分子多肽TAT-siP-PTPN1-HA的考马斯亮蓝染色图。Figure 1 is a Coomassie blue staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
图2为一种小分子多肽TAT-siP-PTPN1-HA的免疫印迹染色图。Figure 2 is a diagram of immunoblot staining of a small molecule polypeptide TAT-siP-PTPN1-HA.
图3为TAT-siP-PTPN1-HA改善Tg2576小鼠学习记忆的水迷宫结果统计图。Figure 3 is a statistical diagram of the water maze results of TAT-siP-PTPN1-HA in improving learning and memory in Tg2576 mice.
图4为TAT-siP-PTPN1-HA改善Tg2576小鼠tau蛋白磷酸化结果统计图。Figure 4 is a graph showing the results of TAT-siP-PTPN1-HA improving the phosphorylation of tau protein in Tg2576 mice.
图5为TAT-siP-PTPN1-HA改善Tg2576小鼠Aβ沉积结果统计图。Figure 5 is a graph showing the improvement of Aβ deposition results in Tg2576 mice by TAT-siP-PTPN1-HA.
图6为TAT-siP-PTPN1-HA改善Tg2576小鼠学习记忆的高尔基染色结果统计图。Figure 6 is a statistical diagram showing the results of Golgi staining of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
图7为TAT-siP-PTPN1-HA改善Tg2576小鼠学习记忆的长时程增强结果统计图。
Figure 7 is a graph showing the results of long-term potentiation of learning and memory in Tg2576 mice by TAT-siP-PTPN1-HA.
实验材料：Experimental Materials:
本发明通过给12月龄Tg2576小鼠(购买自美国杰克逊实验室(The Jackson Laboratory))注射TAT-siP-PTPN1-HA多肽，发现其可以改善老年痴呆模型动物的老年斑、tau蛋白过度磷酸化与学习和/或记忆障碍。The present invention can improve the senile plaque, tau hyperphosphorylation and senile senescence of senile dementia model animals by injecting TAT-siP-PTPN1-HA polypeptide into 12-month-old Tg2576 mice (purchased from The Jackson Laboratory). Learning and / or memory impairment.
多肽TAT-scramble-HA和TAT-siP-PTPN1-HA直接溶解于生理盐水，终浓度为：10mg/kg。The polypeptides TAT-scramble-HA and TAT-siP-PTPN1-HA were directly dissolved in physiological saline to a final concentration of 10 mg/kg.
具体操作步骤如下：The specific steps are as follows:
1.实验对象Experimental object
雄性Tg2576小鼠，SPF级，体重25～32克，20只，常规环境饲养。实验分组：①对照组(野生型对照小鼠Tg2576小鼠)；②模型组(注射了TAT-scramble-HA的Tg2576小鼠)；③治疗组(注射了TAT-siP-PTPN1-HA的Tg2576小鼠)；每组10只。Male Tg2576 mice, SPF grade, weighing 25-32 grams, 20, were housed in a conventional environment. Experimental group: 1 control group (wild type control mouse Tg2576 mice); 2 model group (Tg2576 mice injected with TAT-scramble-HA); 3 treatment group (Tg2576 injected with TAT-siP-PTPN1-HA small) Rat); 10 per group.
2.实验模型制备2. Experimental model preparation
①正常组：12月龄野生型对照小鼠，不处理1 normal group: 12-month-old wild-type control mice, not treated
②模型组：12月龄Tg2576小鼠，通过尾静脉单次注射给予TAT-scramble-HA多肽，剂量为10mg/kg。2 Model group: 12-month-old Tg2576 mice were given a TAT-scramble-HA polypeptide by a single injection in the tail vein at a dose of 10 mg/kg.
③治疗组：12月龄Tg2576小鼠，通过尾静脉单次注射给予TAT-siP-PTPN1-HA多肽，剂量为10mg/kg。3 Treatment group: 12-month-old Tg2576 mice were given TAT-siP-PTPN1-HA polypeptide by single injection in the tail vein at a dose of 10 mg/kg.
3.研究方法和实验结果3. Research methods and experimental results
*P<0.05，与正常组比较*P<0.05, compared with the normal group
**P<0.01，与正常组比较**P<0.01, compared with the normal group
#P<0.05，与模型组比较#P<0.05, compared with the model group
##P<0.01，与模型组比较##P<0.01, compared with the model group
①多肽诱导表达：采用原核表达载体，异丙基硫代半乳糖苷(IPTG)诱导表达，考马斯亮蓝染色鉴定(见图1)
1 polypeptide induced expression: using prokaryotic expression vector, isopropyl thiogalactoside (IPTG) induced expression, identified by Coomassie blue staining (see Figure 1)
多肽的核苷酸序列由武汉擎科公司合成，通过分子生物学技术，将其构建到原核表达载体pGEX-5X-1(购自GE Healthcare中国公司)，在BL21感受态(购自北京全式金公司)中进行转化，扩增。当菌液OD600达到0.6时，加入2mM的IPTG分别诱导0.5小时、1小时和2小时，最后通过SDS-PAGE电泳，并采用考马斯亮蓝染色观察蛋白表达。The nucleotide sequence of the peptide was synthesized by Wuhan Qingke Company, and it was constructed into the prokaryotic expression vector pGEX-5X-1 (purchased from GE Healthcare China) by molecular biology technology, and was competent in BL21 (purchased from Beijing full version). Transformation and amplification in the gold company). When the bacterial solution OD600 reached 0.6, 2 mM IPTG was added to induce 0.5 hour, 1 hour, and 2 hours, respectively, and finally, protein expression was observed by SDS-PAGE electrophoresis and Coomassie blue staining.
结果发现，当诱导2小时后，在目的分子量位置出现大量富集蛋白。图1中1、2、3和4分别表示没有加入IPTG诱导以及诱导0.5小时、1小时和2小时，均没有明显蛋白表达。箭头所指为目的多肽。As a result, it was found that a large amount of enriched protein appeared at the target molecular weight position after induction for 2 hours. 1, 2, 3 and 4 in Fig. 1, respectively, indicate that no significant protein expression was observed without induction of IPTG induction and induction for 0.5 hours, 1 hour and 2 hours. The arrow refers to the polypeptide of interest.
②表达鉴定：通过免疫印迹检测HA表达(见图2)2 Expression identification: HA expression was detected by immunoblotting (see Figure 2)
多肽经诱导表达后，进行纯化，并采用Factor Xa酶(购自NEB公司)进行剪切，分离出目的小分子多肽(约8kDa)。纯化蛋白样品，采用免疫印迹实验，通过HA特异性抗体(购自Abcam公司)检测表达情况。通过识别HA的特异性抗体染色，检测到TAT-siP-PTPN1-HA的表达，该结果证实表达成功。After the polypeptide was induced to express, it was purified, and subjected to shearing using Factor Xa enzyme (purchased from NEB) to isolate the small molecule polypeptide of interest (about 8 kDa). Protein samples were purified and assayed by Western blotting using HA-specific antibodies (purchased from Abcam). Expression of TAT-siP-PTPN1-HA was detected by staining for specific antibodies recognizing HA, and the results confirmed the expression was successful.
③小鼠学习记忆的能力：Morris水迷宫训练及测试(见图3)3 mice's ability to learn and remember: Morris water maze training and testing (see Figure 3)
实验所采用Morris水迷宫测试系统，包括圆形水池直径120cm，高60cm，圆柱形有机玻璃平台直径10cm，高40cm。水池中水面高约45cm，室温及水温均保持在26±2℃，平台放置于某一象限的中心位置，并没于水面下约2cm。训练时将小鼠从任一象限1/2弧度处头面向池壁轻放于水中，检测其潜伏期(即小鼠从入水点起到找到平台所用的时间)、路径作为衡量小鼠学习记忆和测试成绩的指标。每只小鼠每天在4个象限共训练4次，每次游泳时限为60秒，即在60秒内未找到平台者系统自动停止记录，潜伏期记为60秒，由测试者引导其上台，休息30秒后进行下一次训练。首先训练时间为6天，在第7天的时候，记录小鼠找到平台期所需时间，记录小鼠找到平台所需潜伏期。休息一天，去平台，计算1分钟内小鼠在目标象限的停留时间，并分析其在平台所在位置的穿越次数，以评价其记忆能力。The Morris water maze test system was used in the experiment, including a circular pool with a diameter of 120 cm and a height of 60 cm. The cylindrical plexiglass platform has a diameter of 10 cm and a height of 40 cm. The water level in the pool is about 45cm high, and the room temperature and water temperature are kept at 26±2°C. The platform is placed at the center of a certain quadrant and is not about 2cm below the water surface. During training, the mice were placed in the water from the head of the 1/2 radians in either quadrant to the pool wall, and the incubation period (ie, the time from the entry point of the mouse to the platform) was measured. The path was used to measure the learning and memory of the mice. The indicator of test scores. Each mouse was trained 4 times in 4 quadrants each time. The swimming time limit was 60 seconds. That is, the platform was not automatically found within 60 seconds. The incubation period was recorded as 60 seconds. The tester guided him to the stage and rested. The next training will take place after 30 seconds. The first training time was 6 days. On the 7th day, the time required for the mice to find the plateau was recorded, and the latency required for the mice to find the platform was recorded. Take a day off, go to the platform, calculate the dwell time of the mouse in the target quadrant within 1 minute, and analyze the number of crossings at the position of the platform to evaluate its memory ability.
结果显示跟正常组相比，模型组小鼠游泳轨迹混乱，找到平台所需潜伏期时间最长，说明小鼠学习能力受到损伤。在给予TAT-siP-PTPN1-HA后，治疗组小鼠跟模型组相比，轨迹重新恢复简单，潜伏期变短；模型组小鼠穿越平台的时间和距离最短，次数最少，说明模型组老鼠的记忆能力受到损害，不记得平台的位置；治疗组小鼠跟模型组相比穿越平台的时间和距离恢复到
正常一致，穿越平台次数也相应增加，其记忆能力在给予TAT-siP-PTPN1-HA后得到明显恢复。The results showed that compared with the normal group, the swimming trajectory of the model group mice was chaotic, and the incubation period required to find the platform was the longest, indicating that the learning ability of the mice was damaged. After TAT-siP-PTPN1-HA administration, compared with the model group, the mice in the treatment group had simple trajectory recovery and shorter latency; the mice in the model group had the shortest time and distance through the platform, and the number of times was the least, indicating that the mice in the model group Memory ability is impaired, the position of the platform is not remembered; the mice in the treatment group recovered to the time and distance of crossing the platform compared to the model group.
Normally consistent, the number of crossing platforms increased accordingly, and its memory ability was significantly restored after TAT-siP-PTPN1-HA administration.
④海马tau蛋白磷酸化：免疫印迹(见图4)；4 hippocampal tau protein phosphorylation: immunoblotting (see Figure 4);
水迷宫训练完成后，颈椎脱臼断头处死小鼠，迅速取出大脑脑组织置于0-4℃，0.05M Tris缓冲盐溶液(TB，pH7.0)内，快速分离双侧海马，制成10﹪的蛋白匀浆，在1000rmp离心5分钟，取上清，测定蛋白含量后备用。After the water maze training was completed, the mice were sacrificed by decapitation of the cervical vertebrae, and the brain tissue was quickly removed and placed in a 0-4 ° C, 0.05 M Tris buffered saline solution (TB, pH 7.0) to rapidly separate the bilateral hippocampus. The % protein homogenate was centrifuged at 1000 rpm for 5 minutes, the supernatant was taken, and the protein content was determined and used.
分别检测tau蛋白不同位点的磷酸化水平，包括丝氨酸396，丝氨酸404位点。β-actin为内参条带(β-actin购自Abcam公司)，证明蛋白上样量的一致性。通过与总tau抗体tau5比较(抗体tau5购自Abcam公司，用于检测tau的总量)，模型组磷酸化位点磷酸化水平明显增加，tau发生过度磷酸化；治疗组给TAT-siP-PTPN1-HA后，tau的过度磷酸化得到改善。Phosphorylation levels at different sites of tau protein were detected, including serine 396 and serine 404 sites. --actin was an internal reference band (β-actin was purchased from Abcam), demonstrating the consistency of protein loading. By comparison with the total tau antibody tau5 (antibody tau5 was purchased from Abcam for the total amount of tau), the phosphorylation level of the phosphorylation site in the model group was significantly increased, and tau was hyperphosphorylated; the treatment group was given TAT-siP-PTPN1 After HA, the hyperphosphorylation of tau is improved.
结果显示，与正常组相比较，模型组的海马内tau蛋白在丝氨酸396，丝氨酸404位点,磷酸化水平明显增高，TAT-siP-PTPN1-HA处理后，tau磷酸化水平明显下降。The results showed that compared with the normal group, the tau protein in the hippocampus of the model group was significantly increased in serine 396 and serine 404, and the phosphorylation level of tau was significantly decreased after TAT-siP-PTPN1-HA treatment.
⑤海马Aβ含量：酶联免疫吸附法(见图5)5 Hippocampus Aβ content: enzyme-linked immunosorbent assay (see Figure 5)
样品制备同免疫印迹，用Aβ42的抗体(购自Aβeta GmBH,德国)进行包被后漂洗，加入样品，用四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine,TMB)显色，并在分光光度计上波长450nm处读数。Sample preparation was performed with immunoblotting, and the antibody was washed with Aβ42 antibody (purchased from Aβeta GmBH, Germany), and the sample was added to develop color with tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB). And read at a wavelength of 450 nm on a spectrophotometer.
阿尔茨海默病病人的另一病理学特征是脑内Aβ聚集，Aβ的产生是由于淡粉样蛋白前体蛋白(APP)的剪切错误导致的，可以产生两种剪切片段Aβ40和Aβ42。其中，Aβ42具有较高的毒性作用。实验结果显示，模型组老鼠中Aβ42片段的释放明显增多，TAT-siP-PTPN1-HA治疗后，治疗组Aβ42回到正常组水平，进一步证明TAT-siP-PTPN1-HA能够改善阿尔茨海默病模型小鼠出现的病理特征。Another pathological feature of patients with Alzheimer's disease is the accumulation of Aβ in the brain, which is caused by a cleavage error of the light pink protein precursor protein (APP), which can produce two fragments Aβ40 and Aβ42. . Among them, Aβ42 has a high toxic effect. The results showed that the release of Aβ42 fragment in the model group was significantly increased. After treatment with TAT-siP-PTPN1-HA, the Aβ42 in the treatment group returned to the normal group, further demonstrating that TAT-siP-PTPN1-HA can improve Alzheimer's disease. Pathological features of model mice.
⑥海马突触形态：高尔基染色(见图6)6 hippocampal synaptic morphology: Golgi staining (see Figure 6)
水迷宫训练完成后，进行麻醉处理，并依次采用4％甲醛生理盐水、含重铬酸钾媒染液、1％硝酸银溶液灌流，经震荡切片机进行切片，采用梯度酒精脱水，二甲苯透明处理后，中性树胶封片，显微镜下观察树突棘形态。模型组小鼠的大脑海马内树突棘数量减少，蘑菇状树突棘比例下降，治疗组增加了树突棘密度和蘑菇状树突棘含量。
After the water maze training is completed, anesthesia treatment is carried out, and 4% formaldehyde normal saline, potassium dichromate mordant solution and 1% silver nitrate solution are sequentially perfused, sliced by a vibrating slicer, dehydrated by gradient alcohol, and transparently treated with xylene. After that, the neutral gum was sealed and the morphology of dendritic spines was observed under a microscope. In the model group, the number of dendritic spines in the hippocampus of the mice in the model group decreased, and the proportion of mushroom-like dendritic spines decreased. The treatment group increased the density of dendritic spines and the content of mushroom-like dendritic spines.
⑦小鼠电生理：长时程增强电生理记录(见图7)7 mouse electrophysiology: long-term enhanced electrophysiological recording (see Figure 7)
小鼠麻醉后固定于立体定位仪上，施行局部开颅术，将刺激电极和记录电极分别放在海马CA3和CA1区域。刺激CA3区诱发场电位。选择合适的刺激和记录电极位置。绘制I/O曲线，选择诱发fEPSP幅度最大值的40％当作刺激强度，使用一串高频刺激(100Hz，1秒)刺激CA3区Schaeffer collateral-commissural fibers(Schaeffer并行的连合纤维)诱导CA1区长时程增强(LTP)，记录120分钟。The mice were anesthetized and fixed on a stereotaxic instrument, and local craniotomy was performed. The stimulation electrodes and recording electrodes were placed in the hippocampal CA3 and CA1 regions, respectively. Stimulate the field potential induced by the CA3 region. Select the appropriate stimulus and record electrode position. I/O curves were drawn, and 40% of the maximum amplitude of the induced fEPSP was selected as the stimulation intensity. A series of high frequency stimulation (100 Hz, 1 second) was used to stimulate the CA3 Schaeffer collateral-commissural fibers (Schaeffer parallel conjugate fibers) to induce CA1. Long-term time-course enhancement (LTP), recorded for 120 minutes.
与正常组相比较，模型组小鼠的兴奋性突触后电位(EPSP)的幅度和频率都下降，说明模型组出现突触功能障碍。治疗组与模型组相比树突棘密度增加，兴奋性突触后电位(EPSP)的幅度和频率都明显升高。Compared with the normal group, the magnitude and frequency of excitatory postsynaptic potential (EPSP) in the model group decreased, indicating that synaptic dysfunction occurred in the model group. Compared with the model group, the density of dendritic spine increased and the amplitude and frequency of excitatory postsynaptic potential (EPSP) increased significantly.
以上结果显示，尾静脉注射TAT-siP-PTPN1-HA的Tg2576小鼠水迷宫训练及检测过程中平台的潜伏期增加，穿越平台次数明显减少说明小鼠的学习和记忆能力损伤，检测早老性痴呆相关指标tau蛋白磷酸化以及Aβ含量后发现，tau蛋白的磷酸化水平显著上升，Aβ含量也有明显升高，这些早老性痴呆的相关特征的改变，说明Tg2576小鼠模拟了老年痴呆症状。跟模型组相比较，在给与了TAT-siP-PTPN1-HA处理后，检测小鼠水迷宫行为学，其学习和记忆能力得到了有效的改善；同时，早老性痴呆相关指标tau蛋白磷酸化以及Aβ含量和模型组比较也有明显降低，说明TAT-siP-PTPN1-HA能缓减Tg2576小鼠海马区tau的异常过度磷酸化、能减少淀粉样蛋白Aβ沉积并改善动物学习和/或记忆障碍。本发明所述的多肽能用于老年性痴呆的预防和治疗。
The above results showed that the incubation period of the platform in the water maze training and detection of Tg2576 mice injected with TAT-siP-PTPN1-HA in the tail vein increased, and the number of crossing platforms decreased significantly, indicating that the learning and memory impairment of mice was related to the detection of Alzheimer's disease. After the phosphorylation of tau protein and the content of Aβ, it was found that the phosphorylation level of tau protein increased significantly, and the Aβ content also increased significantly. The changes in the correlation characteristics of these Alzheimer's disease indicated that Tg2576 mice mimicked the symptoms of Alzheimer's disease. Compared with the model group, after the treatment of TAT-siP-PTPN1-HA, the behavior of water maze was detected, and the learning and memory ability was effectively improved. Meanwhile, the indicator of tau protein was related to Alzheimer's disease. The Aβ content was also significantly reduced compared with the model group, indicating that TAT-siP-PTPN1-HA can alleviate the abnormal hyperphosphorylation of tau in the hippocampus of Tg2576 mice, reduce amyloid Aβ deposition and improve animal learning and/or memory impairment. . The polypeptide of the present invention can be used for the prevention and treatment of senile dementia.
Claims (10)
- 一种多肽，该多肽选自下组：A polypeptide selected from the group consisting of:(a)一种包括SEQ ID NO:1的氨基酸序列的多肽；(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1;(b)一种包括与SEQ ID NO:1的氨基酸序列的多肽具有至少80％序列同一性的氨基酸序列的多肽；或(b) a polypeptide comprising an amino acid sequence having at least 80% sequence identity to a polypeptide of the amino acid sequence of SEQ ID NO: 1;(c)一种在SEQ ID NO:1的氨基酸序列上包括一个或多个的氨基酸残基的取代、缺失和/或插入的多肽。(c) a polypeptide comprising a substitution, deletion and/or insertion of one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1.
- 权利要求1的多肽，其中该多肽与SEQ ID NO：1所示的氨基酸序列具有至少85％，90％，95％，96％，97％，98％，99％或100％的同一性。The polypeptide of claim 1, wherein the polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 1.
- 权利要求1的多肽，其中该多肽具有SEQ ID NO：1所示的氨基酸序列。The polypeptide of claim 1, wherein the polypeptide has the amino acid sequence set forth in SEQ ID NO: 1.
- 权利要求1-3任一项所述的多肽，其中该多肽能用于治疗或预防老年痴呆。The polypeptide of any one of claims 1 to 3, wherein the polypeptide is useful for treating or preventing senile dementia.
- 一种编码权利要求1-3任一项所述小分子多肽的核苷酸序列。A nucleotide sequence encoding the small molecule polypeptide of any of claims 1-3.
- 根据权利要求5所述的核苷酸序列，其序列为SEQ ID NO：2所示。The nucleotide sequence according to claim 5, which has the sequence of SEQ ID NO: 2.
- 一种核酸构建体，包含权利要求5的核苷酸序列，该核苷酸序列可操作地连接到一种或多种指导所述多肽在合适的宿主中生产的控制序列。A nucleic acid construct comprising the nucleotide sequence of claim 5 operably linked to one or more control sequences which direct production of the polypeptide in a suitable host.
- 一种包含权利要求7所述的核酸构建体的重组表达载体。A recombinant expression vector comprising the nucleic acid construct of claim 7.
- 权利要求1-4任一项所述的小分子多肽在制备治疗或预防老年痴呆药物中的应用。Use of the small molecule polypeptide according to any one of claims 1 to 4 for the preparation of a medicament for treating or preventing Alzheimer's disease.
- 一种药物组合物，包含权利要求1-4任一项所述的小分子多肽。 A pharmaceutical composition comprising the small molecule polypeptide of any one of claims 1-4.
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