WO2017202394A1 - 用于防治阿尔茨海默病的融合蛋白及其制备方法和应用 - Google Patents

用于防治阿尔茨海默病的融合蛋白及其制备方法和应用 Download PDF

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WO2017202394A1
WO2017202394A1 PCT/CN2017/092850 CN2017092850W WO2017202394A1 WO 2017202394 A1 WO2017202394 A1 WO 2017202394A1 CN 2017092850 W CN2017092850 W CN 2017092850W WO 2017202394 A1 WO2017202394 A1 WO 2017202394A1
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p75ntr
ecd
fusion protein
human
seq
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麦俊波
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深圳麦迪科生物技术有限公司
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Definitions

  • the invention relates to the field of biomedical technology, in particular to a fusion protein for preventing and treating Alzheimer's disease, a preparation method and application thereof.
  • AD Alzheimer's disease
  • Alzheimer's disease Since the discovery of Alzheimer's disease in the 20th century, the incidence of AD has increased with the aging of the aging and the rapid growth of the corresponding elderly population. The prevention and treatment of AD has become the focus of attention of governments and medical circles.
  • AD Alzheimer's disease
  • ⁇ -amyloid cascade theory is dominant.
  • a ⁇ amyloid beta-peptide
  • a ⁇ amyloid deposits in the brain
  • a ⁇ is composed of 39-43 amino acid residues, of which A ⁇ 40 and A ⁇ 42 are dominant.
  • a ⁇ is derived from its precursor protein (APP), and A ⁇ is a hydrolysis of APP by ⁇ - and ⁇ -secretase.
  • APP metabolism is abnormal, resulting in excessive production of A ⁇ , or A ⁇ aggregation and precipitation due to A ⁇ clearance disorder to form amyloid plaques.
  • the neurotrophin receptor p75 (p75NTR) combined with A ⁇ and its polymer mediates the neurotoxicity of ⁇ -amyloid peptide, induces the body to produce an inflammatory response, leads to neuronal pathology and apoptosis, promotes the occurrence of AD and further deteriorates the condition.
  • Soluble A ⁇ or its oligomers and insoluble A ⁇ fibrils and plaques have neurotoxic effects. Any method that prevents the overproduction and aggregation of A ⁇ in the brain and blocks the binding of p75NTR to A ⁇ -mediated neurotoxicity can achieve the goal of preventing AD and alleviating AD or preventing AD progression.
  • p75NTR is a p75 neurotrophic factor receptor, a transmembrane glycoprotein with a relative molecular mass of 75 kD. It consists of 427 amino acids and contains a signal peptide consisting of 28 amino acids, an extracellular domain enriched in cysteine, and hydrophobic. The transmembrane region and the intracellular domain of a basic amino acid consisting of 155 amino acids. Its extracellular domain (p75NTR-ECD) consists of four cysteine-rich repeat regions (CRDs), each repeat containing a repeating structure of 40 amino acids and six cysteines, translation After N- and O-glycosylation modification, the second repeat sequence is required for binding to neurotrophic factors (NTs) and A ⁇ .
  • NTs neurotrophic factors
  • TACE TNF-alpha converting enzyme
  • p75NTR-ECD and p75NTR-ECD-FC designed and prepared by Wang Yanjiang and Zhou Xinfu and Wang Yongtang have the following defects; (1) they all use the prokaryotic expression system to prepare the target protein, and the prepared products are not translated sugar. The basic modification, thus failing to maintain the original physical and chemical properties of p75NTR-ECD and the natural spatial conformation of the protein, can not guarantee the effective biological activity of the protein prepared for clinical treatment of AD.
  • p75NTR-ECD-FC is a fusion protein of p75NTR-ECD and FC segment of human immunoglobulin, in which the FC segment can induce antibody-dependent cell-mediated cytotoxicity (ADCC) in vivo, leading to its own immunity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the system attacks the neuronal cells, promotes AD or promotes the progression and deterioration of AD, and does not achieve the purpose of preventing AD.
  • the FC segment of p75NTR-ECD-FC is immunogenic and can induce the body to produce anti-FC antibodies, which will cause the ECD biological activity to fail or be rapidly reduced. solution.
  • p75NTR-ECD and p75NTR-ECD-FC contain only ECD, which can only bind to A ⁇ , failing to induce the body to promote the degradation of A ⁇ , so it can not effectively prevent AD.
  • Interleukin-33 is a semaphore of the interleukin-1 (IL-1) family discovered in 2005. Its gene sequence and IL-1 family member IL -1 ⁇ is similar to IL-18. IL-33 is an important regulator of innate immune response and immune cell infiltration and activation. IL-33 contains 270 amino acids and contains an N-terminal nuclear localization signal, a helix-turn-helix motif and a C-terminus with a molecular weight of approximately 18 kDa.
  • IL-33 binds to a heterodimeric receptor complex composed of ST2 and IL-1RAcP, triggering a cascade of intracellular signaling pathways comprising myeloid differentiation factor 88 (MyD88) and NF- ⁇ B, optionally Type 2 helper T cells, mast cells, neutrophils, and selectively activated macrophages are activated.
  • MyD88 myeloid differentiation factor 88
  • NF- ⁇ B optionally Type 2 helper T cells
  • mast cells optionally activated macrophages
  • macrophages optionally activated macrophages are activated.
  • IL-33 is expressed by oligodendrocytes, while ST2 is mainly expressed by microglia and astrocytes.
  • IL-33 is a pluripotent, pleiotropic cytokine that plays an important regulatory role in infection, inflammation and autoimmune diseases.
  • IL-33 can protect against atherosclerosis, an important risk factor that promotes the development and progression of AD (see “Miller, AM, Xu, D., Asquith, DL, Denby, L., Li, Y., Sattar, N., Baker, AH, McInnes, IB, Liew, FY, 2008. IL-33reduces the development of atherosclerosis. J. Exp. Med. 205, 339246.”). This protective effect may be related to the downregulation of A ⁇ secretion by IL-33. In 2011, Yasuoka, S. et al.
  • IL-33 induces proliferation of microglia and production of cytokines IL-1 ⁇ , TNF ⁇ and IL-10 in a dose-dependent manner (see “Yasuoka, S., Kawanokuchi, J., Parajuli, B., Jin, S., Doi, Y., Noda, M., Sonobe, Y., Takeuchi, H., Mizuno, T., Suzumura, A., 2011. Production and functions of IL -33in the central nervous system.Brain Res.1385,8–17.”). Therefore, the study found that IL-33 can prevent the occurrence of AD and prevent the progression and deterioration of AD, and has potential therapeutic effects on AD prevention.
  • the present invention proposes a fusion protein for preventing and treating Alzheimer's disease, a preparation method and application thereof.
  • a fusion protein for controlling Alzheimer's disease comprising the extracellular domain p75NTR-ECD of human p75NTR, human IL-33, and the carboxy terminus of the p75NTR-ECD and the amino terminus of the human IL-33, respectively.
  • a peptide the amino acid sequence of the p75NTR-ECD is set forth in SEQ ID NO. 1, and the nucleotide sequence is set forth in SEQ ID NO. 2; the amino acid sequence of the human IL-33 is set forth in SEQ ID NO. The nucleotide sequence is shown in SEQ ID NO.
  • a method for constructing the fusion protein comprising the step of linking the carboxy terminus of the extracellular domain p75NTR-ECD of human p75NTR and the amino terminus of human IL-33 via a linker peptide, the extracellular domain p75NTR-ECD of the human p75NTR
  • the amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2; the amino acid sequence of the human IL-33 is shown in SEQ ID NO.
  • the nucleotide sequence is shown in SEQ ID NO.
  • An expression vector for the fusion protein which comprises one of an mRNA, a DNA plasmid vector or a viral vector as a vector.
  • a method for preparing a DNA plasmid vector of the fusion protein comprising the steps of:
  • the gene fragment of the fusion protein p75NTR-ECD-IL-33 was digested and inserted into two corresponding restriction sites between a plasmid vector, transformed into a host strain, and a positive plasmid was extracted to insert the target gene. Sequencing to obtain a DNA plasmid vector containing the gene encoding p75NTR-ECD-IL-33;
  • steps (3) and (4) can be replaced with (3') and (4'), respectively:
  • a gene fragment encoding IL-33-Linker is obtained by a PCR reaction, wherein the amino terminus of IL-33 is linked to the carboxy terminus of Linker, and the gene fragment of Linker is derived from the reverse primer sequence of the PCR reaction;
  • IL-33-Linker (4') The amino terminus of IL-33-Linker was ligated to the carboxy terminus of human p75NTR-ECD by overlapping PCR reaction to obtain the gene fragment of fusion protein p75NTR-ECD-IL-33, wherein IL-33--Linker gene The 5' end of the fragment carries an restriction site, and the 3' end of the p75NTR-ECD gene fragment carries another restriction site.
  • a method for producing a target protein which is one of the extracellular domain p75NTR-ECD of human p75NTR, human IL-33 or the fusion protein p5NTR-ECD-IL-33; comprising the following steps:
  • the protein is the extracellular domain of human p75NTR, p75NTR-ECD or human IL-33, and the expression plasmid of the target protein carrying the Flag tag is obtained by PCR reaction: plasmid VFlag-p75NTR-ECD or plasmid VFlag-IL-33;
  • the fusion protein p5NTR-ECD-IL-33 was obtained by overlapping PCR reaction to obtain the expression plasmid of plasmid-tagged p75NTR-ECD-IL-33: plasmid VFlag-p75NTR-ECD-IL-33;
  • fusion protein or the DNA plasmid vector of the fusion protein for preparing a medicament for preventing and treating Alzheimer's disease.
  • a medicament for controlling Alzheimer's disease which is the fusion protein, or an mRNA liposome expressing the fusion protein, or a viral vector expressing the fusion protein gene.
  • the present invention is designed and constructed for controlling Al A fusion protein for melanoma (also known as p75NTR-ECD-IL-33), which is formed by the extracellular domain p75NTR-ECD of p75NTR and IL-33 linked by a linker (Linker), overcoming p75NTR-ECD or The instability of the fusion protein p75NTR-ECD-FC and the defects of immunogenicity and antibody-dependent cell-mediated cytotoxicity (ADCC) and the mechanism of action of preventing AD (only by binding to A ⁇ ), Combining the functions of both IL-33 and p75NTR-ECD and greatly improving the biological activity of AD by improving the stability and synergy of both.
  • Linker linker
  • p75NTR-ECD-IL-33 has both the biological functions of both p75NTR-ECD and IL-33.
  • the p75NTR-ECD in p75NTR-ECD-IL-33 binds to A ⁇ and its aggregates, prevents p75NTR from binding to A ⁇ and its aggregates, and protects central neurons from the neurotoxicity of A ⁇ .
  • IL-33 in p75NTR-ECD-IL-33 can induce the body to reduce the secretion of A ⁇ and promote the degradation of A ⁇ , prevent the occurrence of AD and prevent the progression and deterioration of AD.
  • p75NTR-ECD-IL-33 is a fusion protein that effectively enhances and prolongs the stability and half-life of p75NTR-ECD and IL-33 contained therein in vitro and in vivo.
  • (3) p75NTR-ECD-IL-33 enables the synergistic effects of p75NTR-ECD and IL-33 contained in the prevention and treatment (ie, prevention and treatment of AD), p75NTR-ECD-IL-33 is combined with A ⁇ by p75NTR-ECD IL-33 has a precise targeting effect on A ⁇ , which induces IL-33 to directly endocytize the body around A ⁇ and its oligomers or produce A ⁇ degrading enzyme to degrade A ⁇ , thereby improving the degradation efficiency of A ⁇ .
  • p75NTR-ECD-IL-33 can be constructed to be expressed in eukaryotic cell lines and humans, allowing p75NTR-ECD and IL-33 in p75NTR-ECD-IL-33 to be fully glycosylated after translation. The modification thus maintains the original physical and chemical properties and biological functions under physiological conditions.
  • p75NTR-ECD-IL-33 abolishes the immunogenicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of FC in p75NTR-ECD-FC, so p75NTR-ECD-IL-33 is expected to serve as A safe and effective drug for the prevention and treatment of AD fills a gap in the prevention and treatment of Alzheimer's disease.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Figure 1 is a schematic diagram showing the construction of the fusion protein p75NTR-ECD-IL-33 in an embodiment of the present invention
  • Figure 2 is a schematic diagram showing the plasmid vector of the fusion protein p75NTR-ECD-IL-33 in the embodiment of the present invention
  • Figure 3 is an electrophoresis pattern of the fusion protein p75NTR-ECD-IL-33, protein p75NTR-ECD and protein IL-33 prepared by HES-293 cell expression line by SDS-PAGE immunoblotting in a specific embodiment of the present invention.
  • Figure 4 is a schematic diagram showing the in vitro stability of the fusion protein p75NTR-ECD-IL-33, protein p75NTR-ECD and IL-33 in a specific embodiment of the present invention
  • Figure 5 is a schematic diagram showing the in vivo stability of the fusion protein p75NTR-ECD-IL-33, protein p75NTR-ECD and IL-33 in a specific embodiment of the present invention
  • Figure 6 is a graph showing changes in the concentration levels of A ⁇ 1-42 in the blood of AD mice after injection of the fusion protein p75NTR-ECD-IL-33 in a specific embodiment of the present invention
  • Figure 7 is a graph showing changes in the content of A? 1-42 in the cerebral cortex of AD mice after injection of the fusion protein p75NTR-ECD-IL-33 in a specific embodiment of the present invention.
  • the present invention designs and constructs a fusion protein for preventing Alzheimer's disease (also referred to as Chimeric antigen receptor protein (CAR-P), which can be named p75NTR-ECD-IL-33).
  • CAR-P Chimeric antigen receptor protein
  • linker means the same concept.
  • p75NTR The p75 neurotrophic factor receptor
  • human interleukin-33 can be abbreviated as “human IL-33” or "IL-33”.
  • the present invention provides a fusion protein for controlling Alzheimer's disease, which comprises the extracellular domain p75NTR-ECD of human p75NTR, human IL-33, and the carboxy terminus of the p75NTR-ECD and the human IL-33, respectively.
  • the amino terminal sequence of the p75NTR-ECD is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2; the amino acid sequence of the human IL-33 is SEQ ID NO. As shown in 3, the nucleotide sequence is shown in SEQ ID NO. specific:
  • amino acid sequence of p75NTR-ECD is (250aa):
  • the nucleotide sequence (750 bp) of p75NTR-ECD is:
  • the amino acid sequence of human IL-33 (269aa) is:
  • Human IL-33 gene nucleotide sequence (including stop codon TAA, 810bp):
  • the method for constructing the fusion protein p75NTR-ECD-IL-33 comprises the steps of linking the carboxy terminus of the extracellular domain p75NTR-ECD of human p75NTR and the amino terminus of human IL-33 via a linker (Linker). 1 is shown.
  • Linker uses a flexible linker peptide or a rigid linker peptide.
  • X is any amino acid
  • X is any one of lysine, alanine or glutamic acid.
  • Linker's amino acid sequence (15aa) is set forth in SEQ ID NO. 5 as: Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser, the Linker gene nucleotide sequence (45 bp) as shown in SEQ ID NO. 6, is:
  • Gly is a non-polar small amino acid that provides flexibility to the fusion protein, allowing the fusion protein to be better folded into a biologically functional natural conformation after translation without affecting p75NTR-ECD- The biological function of p75NTR-ECD and ECD-IL-3 in IL-33;
  • Ser is a polar small amino acid capable of forming hydrogen bonds with H 2 O molecules, increasing the water solubility of the linker peptide and stability in aqueous solution, Linker is prevented from interfering with the biological function of each protein in the fusion protein.
  • Ser can also be replaced with threonine Thr.
  • the fusion protein p75NTR-ECD-IL-33 was constructed using the preferred Linker linkage described above.
  • the amino acid sequence (534aa) is shown in SEQ ID NO. 7, and the nucleotide sequence (containing the start codon and stop codon, 1605 bp) is shown in SEQ ID NO. 8, respectively:
  • the present invention also provides an expression vector for the fusion protein in each of the above embodiments, which can select mRNA, DNA plasmid vector or viral vector (including Lentivirus, adenovirus, adeno-associated virus).
  • mRNA DNA plasmid vector or viral vector (including Lentivirus, adenovirus, adeno-associated virus).
  • viral vector including Lentivirus, adenovirus, adeno-associated virus.
  • adeno-associated virus is used as a carrier.
  • the invention also provides a preparation method of the DNA plasmid vector of the fusion protein, and in a specific embodiment, the following steps are included:
  • the gene fragment of the fusion protein p75NTR-ECD-IL-33 was digested and inserted into two corresponding restriction sites between a plasmid vector, transformed into a host strain, and a positive plasmid was extracted to insert the target gene. Sequencing to obtain a DNA plasmid vector containing the gene encoding p75NTR-ECD-IL-33;
  • steps (3) and (4) can be replaced with (3') and (4'), respectively:
  • a gene fragment encoding IL-33-Linker is obtained by a PCR reaction, wherein the amino terminus of IL-33 is linked to the carboxy terminus of Linker, and the gene fragment of Linker is derived from the reverse primer sequence of the PCR reaction;
  • IL-33-Linker (4') The amino terminus of IL-33-Linker was ligated to the carboxy terminus of human p75NTR-ECD by overlapping PCR reaction to obtain the gene fragment of fusion protein p75NTR-ECD-IL-33, wherein IL-33--Linker gene The 5' end of the fragment carries an restriction site, and the 3' end of the p75NTR-ECD gene fragment carries another restriction site.
  • pcDNA3.1(+) can be selected as the plasmid vector in the step (5); the gene fragment of the fusion protein p75NTR-ECD-IL-33 is inserted into the KpnI of pcDNA3.1(+) Between the XbaI restriction site and the XbaI restriction site.
  • the forward primer F1 shown in SEQ ID NO. 9 ATGGGTGCAGGGGCGACC; the reverse primer R1 shown in SEQ ID NO. 10: GTTGTCTGTCGTCCCTCGTGTTACTACAG.
  • Forward primer F4 (forward primer F3 as shown in SEQ ID NO. 13): CGC GGTACC ATGGGTGCAGGGGCGACC
  • the overlapping PCR target product ie, the gene fragment of the fusion protein p75NTR-ECD-IL-33 with the stop codon TAA at the end of the IL-33 gene fragment
  • the host strain DH5a (provided by ThermoFisher) was transformed, the positive plasmid was extracted, the inserted target gene was sequenced, the correctness of the sequence of the target gene was determined, and finally the recombinant pcDNA3.1 containing the gene encoding p75NTR-ECD-IL-33 was obtained.
  • Plasmid Vp75NTR-ECD-IL-33 Plasmid Vp75NTR-ECD-IL-33.
  • mRNA and viral vectors can be prepared by DNA plasmid vectors.
  • the plasmid Vp75NTR-ECD-IL-33 obtained above is linearized by XbaI, or DraII, or ApaI, or PmeI, and is in vitro transcribed (for example, an mRNA transcription kit can be used (Ambion). mMESSAGE Transcription Kit, AM1344)) can obtain mRNA encoding p75NTR-ECD-IL-33.
  • the plasmid Vp75NTR-ECD-IL-33 can also be used to prepare viral vectors (including Lentivirus, adenovirus, adeno-associated virus, etc.) expressing p75NTR-ECD-IL-33.
  • the present invention also provides a method for producing a target protein which is one of the extracellular domain p75NTR-ECD of human p75NTR, human IL-33 or the fusion protein p5NTR-ECD-IL-33.
  • the method of preparing the protein of interest comprises the steps of:
  • nucleotide sequence is shown in SEQ ID NO. 21:
  • GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG respectively, obtained expression plasmids of plasmid p75NTR-ECD (PCR technology), IL-33 (PCR technology) and p75NTR-ECD-IL-33 (overlapping PCR technology) with plasmid tag: plasmid VFlag-p75NTR-ECD, Plasmid VFlag-IL-33 and plasmid VFlag-p75NTR-ECD-IL-33; that is, the gene fragment of the target protein is used as a template, and a plasmid vector is used as a vector, and two restriction sites of the plasmid vector are used as a target gene.
  • the insertion site is linked to the Flag tag at the amino terminus of the target protein.
  • the expression plasmid of the target protein with Flag tag is obtained by PCR reaction: plasmid VFlag-p75NTR-ECD or plasmid VFlag-IL-33; if the target protein is the fusion protein p5NTR-ECD-IL-33, the expression plasmid containing the Flag-tagged p75NTR-ECD-IL-33 is obtained by overlapping PCR reaction: plasmid VFlag-p75NTR-ECD-IL-33.
  • pcDNA3.1(+) was selected as the vector, and the KpnI and XbaI cleavage sites were used as insertion sites for the target gene, as follows:
  • the forward primer F1 of the p75NTR-ECD gene fragment was cloned to F6 (as shown in SEQ ID NO. 22):
  • the forward primer F4 of the overlapping PCR was replaced by F7 (as shown in SEQ ID NO. 23):
  • the PCR primers are:
  • Forward primer F8 (shown as SEQ ID NO. 24):
  • Reverse primer R8 (shown as SEQ ID NO. 25):
  • the PCR primers are:
  • Forward primer F9 (shown as SEQ ID NO. 26):
  • Reverse primer R9 (shown as SEQ ID NO. 27):
  • HEK-293 cells were treated with 10% (v/v) FBS (Gibco), 100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin in DMEM (Gibco) complete medium, placed at 37 ° C, 5% (volume fraction) CO 2 incubator culture. Cell passage was performed when the cells were 80% full. Before plasmid transfection, HEK-293 cells were planted in 60-mm cell culture dishes and cultured in DMEM (Gibco) medium containing 10% (v/v) FBS (Gibco). The next day, when the cells grew 80% full time for plasmid transfection.
  • plasmid VFlag-p75NTR-ECD plasmid VFlag-IL-33 and plasmid VFlag-p75NTR-ECD-IL-33 were diluted to 0.5 ml with Opti-MEM (Gibco) and gently mixed into a diluent.
  • Opti-MEM Opti-MEM
  • Diluent A and Diluent B were mixed and gently stirred into a liposome transfection mixture and allowed to stand at room temperature for 20 minutes.
  • the liposome transfection mixture was evenly added to the medium in a 60-mm cell culture dish and gently mixed. After the cells were further cultured for 24 hours, the medium was changed to DMEM containing 10% (v/v) FBS (Gibco), 100 ⁇ g/ml penicillin, 100 ⁇ g/ml streptomycin, and 500 ⁇ g/ml neomycin sulfate (Clontech).
  • the screening medium was continued with Gibco) screening medium.
  • the screening medium was changed every 3 days, cells were passaged every 6 days, and cells were collected 3 weeks later, some of which were treated with complete medium containing 5% (v/v) dimethyl sulfoxide (Sigma-Aldrich).
  • the mixture was placed in a liquid nitrogen tank and stored as a stable cell line expressing p75NTR-ECD, IL-33 and p75NTR-ECD-IL-33, CLp75NTR-ECD, CL IL-33 and CLp75NTR-ECD-IL-33, and remaining cells. Extraction and purification of the target proteins p75NTR-ECD, IL-33 and p75NTR-ECD-IL-33, respectively.
  • the collected cells were lysed with CelLytic M cell lysate (Sigma-Aldrich, C2978) containing 10 ⁇ l of protease inhibitor (Sigma-Aldrich, P8340) per 1 ml of lysate, and the lysate supernatant was collected.
  • An affinity chromatography column was prepared by filling the column with an anti-Flag antibody M2 affinity resin (ANTI-FLAG M2affinity resin), and the column was pre-equilibrated with an equilibrium solution.
  • the supernatant was lysed onto the column, and the non-target protein was eluted by washing the column with 10-20 times column volume of TBS buffer (50 mM Tris HCl, with 150 mM NaCl, pH 7.4).
  • the target protein was eluted with a 0.1 M glycine hydrochloride solution of pH 3.5 into pre-frozen tubules prefilled with 15-25 ⁇ l of pH 8.0 in 1 M Tris buffer to obtain p75NTR-ECD, IL-33 and p75NTR-ECD-IL, respectively.
  • 33 target protein crude solution A small sample was taken to determine the protein content.
  • the crude protein of the target protein was directly used for further purification or placed in a -70 ° C freezer for storage.
  • the enterokinase of 0.02 unit/mg fusion protein was mixed and allowed to stand at room temperature for 16 hours. After the end of the reaction, the enterokinase in the reaction mixture was removed using ENTEREKINASE REMOVAL KIT (Sigma-Aldrich, PRK-E) supplied by Sigma-Aldrich to obtain purified target proteins p75NTR-ECD, IL-33 and A solution of p75NTR-ECD-IL-33. A small amount of sample was taken to determine protein content and purity. The target protein solution was dispensed and stored in a -70 ° C freezer or a liquid nitrogen tank.
  • the identification of the target protein is as follows:
  • N-terminal amino acid sequence The target proteins p75NTR-ECD, IL-33 and p75NTR-ECD-IL-33 were transferred to PVDF membrane by SDS-PAGE electrophoresis, and the Shimadzu PPSQ-31A protein automatic sequencer was used. The N-terminal amino acid sequence of the target protein was determined. As a result of the measurement, the N-terminal amino acid sequence of the target protein p75NTR-ECD was determined to be GlyAlaGlyAlaThrGlyArg, and the sequence was identical to the N-terminal amino acid sequence of the native protein human p75NTR-ECD.
  • the N-terminal amino acid sequence of the target protein IL-33 was determined to be: LysProLysMetLysTyrSer, and the sequence was identical to the N-terminal amino acid sequence of the native protein IL-33.
  • the N-terminal amino acid sequence of the target protein p75NTR-ECD-IL-33 was determined to be: GlyAlaGlyAlaThrGlyArg, and the sequence was identical to the N-terminal amino acid sequence of the fusion protein p75NTR-ECD-IL-33.
  • DSC Differential Scanning Calorimetry
  • the Tm value of the target protein p75NTR-ECD-IL-33 was 78.0 ° C
  • the Tm value of the protein p75NTR-ECD was 66.0 ° C
  • the Tm value of the protein IL-33 was 56.0 ° C.
  • the results showed that the recombinant fusion protein p75NTR-ECD-IL-33 significantly increased the stability of the proteins p75NTR-ECD and IL-33. The result is shown in Figure 4.
  • the in vivo stability assay of the target protein p75NTR-ECD-IL-33 was achieved by measuring the half-life of the exogenous protein p75NTR-ECD-IL-33 in vivo. Twenty AD mice were taken, of which 5 were blank control group, 5 were p75NTR-ECD group (intraperitoneal injection of 300 ng p75NTR-ECD), and 5 were IL-33 group (intraperitoneal injection of 300 ng IL-33), 5 Only the p75NTR-ECD-IL-33 group (intraperitoneal injection of 300 ng p75NTR-ECD-IL-33).
  • the venous blood was taken from the tail vein after the mice were injected for 2, 4, 6, 8, 12, 16, 20, and 24 hours, using the ILISA kit of the Biosensis NGFR/p75ECD RapidTM enzyme-linked immunosorbent assay (ELISA) Kit.
  • ELISA enzyme-linked immunosorbent assay
  • the half-lives of p75NTR-ECD, IL-33 and p75NTR-ECD-IL-33 were calculated.
  • the half-life of p75NTR-ECD was 8 hours
  • the half-life of IL-33 was 6 hours
  • the half-life of p75NTR-ECD-IL-33 was 18 hours.
  • the results showed that the recombinant protein p75NTR-ECD-IL-33 significantly increased the stability of p75NTR-ECD and IL-33 in vivo. The result is shown in Figure 5.
  • the in vivo biological function assay of the target protein p75NTR-ECD-IL-33 was achieved by measuring the exogenous protein p75NTR-ECD-IL-33 in vivo to promote the body's secretion of A ⁇ and promote the degradation of A ⁇ .
  • mice Twenty AD mice were taken, of which 5 were blank control group, 5 were p75NTR-ECD group (3 days consecutively, 300 ng p75NTR-ECD was injected intraperitoneally daily), and 5 were IL-33 group (3 consecutive days, daily) 300 ng of IL-33 was injected intraperitoneally and 5 of the p75NTR-ECD-IL-33 group (300 ng p75NTR-ECD-IL-33 was injected intraperitoneally daily for 3 consecutive days).
  • the tail vein blood and cerebral cortex were taken 1 week after the mice began to inject the samples.
  • the cerebral cortex was frozen with dry ice and then homogenized with a protease inhibitor-containing homogenization buffer (250 mM sucrose, 20 mM pH 7.4 Tris-HCl, 1 mM EDTA and 1 mM EGTA) and a Dunes homogenizer to extract the soluble solution using diethylamine.
  • a protease inhibitor-containing homogenization buffer 250 mM sucrose, 20 mM pH 7.4 Tris-HCl, 1 mM EDTA and 1 mM EGTA
  • Dunes homogenizer to extract the soluble solution using diethylamine.
  • a ⁇ The venous blood and soluble A ⁇ solution were measured in the blood and cerebral cortex of A ⁇ 1-42 using the ILISA kit Amyloid beta 42 ELISA Kit, Mouse (Novex, KMB3441) supplied by Thermo Fisher.
  • the recombinant fusion protein p75NTR-ECD-IL-33 fusion p75NTR-ECD and IL-33 has a significant synergistic and promoting effect on the biological function of p75NTR-ECD and IL-33 in the prevention and treatment of AD.
  • the results are shown in Figures 6 and 7.
  • the invention also provides the fusion protein described in the above embodiments, in the preparation of the prevention and treatment of Alzhai
  • the drug for morbidity and the drug for controlling Alzheimer's disease; the drug may be the fusion protein itself described in the above embodiments, or the mRNA liposome expressing the fusion protein, or A viral vector expressing the fusion protein gene is carried.
  • the clinical use of the drug may be: direct injection of the recombinant fusion protein p75NTR-ECD-IL-33 intraperitoneally, intravenously or intracereally, or the expression of the fusion protein p75NTR-ECD-IL-33 by intravenous or intracerebral administration.
  • mRNA liposome or viral vector carrying the p75NTR-ECD-IL-33 gene including Lentivirus, adenovirus, adeno-associated virus, etc.).
  • p75NTR-ECD-IL-33 has both the biological functions of p75NTR-ECD and IL-33.
  • the p75NTR-ECD in p75NTR-ECD-IL-33 binds to A ⁇ and its aggregates, prevents p75NTR from binding to A ⁇ and its aggregates, and protects central neurons from the neurotoxicity of A ⁇ .
  • IL-33 in p75NTR-ECD-IL-33 can induce the body to reduce the secretion of A ⁇ and promote the degradation of A ⁇ , prevent the occurrence of AD and prevent the progression and deterioration of AD.
  • p75NTR-ECD-IL-33 is a fusion protein that effectively enhances and prolongs the stability and half-life of p75NTR-ECD and IL-33 contained therein in vitro and in vivo.
  • p75NTR-ECD-IL-33 enables p75NTR-ECD and IL-33 to play a synergistic role in the prevention and treatment of AD.
  • p75NTR-ECD-IL-33 binds IL-33 via p75NTR-ECD in combination with A ⁇ .
  • a ⁇ has precise targeting, which makes IL-33 induce direct endocytosis around A ⁇ and its oligomers or produce A ⁇ degrading enzyme to degrade A ⁇ , thereby improving the degradation efficiency of A ⁇ .
  • the biological activity of the p75NTR-ECD-IL-33 fusion protein was significantly increased compared to the p75NTR-ECD and IL-33 monomers.
  • p75NTR-ECD-IL-33 was constructed to be expressed in eukaryotic cell lines and in humans, allowing p75NTR-ECD and IL-33 in p75NTR-ECD-IL-33 to be fully glycosylated after translation. The modification thus maintains the original physical and chemical properties and biological functions under physiological conditions.
  • p75NTR-ECD-IL-33 abolishes the immunogenicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of FC in p75NTR-ECD-FC, so p75NTR-ECD-IL-33 can be used as a preventive And safe and effective drugs for the treatment of AD.
  • ADCC antibody-dependent cell-mediated cytotoxicity

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Abstract

一种用于防治阿尔茨海默病的融合蛋白及其制备方法和应用，该融合蛋白包括人p75NTR（p75神经营养因子受体）的胞外域p75NTR-ECD、人IL-33（白细胞介素-33）以及分别连接所述p75NTR-ECD的羧基端和所述人IL-33的氨基端的连接肽；所述p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。上述p75NTR-ECD、人IL-33融合蛋白克服了p75NTR-ECD或其融合蛋白p75NTR-ECD-FC的不稳定和具有免疫原性和抗体依赖性细胞介导的细胞毒作用（ADCC）的缺陷和防治AD的作用机理单一的缺点，同时将IL-33和p75NTR-ECD两者的功能结合在一起并通过提高两者的稳定性和协同作用大大提升防治AD的生物学活性。

Description

用于防治阿尔茨海默病的融合蛋白及其制备方法和应用 技术领域
本发明涉及生物医学技术领域，特别是涉及一种用于防治阿尔茨海默病的融合蛋白及其制备方法和应用。
背景技术
阿尔茨海默病(Alzheimer's disease，AD)，常称“老年痴呆”症。自从20世纪发现阿尔茨海默病以来，AD发病率随着老龄化的加剧和相应的老年人口的快速增长而升高，AD的防治已成为各国政府和医学界关注的焦点。
AD是一种起病隐匿的渐进性发展的神经系统退行性疾病，其确切的发病机理至今未明。AD的病因学说有多种，其中主要以β-淀粉样级联学说占主导地位。大脑出现包含淀粉样β肽(Aβ)淀粉斑的沉积是AD的主要组织病理学特征。淀粉斑的主要成分是Aβ，Aβ由39-43个氨基酸残基组成，而其中以Aβ40和Aβ42为主。Aβ来源于自其前体蛋白(Amyloid precursor protein，APP)，Aβ是APP分别经β-和γ-分泌酶(secretase)水解而成。在病理状态下，APP代谢异常，导致Aβ生成过多，或者由于Aβ清除障碍导致Aβ聚集、沉淀形成淀粉斑。神经营养因子受体p75(p75NTR)结合Aβ及其聚合体介导了β淀粉肽的神经毒性反应，诱导机体产生炎症反应，导致神经元病变和凋亡，促进AD的发生和病情进一步恶化。可溶性Aβ或其寡聚体和不溶性Aβ纤丝和斑块都有神经毒性作用。任何能防止大脑内Aβ的过度产生和聚集以及阻断p75NTR结合Aβ介导神经毒性反应的方法都可达到预防AD发生和缓解AD病情或阻止AD病情恶化的目的。
p75NTR是p75神经营养因子受体，是相对分子质量为75kD的跨膜糖蛋白，由427个氨基酸组成，包含一条由28个氨基酸组成的信号肽、富集半胱氨酸的胞外域、疏水性的跨膜区以及由155个氨基酸组成的富含碱性氨基酸的胞内域。其胞外域(p75NTR extracellular domain，p75NTR-ECD)由4个半胱氨酸富集重复序列区(CRDs)组成，每个重复序列含有40个氨基酸组成的重复结构和6个半胱氨酸，翻译后经N-与O-糖基化修饰，第2个重复序列是结合神经营养因子(NTs)和Aβ所必需的。其跨膜区由含有22个氨基酸的单链组成，跟p75NTR的磷酸化有关。p75NTR被TACE(TNF-alpha converting enzyme)酶所裂解释放其胞外域p75NTR-ECD。
根据游离的p75NTR-ECD可结合Aβ从而阻止p75NTR与Aβ结合产生神经毒性的原理，在中国专利ZL201010561284.3中，王延江和周新富设计p75NTR-ECD和p75NTR-ECD-FC用于AD的防治；在申请号为201210053808.7的中国专利申请中，王永堂等人设计p75NTR-ECD-FC用于促进损伤中枢神经再生与功能恢复。王延江和周新富以及王永堂等人设计和制备的p75NTR-ECD和p75NTR-ECD-FC存在以下缺陷；(1)他们都使用原核表达系制备目标蛋白，所制备的产物都未经过翻译后的糖基化修饰，因而未能保持p75NTR-ECD的原有的理化性质和蛋白质的天然空间构象，所以无法保证其制备的蛋白具有有效的生物活性用于AD的临床治疗。(2)p75NTR-ECD-FC是p75NTR-ECD与人免疫球蛋白的FC段的融合蛋白，其中的FC段能在体内诱导抗体依赖性细胞介导的细胞毒作用(ADCC)，导致自身的免疫系统攻击神经元细胞，促发AD或者促使AD进展和恶化，达不到防治AD的目的。(3)p75NTR-ECD-FC的FC段具有免疫原性，能诱导机体产生抗FC的抗体，促使ECD生物活性失效或者被快速降 解。(4)p75NTR-ECD和p75NTR-ECD-FC只含ECD，只能和Aβ结合，未能诱导机体促进Aβ的降解，所以无法有效防治AD。
白细胞介素-33(Interleukin-33,IL-33)是在2005年发现的白细胞介素-1(Interleukin-1，IL-1)家族的一个信号素，其基因序列与IL-1家族成员IL-1β和IL-18相似。IL-33是协调先天性免疫反应及免疫细胞浸润和活化的重要调节因子。IL-33含有270个氨基酸，包含一个N末端核定位信号、螺旋-转角-螺旋的基元和C端，分子量约18kDa。IL-33与由ST2和IL-1RAcP组成的异源二聚体受体复合物结合，触发包含髓样分化因子88(MyD88)和NF-κB的细胞内信号通道的级联反应，选择性地激活2型辅助性T细胞、肥大细胞，中性粒细胞，和选择性激活的巨噬细胞。在中枢神经系统(CNS)，IL-33由少突胶质细胞表达，而ST2则主要由小胶质细胞和星形胶质细胞表达。IL-33是一种多潜能、多效性的细胞因子，在感染、炎症和自身免疫性疾病中发挥着十分重要的调节作用。2009年，Chapuis J等人通过遗传学研究发现AD患者脑内的IL-33表达显著减少，且IL-33基因的三个单核苷酸多态性(SNPs)与降低AD的发生和进展的风险密切相关(参见“Chapuis J,et al.(2009)Transcriptomic and genetic studies identify IL-33as a candidate gene for Alzheimer’s disease.Mol Psychiatry 14(11):1004–1016.”)。2008年Miller,A.M.等人发现IL-33能对促进AD的发生和进展的重要危险因子动脉粥样硬化起到保护性作用(参见“Miller,A.M.,Xu,D.,Asquith,D.L.,Denby,L.,Li,Y.,Sattar,N.,Baker,A.H.,McInnes,I.B.,Liew,F.Y.,2008.IL-33reduces the development of atherosclerosis.J.Exp.Med.205,339246.”)。这种保护性作用可能与IL-33通过下调Aβ的分泌有关。2011年Yasuoka,S.等人发现IL-33能以呈剂量依赖性方式诱导小胶质细胞的增殖和细胞因子IL-1β、TNFα和IL-10的产生(参见“Yasuoka,S.,Kawanokuchi,J.,Parajuli,B.,Jin,S.,Doi,Y.,Noda,M.,Sonobe,Y.,Takeuchi,H.,Mizuno,T.,Suzumura,A.,2011.Production and functions of IL-33in the central nervous system.Brain Res.1385,8–17.”)。因此，研究发现IL-33能够防止AD的发生和阻止AD病情的进展和恶化，对AD防治具有潜在的治疗作用。
鉴于目前还没有防治AD的有效药物和有效的治疗手段，以及p75NTR-ECD和p75NTR-ECD-FC在开发应用于AD防治方面存在缺陷，找到能够用于防治AD的有效药物至关重要。
发明内容
为了弥补上述现有技术的不足，本发明提出一种用于防治阿尔茨海默病的融合蛋白及其制备方法和应用。
本发明的技术问题通过以下的技术方案予以解决：
一种用于防治阿尔茨海默病的融合蛋白，包括人p75NTR的胞外域p75NTR-ECD、人IL-33以及分别连接所述p75NTR-ECD的羧基端和所述人IL-33的氨基端的连接肽；所述p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
一种所述的融合蛋白的构建方法，包括将人p75NTR的细胞外域p75NTR-ECD的羧基端和人IL-33的氨基端通过连接肽连接起来的步骤，所述人p75NTR的细胞外域p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核 苷酸序列如SEQ ID NO.4所示。
一种所述的融合蛋白的表达载体，其以mRNA，DNA质粒载体或病毒载体中的一种作为载体。
一种所述的融合蛋白的DNA质粒载体的制备方法，包括如下步骤：
(1)克隆得到人p75NTR的细胞外域p75NTR-ECD的基因片段；
(2)克隆得到人IL-33的基因片段；
(3)通过PCR反应得到编码p75NTR-ECD-Linker的基因片段，其中p75NTR-ECD的羧基端与Linker的氨基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
(4)通过重叠PCR反应将p75NTR-ECD-Linker的羧基端与人IL-33的氨基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，p75NTR-ECD-Linker基因片段的5’端带有一酶切位点，IL-33基因片段的3’端带有另一酶切位点；
(5)将融合蛋白p75NTR-ECD-IL-33的基因片段通过酶切后插入一质粒载体中两个相应的酶切位点之间，转化宿主菌，提取阳性质粒，对插入的目的基因进行测序，获得含有编码p75NTR-ECD-IL-33目的基因的DNA质粒载体；
或者，步骤(3)和(4)可以分别用(3’)和(4’)替换：
(3’)通过PCR反应得到编码IL-33-Linker的基因片段，其中IL-33的氨基端与Linker的羧基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
(4’)通过重叠PCR反应将IL-33-Linker的氨基端与人p75NTR-ECD的羧基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，IL-33--Linker基因片段的5’端带有一酶切位点，p75NTR-ECD基因片段的3’端带有另一酶切位点。
一种目标蛋白的制备方法，所述目标蛋白为人p75NTR的胞外域p75NTR-ECD、人IL-33或所述的融合蛋白p5NTR-ECD-IL-33中的一种；包括如下步骤：
(1)以目标蛋白的基因片段为模板，以一质粒载体为载体，以该质粒载体的两个酶切位点为目的基因的插入位点，在目标蛋白的氨基端连接Flag标签，若目标蛋白为人p75NTR的胞外域p75NTR-ECD或人IL-33，则采用PCR反应相应获得带有Flag标签的目标蛋白的表达质粒：质粒VFlag-p75NTR-ECD或质粒VFlag-IL-33；若目标蛋白为融合蛋白p5NTR-ECD-IL-33，则采用重叠PCR反应获得带有Flag标签的p75NTR-ECD-IL-33的表达质粒：质粒VFlag-p75NTR-ECD-IL-33；
(2)将表达质粒转染真核细胞获得表达目标蛋白的稳定细胞系；
(3)从稳定表达目标蛋白的细胞中提取和初步纯化目标蛋白得到目标蛋白粗溶液；
(4)从目标蛋白粗溶液中去除Flag标签获取纯化的目标蛋白。
一种所述的融合蛋白或所述的融合蛋白的DNA质粒载体在制备防治阿尔茨海默病的药物中的应用。
一种用于防治阿尔茨海默病的药物，所述药物为所述的融合蛋白，或者表达所述的融合蛋白的mRNA脂质体，或者携带表达所述的融合蛋白基因的病毒载体。
本发明与现有技术对比的有益效果包括：本发明设计和构建了用于防治阿尔 茨海默病的融合蛋白(也可以命名为p75NTR-ECD-IL-33)，其由p75NTR的胞外域p75NTR-ECD和IL-33通过连接肽(Linker)连接而成，克服了p75NTR-ECD或其融合蛋白p75NTR-ECD-FC的不稳定和具有免疫原性和抗体依赖性细胞介导的细胞毒作用(ADCC)的缺陷和防治AD的作用机理单一(只通过和Aβ结合)的缺点，同时将IL-33和p75NTR-ECD两者的功能结合在一起并通过提高两者的稳定性和协同作用大大提升防治AD的生物学活性。具体来说，具有以下优点：(1)p75NTR-ECD-IL-33同时具有p75NTR-ECD和IL-33两者的生物学功能。p75NTR-ECD-IL-33中的p75NTR-ECD能与Aβ及其聚合体结合，阻止p75NTR与Aβ及其聚合体结合，保护中枢神经元免受Aβ的神经毒性的损害。p75NTR-ECD-IL-33中的IL-33能诱导机体降低Aβ的分泌和促进Aβ的降解，防止AD的发生和阻止AD病情的进展和恶化。(2)p75NTR-ECD-IL-33是融合蛋白，能有效提高和延长其中包含的p75NTR-ECD和IL-33在体内外的稳定性和半衰期。(3)p75NTR-ECD-IL-33能使其中包含的p75NTR-ECD和IL-33在防止和治疗(即防治)AD方面发挥协同作用，p75NTR-ECD-IL-33通过p75NTR-ECD结合Aβ使IL-33对Aβ具有精准的靶向性，使IL-33在Aβ及其寡聚体周围诱导机体直接内吞或者产生Aβ降解酶降解Aβ，从而提高机体对Aβ的降解效率。结果，使p75NTR-ECD-IL-33融合蛋白的生物学活性较p75NTR-ECD和IL-33单体大幅度提高。(4)p75NTR-ECD-IL-33可被构建成在真核细胞系和人体内表达，使p75NTR-ECD-IL-33中的p75NTR-ECD和IL-33在翻译后得到充分的糖基化修饰因而保持原有的理化性质和生理状态下的生物学功能。(5)p75NTR-ECD-IL-33消除了p75NTR-ECD-FC中的FC的免疫原性和抗体依赖性细胞介导的细胞毒作用(ADCC)，所以预期p75NTR-ECD-IL-33可以作为预防和治疗AD的安全有效药物，填补了防治阿尔茨海默病至今还没有有效药物的空白。
附图说明
图1是本发明具体实施方式中的融合蛋白p75NTR-ECD-IL-33的构建示意图；
图2是本发明具体实施方式中的融合蛋白p75NTR-ECD-IL-33的质粒载体示意图；
图3是本发明具体实施方式中SDS-PAGE免疫印迹测定用HEK-293细胞表达系所制备的融合蛋白p75NTR-ECD-IL-33、蛋白p75NTR-ECD和蛋白IL-33的电泳图。
图4是本发明具体实施方式中融合蛋白p75NTR-ECD-IL-33、蛋白p75NTR-ECD和IL-33的体外稳定性测定示意图；
图5是本发明具体实施方式中融合蛋白p75NTR-ECD-IL-33、蛋白p75NTR-ECD和IL-33的体内稳定性测定示意图；
图6是本发明具体实施方式中经注射融合蛋白p75NTR-ECD-IL-33后AD小鼠血液中Aβ1-42的浓度水平变化图；
图7是本发明具体实施方式中经注射融合蛋白p75NTR-ECD-IL-33后AD小鼠大脑皮层中Aβ1-42的含量变化图。
具体实施方式
下面对照附图并结合优选的实施方式对本发明作进一步说明。
本发明设计和构建了用于防治阿尔茨海默病的融合蛋白(也可称为嵌合抗原受体蛋白(Chimeric antigen receptor Protein,CAR-P)，可命名为p75NTR-ECD-IL-33)。在本文中“连接肽”“linker”表示的是同一个概念。“人 p75神经营养因子受体”可缩写为“p75NTR”；“人白细胞介素-33”可缩写为“人IL-33”或“IL-33”。
本发明提供一种用于防治阿尔茨海默病的融合蛋白，其包括人p75NTR的胞外域p75NTR-ECD、人IL-33以及分别连接所述p75NTR-ECD的羧基端和所述人IL-33的氨基端的连接肽；所述p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。具体的：
p75NTR-ECD的氨基酸序列为(250aa)：
Figure PCTCN2017092850-appb-000001
p75NTR-ECD的核苷酸序列(750bp)为：
Figure PCTCN2017092850-appb-000002
人IL-33的氨基酸序列(269aa)为：
Figure PCTCN2017092850-appb-000003
Figure PCTCN2017092850-appb-000004
人IL-33的基因核苷酸序列(含终止密码子TAA，810bp)：
Figure PCTCN2017092850-appb-000005
融合蛋白p75NTR-ECD-IL-33的构建方法包括：将人p75NTR的胞外域p75NTR-ECD的羧基端和人IL-33的氨基端通过连接肽(Linker)连接起来的步骤，具体连接方法如图1所示。
在较优的实施例中，Linker选用柔性连接肽或者刚性连接肽。其中，优选地，柔性连接肽选择：(Gly Gly Gly Gly Ser)n，n＝1～6之间的整数；或者(Gly Gly Gly Gly Thr)n，n＝1～6之间的整数。刚性连接肽选择：(Glu Ala Ala Ala Lys)m，m＝1～6之间的整数，或者富含脯氨酸序列(XPro)q，q＝3～10之间的整数，X为任意氨基酸，较优的是X为赖氨酸、丙氨酸或者谷氨酸中的任一种。
在一个更优的实施例中，Linker的氨基酸序列(15aa)如SEQ ID NO.5所示，为：Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser，该Linker基因核苷酸序列(45bp)如SEQ ID NO.6所示，为：
Figure PCTCN2017092850-appb-000006
选用的该优选Linker中，Gly是非极性的小氨基酸，可以提供融合蛋白的柔性，便于使融合蛋白在翻译后更好地进行折叠成具有生物学功能活性的天然构象，不影响p75NTR-ECD-IL-33中p75NTR-ECD和ECD-IL-3的生物学功能；Ser是极性的小氨基酸，能和H2O分子形成氢键，增加连接肽的水溶性和在水溶液中的稳定性，防止Linker干扰融合蛋白中各个蛋白的生物学功能，当然，在其他实施例中，Ser也可以用苏氨酸Thr代替。
相应的，用上述优选的Linker连接的构建的融合蛋白p75NTR-ECD-IL-33 的氨基酸序列(534aa)如SEQ ID NO.7所示，核苷酸序列(含有起始密码子和终止密码子，1605bp)如SEQ ID NO.8所示，分别为：
Figure PCTCN2017092850-appb-000007
Figure PCTCN2017092850-appb-000008
本发明还提供一种上述各实施例方式中的融合蛋白的表达载体，该表达载体可以选择mRNA，DNA质粒载体或病毒载体(包括慢病毒(Lentivirus)，腺病毒(adenovirus)，腺相关病毒(adeno-associated virus)等)中的一种作为载体。
本发明还提供所述的融合蛋白的DNA质粒载体的制备方法，在具体实施方式中，包括如下步骤：
(1)克隆得到人p75NTR的细胞外域p75NTR-ECD的基因片段；
(2)克隆得到人IL-33的基因片段；
(3)通过PCR反应得到编码p75NTR-ECD-Linker的基因片段，其中p75NTR-ECD的羧基端与Linker的氨基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
(4)通过重叠PCR反应将p75NTR-ECD-Linker的羧基端与人IL-33的氨基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，p75NTR-ECD-Linker基因片段的5’端带有一酶切位点，IL-33基因片段的3’端带有另一酶切位点；
(5)将融合蛋白p75NTR-ECD-IL-33的基因片段通过酶切后插入一质粒载体中两个相应的酶切位点之间，转化宿主菌，提取阳性质粒，对插入的目的基因进行测序，获得含有编码p75NTR-ECD-IL-33目的基因的DNA质粒载体；
或者，步骤(3)和(4)可以分别用(3’)和(4’)替换：
(3’)通过PCR反应得到编码IL-33-Linker的基因片段，其中IL-33的氨基端与Linker的羧基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
(4’)通过重叠PCR反应将IL-33-Linker的氨基端与人p75NTR-ECD的羧基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，IL-33--Linker基因片段的5’端带有一酶切位点，p75NTR-ECD基因片段的3’端带有另一酶切位点。
在优选的实施方式中：可以选择pcDNA3.1(+)作为所述步骤(5)中的质粒载体；所述融合蛋白p75NTR-ECD-IL-33的基因片段插入pcDNA3.1(+)的KpnI 和XbaI酶切位点之间。
以下具体描述一个优选的实施方式来说明p75NTR-ECD-IL-33目的基因的DNA质粒载体的构建和制备过程：
(1)p75NTR-ECD的基因片段(含有起始密码子ATG，未含终止密码子TAA)克隆自Human NGFR Gene cDNA，克隆所需的引物(5'-3')如下：
SEQ ID NO.9所示的正向引物F1：ATGGGTGCAGGGGCGACC；SEQ ID NO.10所示的反向引物R1：GTTGTCTGTCGTCCCTCGTGTTACTACAG。
(2)人IL-33的基因片段(未含起始密码子ATG，含有终止密码子TAA)克隆自Human IL33_Gene cDNA。克隆所需的引物(5'-3')如下：SEQ ID NO.11所示的正向引物F2：AAACCTAAAATGAAATATTCGACTAACAAAATTAGTACCGCG；SEQ ID NO.12所示的反向引物R2：
Figure PCTCN2017092850-appb-000009
(3)通过PCR反应得到编码p75NTR-ECD-Linker的基因片段，其中p75NTR-ECD的羧基端与Linker的氨基端连接，Linker的基因片段来自PCR反应的反向引物(R3)序列；PCR反应的引物序列(5'-3')为：
SEQ ID NO.13所示的正向引物F3：
Figure PCTCN2017092850-appb-000010
KpnI酶切位点
SEQ ID NO.14所示的反向引物R3：
Figure PCTCN2017092850-appb-000011
Linker核苷酸序列
Figure PCTCN2017092850-appb-000012
(4)通过重叠PCR反应将p75NTR-ECD-Linker的羧基端与IL-33的氨基端连接，p75NTR-ECD-Linker基因片段的5’端带有KpnI酶切位点，IL-33基因片段的3’端带有XbaI酶切位点。重叠PCR反应的两对引物序列(5'-3')为：
正向引物F4(同SEQ ID NO.13所示的正向引物F3)：CGCGGTACCATGGGTGCAGGGGCGACC
KpnI酶切位点
SEQ ID NO.15所示的反向引物R4：
Figure PCTCN2017092850-appb-000013
IL-33基因片段负链3’端序列
Figure PCTCN2017092850-appb-000014
p75NTR-ECD-Linker基因片段负链5’端序列
SEQ ID NO.16所示的正向引物F5：
Figure PCTCN2017092850-appb-000015
p75NTR-ECD-Linker基因片段正链3’端序列
Figure PCTCN2017092850-appb-000016
IL-33基因片段正链5’端序列
SEQ ID NO.17所示的反向引物R5：
Figure PCTCN2017092850-appb-000017
XbaI酶切位点
(5)将重叠PCR目标产物(即融合蛋白p75NTR-ECD-IL-33的基因片段，其IL-33基因片段末端带有终止密码子TAA)经KpnI和XbaI酶切后后插入pcDNA3.1(+)的KpnI和XbaI酶切位点之间。转化宿主菌DH5a(由ThermoFisher公司提供)，提取阳性质粒，对插入的目的基因进行测序，确定目的基因序列的正确性，最后获得含有编码p75NTR-ECD-IL-33目的基因的重组pcDNA3.1(+)质粒Vp75NTR-ECD-IL-33。
通过DNA质粒载体可以制备得到mRNA和病毒载体。在一个优选的实施方式中，上述得到的质粒Vp75NTR-ECD-IL-33经过XbaI、或DraII、或ApaI、或PmeI单酶切线性化后，经体外转录(例如可以使用mRNA转录试剂盒(Ambion的mMESSAGE
Figure PCTCN2017092850-appb-000018
Transcription Kit，AM1344))可以得到编码p75NTR-ECD-IL-33的mRNA。质粒Vp75NTR-ECD-IL-33也可用于制备表达p75NTR-ECD-IL-33的病毒载体(包括慢病毒(Lentivirus)，腺病毒(adenovirus)，腺相关病毒(adeno-associated virus)等)。
本发明还提供一种目标蛋白的制备方法，所述目标蛋白为人p75NTR的胞外域p75NTR-ECD、人IL-33或所述的融合蛋白p5NTR-ECD-IL-33中的一种。在优选的实施例中，目标蛋白的制备方法包括如下步骤：
(1)用于制备p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的表达质粒的构建和制备：为了方便提取和纯化目标蛋白，分别在p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的氨基端连接Flag标签(优选的，本例中采用的Flag标签的氨基酸序列如SEQ ID NO.18所示:AspTyrLysAspAspAspAspLys，核苷酸序列如SEQ ID NO.19所示：GATTACAAAGATGACGATGATAAA；或者在其他优选实施例中，还可以选择Flag标签的氨基酸序列如SEQ ID NO.20所示：
AspTyrLysAspHisAspGlyAspTyrLysAspHisAspIleAspTyrLysAspAspAspAspLys，核苷酸序列如SEQ ID NO.21所示：
GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG)，分别获得都带有Flag标签的p75NTR-ECD(PCR技术)、IL-33(PCR技术)和p75NTR-ECD-IL-33(重叠PCR技术)的表达质粒：质粒VFlag-p75NTR-ECD，质粒VFlag-IL-33和质粒VFlag-p75NTR-ECD-IL-33；也即以目标蛋白的基因片段为模板，以一质粒载体为载体，以该质粒载体的两个酶切位点为目的基因的插入位点，在目标蛋白的氨基端连接Flag标签，若目标蛋白为人p75NTR的胞外域p75NTR-ECD或人IL-33，则采用PCR反应相应获得带有Flag标签的目标蛋白的表达质粒：质粒VFlag-p75NTR-ECD或质粒VFlag-IL-33；若目标蛋白为融合蛋白p5NTR-ECD-IL-33，则采用重叠PCR反应获得带有Flag标签的p75NTR-ECD-IL-33的表达质粒：质粒VFlag-p75NTR-ECD-IL-33。在本例中，选择pcDNA3.1(+)为载体，以KpnI和XbaI酶切位点为目的基因的插入位点，分别如下：
a、制备质粒VFlag-p75NTR-ECD-IL-33时，按构建和制备质粒Vp75NTR-ECD-IL-33的方法获得，其中两个正向引物更换如下：
p75NTR-ECD的基因片段克隆时的正向引物F1换成F6(如SEQ ID NO.22所示)：
Figure PCTCN2017092850-appb-000019
Flag标签正链5’端序列
重叠PCR的正向引物F4换成F7(如SEQ ID NO.23所示)：
Figure PCTCN2017092850-appb-000020
KpnI酶切位点   Flag标签序列
b、制备质粒V Flag-p75NTR-ECD时，PCR引物为：
正向引物F8(如SEQ ID NO.24所示)：
Figure PCTCN2017092850-appb-000021
KpnI酶切位点  Flag标签序列
ACCGGT
p75NTR-ECD基因正链5’端序列
反向引物R8(如SEQ ID NO.25所示)：
Figure PCTCN2017092850-appb-000022
XbaI酶切位点    p75NTR-ECD基因负链5’端序列
c、制备质粒V Flag-IL-33时，PCR引物为：
正向引物F9(如SEQ ID NO.26所示)：
Figure PCTCN2017092850-appb-000023
KpnI酶切位点  Flag标签序列
Figure PCTCN2017092850-appb-000024
IL-33基因片段正链5’端序列
反向引物R9(如SEQ ID NO.27所示)：
Figure PCTCN2017092850-appb-000025
XbaI酶切位点   IL-33基因片段负链5’端序列
(2)将表达质粒转染真核细胞获得表达目标蛋白的稳定细胞系。优选采用哺乳动物细胞，本例中更优选采用人HEK-293细胞，具体：
HEK-293细胞用含10％(v/v)FBS(Gibco)，100μg/ml青霉素，100μg/ml链霉素的DMEM(Gibco)完全培养基，置于37℃，5％(体积分数)CO2的培养箱培养。当细胞长到80％满时进行细胞传代。做质粒转染前，将HEK-293细胞植于60-mm细胞培养皿，用含10％(v/v)FBS(Gibco)的DMEM(Gibco)培养基培养，第二天，当细胞长到80％满时用于质粒转染。转染时，分别将8.0μg质粒VFlag-p75NTR-ECD、质粒VFlag-IL-33和质粒VFlag-p75NTR-ECD-IL-33用Opti-MEM(Gibco)稀释至0.5ml并轻柔搅匀为稀释液A。将20μl Lipofectamine 2000(Invitrogen)用Opti-MEM(Gibco)稀释至0.5ml并轻柔搅匀为稀释液B，在室温下(20-25℃，本例中的室温为25℃，下同)孵育5分钟。将稀释液A和稀释液B混合并轻柔搅匀为脂质体转染混合液，在室温下放置20分钟。将脂质体转染混合液均匀加入60-mm细胞培养皿内的培养基内并轻柔混匀。细胞继续培养24小时后，将培养基更换为含10％(v/v)FBS(Gibco)，100μg/ml青霉素，100μg/ml链霉素，500μg/ml硫酸新霉素(Clontech)的DMEM(Gibco)筛选培养基继续进行筛选培养。每隔3天换一次筛选培养基，每隔6天细胞传代一次，3周后收集细胞，其中部分细胞用含5％(v/v)二甲基亚砜(Sigma-Aldrich)的完 全培养基混匀置于液氮罐中保存分别作为表达p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的稳定细胞系CLp75NTR-ECD，CL IL-33和CLp75NTR-ECD-IL-33，剩余细胞分别用于目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的提取和纯化。
(3)从稳定表达目标蛋白的细胞中提取和初步纯化目标蛋白得到目标蛋白粗溶液。本例中，具体为：使用Sigma-Aldrich提供的提取和纯化带有Flag标签蛋白的试剂盒
Figure PCTCN2017092850-appb-000026
M2 Affinity Gel(Sigma-Aldrich,A2220)从稳定表达目标蛋白的细胞中提取和纯化目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33。按产品说明书的操作程序进行操作。用含有每1ml裂解液10μl蛋白酶抑制剂(Sigma-Aldrich，P8340)的CelLytic M细胞裂解液(Sigma-Aldrich，C2978)裂解所收集的细胞，收集裂解上清液。用抗Flag抗体M2的亲和树脂(ANTI-FLAG M2affinity resin)填充层析柱制备亲和层析柱，用平衡液预平衡层析柱。将裂解上清液上柱，用10-20倍层析柱体积的TBS缓冲液(50mM Tris HCl,with 150mM NaCl,pH 7.4)洗层析柱将非目标蛋白洗脱掉。用pH 3.5的0.1M甘氨酸盐酸溶液将目标蛋白洗脱进预先装有15-25μl pH 8.0的1M Tris缓冲液的预冷冻的小管内分别获得p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的目标蛋白粗溶液。取小量样品测定蛋白质含量。将目标蛋白粗溶液直接用于进一步纯化或者置于-70℃冷柜冻存备用。
(4)从目标蛋白粗溶液中去除Flag标签获取纯化的目标蛋白。本例中，具体为：使用Sigma-Aldrich提供的重组牛肠肠激酶Enterokinase(Sigma-Aldrich，E4906)酶切去除目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的氨基端的Flag标签。将目标蛋白粗溶液用含有500mM Tris-HCl,pH 8.0，2.0mM CaCl2和1％(v/v)TWEEN20的溶液调至浓度为1.5mg/ml，pH值7.0-8.0作为反应溶液，再加入0.02单位/mg融合蛋白的肠激酶并混匀，于室温下放置16小时。反应结束后，使用Sigma-Aldrich提供的去除肠激酶试剂盒ENTEROKINASE REMOVAL KIT(Sigma-Aldrich，PRK-E)将反应混合物中的肠激酶去除掉，获得纯化的目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的溶液。取少量的样品测定蛋白质含量和纯度。将目标蛋白溶液分装并置于-70℃冷柜或者液氮罐冻存。
一、目标蛋白的鉴定如下：
1、N-末端氨基酸序列的测定：将目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33经SDS-PAGE电泳转移至PVDF膜上，用岛津PPSQ-31A蛋白自动测序仪对目标蛋白N-末端氨基酸序列进行测定。测定结果显示，目标蛋白p75NTR-ECD N-末端氨基酸序列测定结果为：GlyAlaGlyAlaThrGlyArg，序列与天然蛋白人p75NTR-ECD的N-末端氨基酸序列相同。目标蛋白IL-33的N-末端氨基酸序列测定结果为：LysProLysMetLysTyrSer，序列与天然蛋白IL-33的N-末端氨基酸序列相同。目标蛋白p75NTR-ECD-IL-33的N-末端氨基酸序列测定结果为：GlyAlaGlyAlaThrGlyArg，序列与融合蛋白p75NTR-ECD-IL-33的N-末端氨基酸序列相同。
2、Western Blot的免疫鉴定：将目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33经SDS-PAGE电泳转移至PVDF膜上，用特异性识别p75NTR-ECD的抗体Anti-p75NGF Receptor antibody[NGFR5]-N-terminal(由Abcam提供，ab192774)和特异性识别IL-33抗体Anti-IL33antibody(由Abcam提供，ab83873)做免疫印记鉴定。测定结果显示，Anti-p75NGF Receptor antibody 抗体特异性识别目标蛋白p75NTR-ECD和p75NTR-ECD-IL-33，Anti-IL33antibody抗体特异性识别目标蛋白IL-33和p75NTR-ECD-IL-33。结果如图3所示。
二、目标蛋白p75NTR-ECD-IL-33体外稳定性测定：
差示扫描量热法(DSC)是一种用于直接鉴定蛋白质生物分子在自然状态下的稳定性的技术，溶液中的蛋白质生物分子在其自然(折叠)和变性(展开)构象之间保持平衡，其热跃迁中点(Tm)越高，分子就越稳定。将目标蛋白p75NTR-ECD、IL-33和p75NTR-ECD-IL-33通过差示扫描量热仪MicroCal VP-Capillary DSC系统进行测定其Tm值。测得：目标蛋白p75NTR-ECD-IL-33的Tm值为78.0℃，蛋白p75NTR-ECD的Tm值为66.0℃，蛋白IL-33的Tm值为56.0℃。测定结果显示，重组融合蛋白p75NTR-ECD-IL-33能显著提高蛋白p75NTR-ECD和IL-33的稳定性。结果如图4所示。
三、目标蛋白p75NTR-ECD-IL-33体内稳定性测定：
目标蛋白p75NTR-ECD-IL-33体内稳定性测定通过测定外源蛋白p75NTR-ECD-IL-33在体内的半衰期来实现。取AD小鼠20只，其中5只为空白对照组，5只为p75NTR-ECD组(腹膜内注射300ng p75NTR-ECD)，5只为IL-33组(腹膜内注射300ng IL-33)，5只为p75NTR-ECD-IL-33组(腹膜内注射300ng p75NTR-ECD-IL-33)。在小鼠注射样品2、4、6、8、12、16、20、24小时后分别通过尾静脉取静脉血，用Biosensis公司的ILISA试剂盒NGFR/p75ECD RapidTM enzyme-linked immunosorbent assay(ELISA)Kit(Biosensis，BEK-2219-1P)测定p75NTR-ECD和p75NTR-ECD-IL-33的血液浓度，用Boster公司的ILISA试剂盒Human IL-33 PicoKineTMELISA Kit(Boster，EK0929)测定IL-33和p75NTR-ECD-IL-33的血液浓度。计算p75NTR-ECD、IL-33和p75NTR-ECD-IL-33的半衰期。测得：p75NTR-ECD的半衰期为8小时，IL-33的半衰期为6小时，p75NTR-ECD-IL-33的半衰期为18小时。测定结果显示，重组蛋白p75NTR-ECD-IL-33显著地提高p75NTR-ECD和IL-33在体内的稳定性。结果如图5所示。
四、目标蛋白p75NTR-ECD-IL-33防治AD的生物学功能活性测定：
目标蛋白p75NTR-ECD-IL-33体内生物学功能测定通过测定外源蛋白p75NTR-ECD-IL-33在体内促进机体降低Aβ的分泌和促进Aβ的降解来实现。取AD小鼠20只，其中5只为空白对照组，5只为p75NTR-ECD组(连续3天，每天腹膜内注射300ng p75NTR-ECD)，5只为IL-33组(连续3天，每天腹膜内注射300ng IL-33)，5只为p75NTR-ECD-IL-33组(连续3天，每天于腹膜内注射300ng p75NTR-ECD-IL-33)。在小鼠开始注射样品1周后分别取尾静脉血和大脑皮层。大脑皮层用干冰速冻，然后用含有蛋白酶抑制剂的匀浆缓冲液(250mM蔗糖,20mM pH 7.4Tris-HCl,1mM EDTA和1mM EGTA)和杜恩斯匀浆器匀浆，使用二乙胺提取可溶性Aβ。静脉血和可溶性Aβ溶液用Thermo Fisher公司提供的ILISA试剂盒Amyloid beta 42 ELISA Kit,Mouse(Novex，KMB3441)测定Aβ1-42的血液和大脑皮层中的含量。测定结果显示，相对于p75NTR-ECD和IL-33，使用重组融合蛋白p75NTR-ECD-IL-33更显著地促进Aβ在体内的降解和清除。重组融合蛋白p75NTR-ECD-IL-33融合p75NTR-ECD和IL-33后对p75NTR-ECD和IL-33在防治AD方面的生物学功能活性具有显著协同和促进作用。结果如图6和图7所示。
本发明还提供一种以上各实施方式中所述的融合蛋白在制备防治阿尔茨海 默病的药物中的应用以及用于防治阿尔茨海默病的药物；所述药物可以为以上各实施方式中所述的融合蛋白本身，或者表达所述的融合蛋白的mRNA脂质体，或者携带表达所述的融合蛋白基因的病毒载体。所述药物的临床使用方法可以为：可经腹膜内、静脉或者脑内直接注射重组融合蛋白p75NTR-ECD-IL-33，或者可经静脉或者脑内施用表达融合蛋白p75NTR-ECD-IL-33的mRNA脂质体或者携带p75NTR-ECD-IL-33基因的病毒载体(包括慢病毒(Lentivirus)，腺病毒(adenovirus)，腺相关病毒(adeno-associated virus)等)。
通过上述实验和测定，本发明实施例中得到的p75NTR-ECD-IL-33的优点包括：
(1)p75NTR-ECD-IL-33同时具有p75NTR-ECD和IL-33两者的生物学功能。p75NTR-ECD-IL-33中的p75NTR-ECD能与Aβ及其聚合体结合，阻止p75NTR与Aβ及其聚合体结合，保护中枢神经元免受Aβ的神经毒性的损害。p75NTR-ECD-IL-33中的IL-33能诱导机体降低Aβ的分泌和促进Aβ的降解，防止AD的发生和阻止AD病情的进展和恶化。
(2)p75NTR-ECD-IL-33是融合蛋白，能有效提高和延长其中包含的p75NTR-ECD和IL-33在体内外的稳定性和半衰期。
(3)p75NTR-ECD-IL-33能使其中包含的p75NTR-ECD和IL-33在防止和治疗AD方面发挥协同作用，p75NTR-ECD-IL-33通过p75NTR-ECD结合Aβ使IL-33对Aβ具有精准的靶向性，使IL-33在Aβ及其寡聚体周围诱导机体直接内吞或者产生Aβ降解酶降解Aβ，从而提高机体对Aβ的降解效率。结果，使p75NTR-ECD-IL-33融合蛋白的生物学活性较p75NTR-ECD和IL-33单体大幅度提高。
(4)p75NTR-ECD-IL-33被构建成在真核细胞系和在人体内表达，使p75NTR-ECD-IL-33中的p75NTR-ECD和IL-33在翻译后得到充分的糖基化修饰因而保持原有的理化性质和生理状态下的生物学功能。
(5)p75NTR-ECD-IL-33消除了p75NTR-ECD-FC中的FC的免疫原性和抗体依赖性细胞介导的细胞毒作用(ADCC)，所以p75NTR-ECD-IL-33可以作为预防和治疗AD的安全有效药物。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的技术人员来说，在不脱离本发明构思的前提下，还可以做出若干等同替代或明显变型，而且性能或用途相同，都应当视为属于本发明的保护范围。
Figure PCTCN2017092850-appb-000027
Figure PCTCN2017092850-appb-000028
Figure PCTCN2017092850-appb-000029
Figure PCTCN2017092850-appb-000030
Figure PCTCN2017092850-appb-000031
Figure PCTCN2017092850-appb-000032
Figure PCTCN2017092850-appb-000033
Figure PCTCN2017092850-appb-000034
Figure PCTCN2017092850-appb-000035
Figure PCTCN2017092850-appb-000036

Claims (10)

  1. 一种用于防治阿尔茨海默病的融合蛋白，其特征在于，包括人p75NTR的胞外域p75NTR-ECD、人IL-33以及分别连接所述p75NTR-ECD的羧基端和所述人IL-33的氨基端的连接肽；所述p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
  2. 如权利要求1所述的融合蛋白，其特征在于：所述连接肽为柔性连接肽，
    所述柔性连接肽的氨基酸序列为(Gly Gly Gly Gly Y)n，其中，n为1～6之间的整数，Y表示Ser或者Thr；优选地：所述连接肽的氨基酸序列如SEQ ID NO.5所示，核苷酸序列如SEQ ID NO.6所示；相应地，所述融合蛋白的氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID NO.8所示。
  3. 如权利要求1所述的融合蛋白，其特征在于：所述连接肽为刚性连接肽，所述刚性连接肽为(Glu Ala Ala Ala Lys)m，其中，m为1～6之间的整数；或者所述刚性连接肽为富含脯氨酸序列(X Pro)q，其中q为3～10之间的整数，X为任意氨基酸，其中，X优选赖氨酸、丙氨酸或者谷氨酸中的任一种。
  4. 一种权利要求1-3任意一项所述的融合蛋白的构建方法，其特征在于：包括将人p75NTR的细胞外域p75NTR-ECD的羧基端和人IL-33的氨基端通过连接肽连接起来的步骤，所述人p75NTR的细胞外域p75NTR-ECD的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示；所述人IL-33的氨基酸序列如SEQ ID NO.3所示，核苷酸序列如SEQ ID NO.4所示。
  5. 一种权利要求1-3任意一项所述的融合蛋白的表达载体，其特征在于：所述表达载体以mRNA，DNA质粒载体或病毒载体中的一种作为载体。
  6. 一种权利要求1-3任意一项所述的融合蛋白的DNA质粒载体的制备方法，其特征在于，包括如下步骤：
    (1)克隆得到人p75NTR的细胞外域p75NTR-ECD的基因片段；
    (2)克隆得到人IL-33的基因片段；
    (3)通过PCR反应得到编码p75NTR-ECD-Linker的基因片段，其中p75NTR-ECD的羧基端与Linker的氨基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
    (4)通过重叠PCR反应将p75NTR-ECD-Linker的羧基端与人IL-33的氨基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，p75NTR-ECD-Linker基因片段的5’端带有一酶切位点，IL-33基因片段的3’端带有另一酶切位点；
    (5)将融合蛋白p75NTR-ECD-IL-33的基因片段通过酶切后插入一质粒载体中两个相应的酶切位点之间，转化宿主菌，提取阳性质粒，对插入的目的基因进行测序，获得含有编码p75NTR-ECD-IL-33目的基因的DNA质粒载体；
    或者，步骤(3)和(4)可以分别用(3’)和(4’)替换：
    (3’)通过PCR反应得到编码IL-33-Linker的基因片段，其中IL-33的氨基端与Linker的羧基端连接，所述Linker的基因片段来自PCR反应的反向引物序列；
    (4’)通过重叠PCR反应将IL-33-Linker的氨基端与人p75NTR-ECD的羧基端连接，获得融合蛋白p75NTR-ECD-IL-33的基因片段，其中，IL-33-Linker基因片段的5’端带有一酶切位点，p75NTR-ECD基因片段的3’端带有另一酶切位点。
  7. 如权利要求6所述的制备方法，其特征在于：选择pcDNA3.1(+)作为所述步骤(5)中的质粒载体；所述融合蛋白p75NTR-ECD-IL-33的基因片段插入pcDNA3.1(+)的KpnI和XbaI酶切位点之间。
  8. 一种目标蛋白的制备方法，其特征在于：所述目标蛋白为人p75NTR的胞外域p75NTR-ECD、人IL-33或权利要求1-3任意一项所述的融合蛋白p5NTR-ECD-IL-33中的一种；包括如下步骤：
    (1)以目标蛋白的基因片段为模板，以一质粒载体为载体，以该质粒载体的两个酶切位点为目的基因的插入位点，在目标蛋白的氨基端连接Flag标签，若目标蛋白为人p75NTR的胞外域p75NTR-ECD或人IL-33，则采用PCR反应相应获得带有Flag标签的目标蛋白的表达质粒：质粒VFlag-p75NTR-ECD或质粒VFlag-IL-33；若目标蛋白为融合蛋白p5NTR-ECD-IL-33，则采用重叠PCR反应获得带有Flag标签的p75NTR-ECD-IL-33的表达质粒：质粒VFlag-p75NTR-ECD-IL-33；(2)将表达质粒转染真核细胞获得表达目标蛋白的稳定细胞系；
    (3)从稳定表达目标蛋白的细胞中提取和初步纯化目标蛋白得到目标蛋白粗溶液；
    (4)从目标蛋白粗溶液中去除Flag标签获取纯化的目标蛋白；
    优选所述质粒载体为pcDNA3.1(+)，以KpnI和XbaI酶切位点为目的基因的插入位点；
    优选所述真核细胞为哺乳动物细胞；进一步优选所述哺乳动物细胞为人HEK-293细胞；
    优选所述Flag标签的氨基酸序列如SEQ ID NO.18所示，核苷酸序列SEQ ID NO.19所示；或优选所述Flag标签的氨基酸序列如SEQ ID NO.20所示，核苷酸序列SEQ ID NO.21所示。
  9. 一种权利要求1-3任意一项所述的融合蛋白或由权利要求6制备得到的融合蛋白的DNA质粒载体在制备防治阿尔茨海默病的药物中的应用。
  10. 一种用于防治阿尔茨海默病的药物，其特征在于：所述药物为权利要求1-3任意一项所述的融合蛋白，或者表达权利要求1-3任意一项所述的融合蛋白的mRNA脂质体，或者携带表达权利要求1-3任意一项所述的融合蛋白基因的病毒载体。
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