WO2017008638A1 - 一种嵌合诺如病毒p颗粒及其制备和应用 - Google Patents

一种嵌合诺如病毒p颗粒及其制备和应用 Download PDF

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WO2017008638A1
WO2017008638A1 PCT/CN2016/087643 CN2016087643W WO2017008638A1 WO 2017008638 A1 WO2017008638 A1 WO 2017008638A1 CN 2016087643 W CN2016087643 W CN 2016087643W WO 2017008638 A1 WO2017008638 A1 WO 2017008638A1
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protein
seq
recombinant
peptide
10copy
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French (fr)
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孔维
吴慧
姜春来
于湘晖
付璐
李瑛楠
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长春百克生物科技股份公司
吉林大学
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Priority to EP16823785.7A priority Critical patent/EP3323885B8/en
Priority to US15/744,585 priority patent/US11116831B2/en
Publication of WO2017008638A1 publication Critical patent/WO2017008638A1/zh

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/385Haptens or antigens, bound to carriers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0043Nose
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • C07ORGANIC CHEMISTRY
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    • C07K19/00Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N15/09Recombinant DNA-technology
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    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04Inactivation or attenuation; Producing viral sub-units
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55561CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • the invention relates to the field of molecular biology and immunology.
  • the present invention relates to recombinant P particles formed from a Norovirus capsid P protein of a chimeric A ⁇ 1-m peptide, wherein the recombinant P particles form an ordered and repetitive array of antigens, wherein m is selected An integer from 6-15.
  • AD Alzheimer's disease
  • AD is a progressive neurodegenerative disease whose main clinical features are progressive memory loss, cognitive dysfunction and behavioral abnormalities, and ultimately loss of independence. Life ability, 10 to 20 years after the onset of death due to complications. At present, the most common neurodegenerative disease cannot be cured, and there is no effective means of delay, which seriously endangers the physical and mental health and quality of life of the elderly.
  • amyloid- ⁇ protein in brain tissue is the main cause of neuronal damage and cognitive dysfunction.
  • a ⁇ is derived from its precursor substance ⁇ -Amyloid precursor protein ( ⁇ APP).
  • ⁇ APP is a transmembrane protein that is widely present in various tissues in the body and has the highest expression in brain tissue.
  • ⁇ APP can be cleaved into A ⁇ proteins ranging in length from 38 to 43 amino acids, of which A ⁇ 1-42 protein is the main type of amyloid deposition.
  • the A ⁇ active immunization vaccine for the purpose of stimulating the body to produce A ⁇ antibody to remove A ⁇ 1-42 deposition in the brain is an important method for treating Alzheimer's disease.
  • a ⁇ 1-42 polypeptide vaccine showed a good therapeutic effect in delaying memory loss in the AD transgenic mouse model.
  • 6% of AD patients developed side effects of meningitis and the patient produced lower levels of A ⁇ 1-42 antibodies.
  • the polypeptide A ⁇ 1-15 which only contains the first 15 amino acids of the N-terminus of the A ⁇ polypeptide, contains only B cell epitopes and does not contain T cell epitopes, and the polypeptide vaccine can stimulate the body to produce A ⁇ 1-42.
  • the protein has a specific immune response and shows good safety in human trials. Therefore, A ⁇ 1-15 has become an antigenic peptide with great research potential.
  • the A ⁇ 1-15 peptide acts as a hapten, and its immune effect and the persistence of the induced antibody are not very good. Therefore, there is a need for a vaccine that is safer and has a good immune effect against Alzheimer's disease.
  • the prior art it is generally preferred to separately synthesize virus-like particles and A ⁇ polypeptides, and then couple the two To obtain a highly immunogenic vaccine.
  • the vaccine obtained by the coupling method or the like is difficult to control the number of inserted epitopes, and it is also difficult to efficiently purify the vaccine.
  • the capsid P protein of Norovirus is a very ideal antigen display platform.
  • a ⁇ 1-m m is an integer selected from 6-15
  • peptide coding sequence is inserted into the loop structure of the DNA sequence encoding the Norovirus capsid P protein, A ⁇ 1 -m can be displayed on the outermost surface of recombinant P particles formed by recombinant P protein, thereby facilitating the maximum stimulation of the body to produce a specific immune response against A ⁇ 1-42 protein.
  • the recombinant P particle of the invention has simple preparation method, controllable number of inserted epitopes, simple purification process, low manufacturing cost, good safety, high immunogenicity and high antibody titer induced.
  • the present invention utilizes genetic engineering means to insert a plurality of copies of DNA encoding the antigen peptide A ⁇ 1-m (m is an integer selected from 6-15) into the DNA encoding loop 1, loop 2 and/or loop 3 of the P protein.
  • recombinant P particles capable of fully displaying A ⁇ 1-m (m is an integer selected from 6-15) epitopes are formed, thereby enhancing the effectiveness, persistence and safety of vaccine immunization. Sex.
  • the present invention provides a recombinant P particle formed from a Norovirus P protein of a chimeric A ⁇ 1-m peptide, wherein m is an integer selected from 6-15. In an embodiment of the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the recombinant P particles form an ordered and repetitive array of antigens.
  • the amino acid sequence of at least one of the A ⁇ 1-m peptides is inserted into loop1, loop2 and/or loop3 of the Norovirus capsid P protein.
  • the amino acid sequence of the Norovirus capsid P protein is set forth in SEQ ID NO: 1.
  • the N1 A ⁇ 1-m peptide sequences are inserted after one or more amino acid sites selected from the group consisting of the 70th to the 74th amino acids of SEQ ID NO: 1, ie, I70, A71, G72, T73 and Q74;
  • N2 A ⁇ 1-m peptide sequences are inserted after one or more amino acid positions selected from: 148th to 151th amino acids of SEQ ID NO: 1, ie, T148, S149, N150 and D151 N3
  • a ⁇ 1-m peptide sequences are inserted after one or more amino acid positions selected from: 168th to 171th amino acids of SEQ ID NO: 1, ie, D168, G169, S170 and T171; wherein N1 And N2 and N3 are each an integer independently selected from 0 to 40, and N1+N2+N3
  • N1, N2 and N3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+ N3 ⁇ 1.
  • a ⁇ 1-m peptide sequence is inserted after an amino acid site means that the N-terminus of the A ⁇ 1-m peptide sequence is directly linked to the C-terminus of the amino acid residue via a peptide bond or to the amino acid via an amino acid linker.
  • the plurality of consecutive A ⁇ 1-m peptide sequences inserted into SEQ ID NO: 1 may be directly linked or may be linked by an amino acid linker.
  • the A ⁇ 1-m peptide sequence means a polypeptide consisting of the first m amino acids of the N-terminus of the entire A ⁇ 1-42 protein, wherein m is an integer selected from 6-15.
  • the A ⁇ 1-m peptide in the present invention may be the 1-6 position, 1-7 position, 1-8 position, 1-9 position, 1-10 position, 1-11 position, 1 of the N-terminus of the intact A ⁇ 1-42 protein.
  • the A ⁇ 1-m peptide sequence of the present invention may be an A ⁇ 1-m peptide of human, mouse, primate, rabbit, scorpion, rat, guinea pig.
  • the amino acid sequences of the A ⁇ 1-15 peptides of human, mouse, primate, rabbit, scorpion, rat, guinea pig are shown in SEQ ID NO: 2-SEQ ID NO: 8, respectively. Based on these sequences, those skilled in the art can readily determine the A ⁇ 1-m peptide of the present invention (m is an integer selected from 6-15).
  • the A ⁇ 1-m peptide is a human A ⁇ 1-m peptide.
  • the amino acid sequence of the Norovirus capsid P protein of the chimeric A ⁇ 1-m peptide is SEQ ID NO: 15-141. More preferably, the amino acid sequence of the Norovirus capsid P protein of the chimeric A ⁇ 1-m peptide is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 57, SEQ ID NO: 73, SEQ ID NO: 92, SEQ ID NO: 117, SEQ ID NO: 132, SEQ ID NO: 133, and SEQ ID NO: 134.
  • the present invention also provides a nucleotide sequence encoding a Norovirus capsid P protein of the chimeric A ⁇ 1-m peptide forming the recombinant P particle of the first aspect, wherein m is an integer selected from 6-15 . In an embodiment of the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the nucleotide sequence comprises a nucleotide sequence encoding a Norovirus capsid P protein and at least one copy of a nucleotide sequence encoding an A ⁇ 1-m peptide, wherein the at least one copy of the core encoding the A ⁇ 1-m peptide
  • the nucleotide sequence is inserted into a nucleotide sequence encoding a loop region of the Norovirus capsid P protein, wherein the inserted nucleotide sequence encoding the A ⁇ 1-m peptide does not cause a frameshift mutation of the Norovirus P protein .
  • the at least one copy of the nucleotide sequence encoding the A ⁇ 1-m peptide can be inserted into a nucleotide sequence encoding the same loop region of the Norovirus capsid P protein or inserted into the encoding Noro The nucleotide sequence of the different circular regions of the viral capsid P protein.
  • the nucleotide sequence of the Norovirus capsid P protein is SEQ ID NO: 9
  • Loop1 corresponds to nucleotides 208-222
  • Loop2 corresponds to nucleotides 442-453
  • Loop3 corresponds to 502- 513 nucleotides.
  • the number of nucleotide sequences encoding the A ⁇ 1-m peptide inserted into Loop1, Loop2 and Loop3 is N1, N2 and N3, respectively;
  • N1, N2 and N3 are each independently selected from 0-40 Integer, and N1+N2+N3 ⁇ 1.
  • N1, N2 and N3 are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+ N3 ⁇ 1.
  • the 5'-terminal nucleotide of the nucleotide sequence encoding the A ⁇ 1-m peptide is directly linked to the Norovirus capsid P protein ring by a 3',5'-phosphodiester bond.
  • the nucleotides of the region are ligated or linked to the nucleotide encoding the loop region of the Norovirus capsid P protein via a DNA linker. Whether the insertion is inserted directly or through a DNA linker does not cause a frameshift in the translation of the nucleotide sequence.
  • a plurality of consecutive nucleotide sequences encoding A ⁇ 1-m peptides inserted into a nucleotide sequence encoding a Norovirus capsid P protein are passed between 3', 5'-phosphate
  • the diester bonds are directly linked or linked by a DNA linker. Whether the insertion is inserted directly or through a DNA linker does not cause a frameshift in the translation of the nucleotide sequence.
  • the A ⁇ 1-m peptide nucleotide sequence is a nucleotide sequence encoding an A ⁇ 1-m peptide of human, mouse, primate, rabbit, scorpion, rat, guinea pig. More preferably, the A ⁇ 1-m peptide nucleotide sequence is a nucleotide sequence encoding a human A ⁇ 1-m peptide.
  • the nucleotide sequence of the Norovirus capsid P protein encoding the chimeric A ⁇ 1-m peptide is SEQ ID NO: 142-268. More preferably, the nucleotide sequence of the Norovirus capsid P protein encoding the chimeric A ⁇ 1-m peptide is selected from the group consisting of: SEQ ID NO: 151, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO 184, SEQ ID NO: 200, SEQ ID NO: 219, SEQ ID NO: 244, SEQ ID NO: 259, SEQ ID NO: 260, and SEQ ID NO: 261.
  • the present invention also provides a pharmaceutical composition for preventing or treating Alzheimer's disease comprising the recombinant P particles of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is preferably used in mammals, more preferably in humans.
  • the pharmaceutical composition is a vaccine composition, more preferably the vaccine composition further comprises an adjuvant.
  • the adjuvant is CpG.
  • the adjuvant is an aluminum adjuvant.
  • the vaccine composition is preferably immunized by subcutaneous or nasal route, more preferably by subcutaneous route.
  • the present invention provides the recombinant p particle of the first aspect of the present invention for use in the preparation of a therapeutic Or the use of drugs to prevent Alzheimer's disease.
  • the drug is a vaccine.
  • the medicament is preferably for use in a mammal, more preferably for a human.
  • the present invention also provides a method for preparing the recombinant P particles of the first aspect of the invention, the method comprising the steps of:
  • the method further comprises a separation and purification step.
  • purification can be carried out using cation exchange chromatography and/or hydrophobic chromatography.
  • Figure 1 shows a map of each recombinant pET26b plasmid construct.
  • A is a schematic diagram of the constructed pET26b-Pprotein plasmid
  • B is a schematic diagram of the pET26b-Pprotein-mEagI plasmid with the mEagI cleavage site obtained by mutation;
  • C is a schematic diagram of the pET26b-Pprotein-mEagI&mKpnI plasmid with the mEagI and mKpnI cleavage sites obtained by mutation;
  • D is a schematic diagram of pET26b-Pprotein-10copy-A ⁇ 1-6-loop1G72 plasmid, carrying the target gene fragment P protein-10copy-A ⁇ 1-6-loop1G72 between the mKpnI restriction site and the SalI restriction site;
  • E is a schematic diagram of pET26b-P protein-1copy-A ⁇ 1-6-loop2S149 plasmid, carrying the target gene fragment P protein-1copy-A ⁇ 1-6-loop2S149 between the mEagI cleavage site and the SalI restriction site;
  • F is a schematic diagram of pET26b-P protein-10copy-A ⁇ 1-6-loop2S149 plasmid, carrying the target gene fragment P protein-10copy-A ⁇ 1-6-loop2S149 between the mEagI cleavage site and the SalI restriction site;
  • G is a schematic diagram of pET26b-P protein-20copy-A ⁇ 1-6-loop3G169 plasmid, carrying the target gene fragment P protein-20copy-A ⁇ 1-6-loop3G169 between the mEagI cleavage site and the SalI restriction site;
  • H is a schematic diagram of pET26b-P protein-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 plasmid, and the target gene fragment Pprotein-10copy-A ⁇ 1-15- is carried between the mEagI cleavage site and the mKpnI restriction site. loop1G72-10copy-A ⁇ 1-6-loop2S149;
  • I is pET26b-P protein-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 Schematic diagram, carrying the target gene fragment Pprotein-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 between the mEagI cleavage site and the mKpnI restriction site;
  • J is a schematic diagram of pET26b-P protein-1copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop3G169 plasmid, and the target gene fragment P protein-1copy-A ⁇ 1-6 is carried between the mEagI restriction site and the mKpnI restriction site. -loop1G72-10copy-A ⁇ 1-6-loop3G169;
  • K is a plasmid map of pET26b-Pprotein-1copy-A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1copy-A ⁇ 1-6-loop3G169, carrying the target gene fragment Pprotein between the mEagI cleavage site and the mKpnI restriction site -1copy-A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1copy-A ⁇ 1-6-loop3G169.
  • L is a plasmid map of pET26b-Pprotein-3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169, carrying the target gene fragment Pprotein between the mEagI cleavage site and the mKpnI restriction site -3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169.
  • M is a schematic diagram of pET26b-P protein-10copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop2S149-10copy-A ⁇ 1-6-loop3G169 plasmid, carrying the target gene fragment between the mEagI cleavage site and the mKpnI restriction site Pprotein-10copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop2S149-10copy-A ⁇ 1-6-loop3G169.
  • Figure 2 shows a schematic diagram of the structure of a gene fragment of interest.
  • A is a three-loop structure of P protein - a schematic diagram of the position of loop1, loop2, loop3, and a site-directed mutation site and a P-protein gene with an enzyme cleavage site;
  • FIG. B is a schematic diagram of the P gene target gene fragment of loop A, which has ten copies of A ⁇ 1-6 gene, which is referred to as P protein-10copy-A ⁇ 1-6-loop1G72;
  • C is a schematic diagram of a target fragment of a P particle of a copy of the A ⁇ 1-6 gene, which is referred to as P protein-1copy-A ⁇ 1-6-loop2S149;
  • D is a schematic diagram of a P-particle target gene fragment of loop A, with a copy of ten copies of the A ⁇ 1-6 gene, which is referred to as P protein-10copy-A ⁇ 1-6-loop2S149;
  • E is a schematic diagram of a P-particle target gene fragment of 20 copies of A ⁇ 1-6 gene, which is referred to as P protein-20copy-A ⁇ 1-6-loop3G169, on loop3.
  • F is a loop1, a ten copy of the A ⁇ 1-15 gene is ligated after the site G72, and a copy of the P fragment of the P-particle of the A ⁇ 1-6 gene is inserted into the loop S149.
  • G is loop2, and after the site S149, three copies of the A ⁇ 1-12 gene are ligated, and on loop3,
  • a schematic diagram of a P fragment of the P particle of the A ⁇ 1-6 gene, which is submerged with G169, is referred to as P protein-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169;
  • H is a loop1, a copy of the A ⁇ 1-6 gene is ligated to the site G72, and a copy of the P fragment of the P-particle of the A ⁇ 1-6 gene is inserted into the loop G3.
  • K is loop1, and ten copies of A ⁇ 1-6 gene are ligated after site G72. On loop2, ten copies of A ⁇ 1-6 gene are ligated after site S149, and loop G3 is inserted after site G169.
  • a schematic diagram of ten copies of the P particle target gene fragment of the A ⁇ 1-6 gene abbreviated as P protein-10copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop2S149-10copy-A ⁇ 1-6-loop3G169.
  • Figure 3 shows a schematic of 10 recombinant P particles.
  • A is a schematic diagram of P protein-10copy-A ⁇ 1-6-loop1G72;
  • B is a schematic diagram of P protein-1copy-A ⁇ 1-6-loop2S149;
  • C is a schematic diagram of P protein-10copy-A ⁇ 1-6-loop2S149;
  • D is P protein-20copy-A ⁇ 1 Schematic diagram of -6-loop3G169;
  • E is a schematic diagram of P protein-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149;
  • F is a schematic diagram of P protein-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169;
  • G is a schematic diagram of P protein-1copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop3G169;
  • H is P protein-1copy-A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1
  • Figure 4 shows the characterization of the purification and properties of recombinant P particles.
  • A is the SDS-PAGE and non-denatured protein electropherogram of PP-10copy-A ⁇ 1-6-loop1G72 protein, and electron micrograph;
  • B is SDS-PAGE and non-denatured protein electrophoresis map of PP-1copy-A ⁇ 1-6-loop2S149 protein, and electron microscope Photo;
  • C is PP-10copy-A ⁇ 1-6-loop2S149 protein SDS-PAGE and non-denatured protein electropherogram,
  • electron micrograph D is PP-20copy-A ⁇ 1-6-loop3G169 protein SDS-PAGE and non-denatured protein electropherogram, and electron micrograph;
  • E is PP-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 protein SDS -PAGE and non-denatured protein electropherograms, and electron micrographs;
  • F is PP-3copy-
  • Figure 5 shows the determination of the optimal immunization dose and optimal immunoadjuvant for the PP-10copy-A ⁇ 1-6-loop2S149 protein vaccine in the C57BL/6J mouse model.
  • A is the detection of anti-A ⁇ 42 antibody in mouse immune serum after immunization with PP-10copy-A ⁇ 1-6-loop2S149 protein vaccine stimulated with different doses and different immune adjuvants.
  • B is the detection of T cell response in mouse spleen lymphocytes after immunization with different forms of PP-10copy-A ⁇ 1-6-loop2S149 protein vaccine.
  • Figure 6 shows the comparison of mouse anti-A ⁇ 42 antibody ELISA and mouse A ⁇ 42 antibody levels in different immunized groups after immunization of mice with three different forms of protein vaccine.
  • A is the statistical data of four immunization results in the subcutaneous immunization group of PBS;
  • B is the statistical data of four immunization results in the PBS nasal immunization group;
  • C is the statistical data of four immunization results in the CpG subcutaneous immunization group;
  • D is the statistical result of four immunizations in the CpG nasal immunization group.
  • E is the statistical data of four immunization results of PP-1copy-A ⁇ 1-6-loop2S149 subcutaneously immunized with CpG as adjuvant
  • F is the statistical result of four immunizations of PP-1copy-A ⁇ 1-6-loop2S149 with CpG as adjuvant in nasal immunization group.
  • Figure G PP-10copy-A ⁇ 1-6-loop2S149 with CpG as an adjuvant subcutaneous immunization group four times immunization results statistical chart; H is PP-10copy-A ⁇ 1-6-loop2S149 with CpG as adjuvant adjuvant nasal immunization group four immunization Results statistical chart; I is PP-3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169 is the statistical analysis of the four immunization results of the subcutaneous immunization group; J is PP-3copy- A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169 is a statistical chart of four immunization results in adjuvant nasal immunization group; K is the comparison chart of the results after the fourth immunization of each group, PP-3copy -A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S
  • SEQ ID NO: 1 is the amino acid sequence of the Norovirus P protein.
  • SEQ ID NOS: 2-8 are the amino acid sequences of A ⁇ 1-15 peptides of human, mouse, primate, rabbit, scorpion, rat, guinea pig, respectively.
  • SEQ ID NO: 9 is the nucleotide sequence encoding the Norovirus P protein.
  • SEQ ID NO: 10 is the nucleotide sequence encoding the human A ⁇ 1-15 peptide.
  • SEQ ID NO: 11 is a site directed mutagenesis forward primer used in the process of obtaining the pET26b-P protein-mEagI plasmid.
  • SEQ ID NO: 12 is a site-directed mutagenesis reverse primer used in the process of obtaining the pET26b-P protein-mEagI plasmid.
  • SEQ ID NO: 13 is a site directed mutagenesis forward primer used in the process of obtaining the pET26b-P protein-mEagI&mKpnI plasmid.
  • SEQ ID NO: 14 is a site-directed mutagenesis reverse primer used in the process of obtaining the pET26b-P protein-mEagI&mKpnI plasmid.
  • SEQ ID NO: 15 - SEQ ID NO: 141 is the nucleic acid sequence encoding the Norovirus capsid P protein of the chimeric A ⁇ 1-m peptide prepared in the examples of the present invention.
  • SEQ ID NO: 142 - SEQ ID NO: 151 is a nucleotide sequence of a synthetic DNA fragment of interest in the process of constructing a recombinant plasmid expressing the preferred 10 recombinant P proteins, corresponding to 10 copy-A ⁇ 1-6-loop1G72, 1copy, respectively -A ⁇ 1-6-loop2S149, 10copy-A ⁇ 1-6-loop2S149, 20copy-A ⁇ 1-6-loop3G169, 3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169, 10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1 -6-loop2S149, 1copy-A ⁇ 1-6-loop1-G72-10copy-A ⁇ 1-6-loop3G169, 1copy-A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1copy-A ⁇ 1-6-loop3G169, 3copy-A ⁇ 1 -6-loop1G72-3copy
  • An ordered and repetitive array of antigens means that a plurality of A ⁇ 1-m peptides (m is an integer selected from 6-15) are repeatedly and orderedly arranged on the surface of Norovirus recombinant P particles.
  • P protein refers to the P protein in the capsid protein of Norovirus, which can be self-assembled into P particles in vitro.
  • Pprotein is used at the gene level, meaning a gene fragment encoding a P protein, a nucleotide sequence, a plasmid, etc.; Pprotein is used at the protein level, meaning a P protein monomer or a dimer.
  • the protein schematics are all Pproteins, such as the plasmid encoding the P protein shown in Figures 1, 2 and 3, and the P protein monomer shown in the denaturing electrophoresis of Figure 4.
  • P particle (PP) refers to protein particles self-assembled by P protein in Norovirus, which is most common in 24-mer form.
  • P species (PP) is used only at the protein level, meaning a form of multimer (eg, 24mer, etc.), including various proteins for detection of properties and proteins for immunization, as shown in the figure.
  • Pprotein-N1copy-A ⁇ 1-m-loop1Ak1-N2copy-A ⁇ 1-m-loop2Ak2-N3copy-A ⁇ 1-m-loop3Ak3 means that N1 copies of A ⁇ 1-m are chimeric after the K1 amino acid in the loop1 loop of the P protein. N2 copies of A ⁇ 1-m are ligated after the K2 amino acid in the loop 2 loop, and N3 copies of A ⁇ 1-m are ligated after the K3 amino acid in the loop 3 loop, and m is independently selected from 1 An integer of -40. Unless otherwise indicated, A ⁇ 1-m and P proteins are linked by a polypeptide linker having three glycines.
  • Pprotein-1copy-A ⁇ 1-6-loop2T148-1copy-A ⁇ 1-6-loop2S149-1copy-A ⁇ 1-6-loop2N150 means the amino acid at position 148 in the loop2 loop of the P protein, and the 149th position After the amino acid, and after the amino acid at position 150, 1 copy of A ⁇ 1-6 was respectively inserted, and all A ⁇ 1-6 and P proteins were linked by an amino acid linker having three GGA-encoded glycines.
  • P protein-10copy-A ⁇ 1-6-loop2S149(GGA)0 means that 10 copies of A ⁇ 1-6 are chimeric after the amino acid at position 149 in the loop2 loop of the P protein, and A ⁇ 1-6 and P protein are There is no linker, and there is no amino acid linker between each A ⁇ 1-6, that is, A ⁇ 1-6 and P protein and each A ⁇ 1-6 are directly linked by a peptide bond.
  • P protein-10copy-A ⁇ 1-6-loop2S149(GGC)3 means that 10 copies of A ⁇ 1-6 are chimeric after the amino acid at position 149 in the loop2 loop of the P protein, and A ⁇ 1-6 and P protein are involved. Interlinked by an amino acid linker having three GGC-encoded glycines, and each A ⁇ 1-6 is linked by an amino acid linker having three GGC-encoded glycines.
  • the present invention provides 127 recombinant P particles in which a plurality of copies of A ⁇ 1-m peptide (m is an integer selected from 6-15) are ligated in the loop1, loop2 and/or loop3 regions of the P protein to form the 127 species.
  • Recombinant P proteins of recombinant P particles are shown in Table 1.
  • the recombinant P protein or recombinant P particles of the group numbers in the following tables correspond to the corresponding group number of recombinant P proteins in Table 1, respectively.
  • the invention also provides a method of preparing the recombinant P protein particle, comprising the steps of:
  • a synthetic DNA fragment comprising a DNA fragment encoding a P protein loop1, loop2 and/or loop3 domain in which a multicopy A ⁇ 1-m peptide is chimeric;
  • the site-directed mutagenesis was carried out before and after loop1 of the P protein without changing the amino acid sequence, and new restriction sites mKpnI and mEagI were obtained (as shown in Fig. 1B);
  • the synthetic coding obtained in step 1 is chimeric with multiple copies of A ⁇ 1.
  • a DNA fragment of the P protein loop1, loop2 and/or loop3 domain of the -m peptide is used to replace a plurality of wild-type circular structure DNAs, and a plurality of recombinant P protein expression plasmids each carrying a multicopy A ⁇ 1-m peptide are constructed (eg Figure 1D-1M);
  • the expression plasmid obtained in the step 4 was transferred into Escherichia coli, and the P protein in which the A ⁇ 1-m immunogen was chimeric was stably expressed, and self-assembled to form P particles in a 24-mer form.
  • mice can be stimulated to produce the highest titers of specific antibodies against A[beta] 1-42, while the protein vaccine does not cause mice to produce T cell responses against A[beta] 1-42 (as shown in Figure 5).
  • the protein vaccine is an immunogen, and CpG is used as an immunological adjuvant.
  • the immune effects of the three protein vaccines are compared in a mouse model, and the effects of subcutaneous and nasal immunological methods on the immune effect of the protein vaccine are analyzed (results shown in Fig. 6). ).
  • the experimental results show that subcutaneous immunization is more conducive to protein vaccine-induced antibody production than nasal immunization.
  • the nucleotide sequence of the P protein represented by SEQ ID NO: 9 is synthesized by a method of gene synthesis, and the synthesized gene fragment is subjected to Nde I/Xho I double digestion by the same double digestion method as described above, and the product is subjected to digestion. Agarose gel electrophoresis was carried out, and recovery was carried out using a recovery column to obtain a target gene fragment having a double-digested cohesive end.
  • the fixed point mutation method is as follows:
  • 5'CCGCCG3' was mutated to 5'CGGCCG3' to obtain a pET26b-P protein-mEagI plasmid containing the mEagI cleavage site, and the amino acid sequence was not changed.
  • the specific method is as follows: using a pair of fully complementary bidirectional primers containing a mutation site:
  • the whole plasmid was subjected to PCR reaction.
  • the PCR reaction system was KOD-Plus DNA polymerase system (purchased from TOYOBO Co., Ltd.), and the total volume of the reaction system was 50 ⁇ l (buffer 5 ⁇ l, dNTP 0.2 mM, magnesium sulfate 1 mM, upstream and downstream primers 0.3 each) ⁇ M, template DNA 50ng, KOD enzyme 1 ⁇ l, add water to the final volume of 50 ⁇ l) PCR according to the reaction system instructions, to obtain 20 ⁇ l PCR product, add 1 ⁇ l DpnI enzyme (purchased from NEB company) to the product, digest at 37 °C for 1h, take 10 ⁇ l The digested product was added to Tran1-Blue competent cells (purchased from Beijing Quanjin Company), placed on ice for 30 min, then heat-shocked at 42 °C for 45 s, placed on ice for 2 min, and then added to 600 ⁇ l of non-resistant liquid LB medium.
  • the pET26b-P protein-mEagI&mKpnI plasmid was obtained by the same construction method as above, and the sequencing verification sequence was correct.
  • Example 3 Synthesis of a gene fragment of a P particle having a chimeric human A ⁇ 1-m gene
  • the first is a gene fragment of the P protein loop structure of the human A ⁇ 1-m gene located between the mKpnI restriction site and the SalI restriction site, that is, the N1 copy of the A ⁇ 1-m gene is only chimerized on loop1.
  • the P protein prepared by the method is abbreviated as P protein-N 1copy-A ⁇ 1-m-loop 1;
  • the second is a gene fragment of the P protein loop structure of the human A ⁇ 1-m gene located between the SalI cleavage site and the mEagI cleavage site, ie, (1) chimeric N2 copies of A ⁇ 1 only on loop2 -m gene, the P protein prepared by this method is abbreviated as P protein-N2copy-A ⁇ 1-m-loop2; (2) only N3 copies of A ⁇ 1-m gene are chimerized on loop3, and the P protein prepared by this method is abbreviated as P protein-N3copy-A ⁇ 1-m-loop3; (3) chimeric N2 copies of A ⁇ 1-m gene on loop2 and N3 copies of A ⁇ 1-m gene on loop3, P protein prepared by this method is called P protein -N2copy-A ⁇ 1-m-loop2-N3copy-A ⁇ 1-m-loop3.
  • m is an integer independently selected from 1 to 40.
  • the third is a gene fragment of the P protein loop structure of the chimeric human A ⁇ 1-m gene located between the mKpnI cleavage site and the mEagI cleavage site, ie, (1) chimeric N1 copies of A ⁇ 1- on loop1 m gene, and chimeric N2 copies of A ⁇ 1-m gene on loop2, P protein prepared by this method is abbreviated as P protein-N1copy-A ⁇ 1-m-loop1-N2copy-A ⁇ 1-m-loop2; (2) on loop1 Chimeric N1 copies of the A ⁇ 1-m gene, and chimeric N3 copies of the A ⁇ 1-m gene on loop3.
  • the P protein prepared by this method is abbreviated as P protein-N1copy-A ⁇ 1-m-loop1-N3copy-A ⁇ 1-m- Loop3; (3) chimeric N 1 copy of the A ⁇ 1-m gene on loop1, and chimeric N2 copies of the A ⁇ 1-m gene on loop2, and chimeric N3 copies of the A ⁇ 1-m gene on loop3,
  • the P protein prepared by this method is abbreviated as P protein-N1copy-A ⁇ 1-m-loop1-N2copy-A ⁇ 1-m-loop2-N3copy-A ⁇ 1-m-loop3.
  • m is an integer independently selected from 1 to 40.
  • P protein-10copy-A ⁇ 1-6-loop1G72 was synthesized with the loop1 gene fragment encoding the DNA of 10 copies of the A ⁇ 1-6 peptide (as shown in Fig. 2B), and the synthesized gene fragment sequence was SEQ ID NO: 269. Shown.
  • P protein-1copy-A ⁇ 1-6-loop2S149 is synthesized with a loop2 gene fragment encoding DNA of 1 copy of A ⁇ 1-6 peptide (as shown in Fig. 2C), and the synthesized gene fragment sequence is SEQ ID NO: 270 Shown.
  • P protein-10copy-A ⁇ 1-6-loop2S149 is synthesized with a loop2 gene fragment encoding DNA of 10 copies of A ⁇ 1-6 peptide (as shown in Fig. 2D), and the synthesized gene fragment sequence is SEQ ID NO: 271 Shown.
  • P protein-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 is ligated with loop2 of DNA encoding 3 copies of A ⁇ 1-12 peptide and loop3 of DNA encoding 3 copies of A ⁇ 1-6 peptide
  • the synthesis of the gene fragment (as shown in Figure 2G), the sequence of the synthesized gene fragment is shown in SEQ ID NO:273.
  • P protein-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 is ligated with loop1 of DNA encoding 10 copies of A ⁇ 1-15 peptide and loop2 of DNA encoding 10 copies of A ⁇ 1-6 peptide, respectively.
  • the gene fragment shown in Figure 2F
  • the sequence of the synthesized gene fragment is shown in SEQ ID NO:274.
  • P protein-1copy-A ⁇ 1-6-loop1-G72-10copy-A ⁇ 1-6-loop3G169 is ligated with loop1 of DNA encoding 1 copy of A ⁇ 1-6 peptide and DNA encoding 10 copies of A ⁇ 1-6 peptide, respectively
  • the synthesis of the gene fragment of loop3 (shown in Figure 2H), the sequence of the synthesized gene fragment is shown in SEQ ID NO:275.
  • P protein-3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169 was ligated with the loop1, loop2, and loop3 gene fragments encoding the DNA of 3 copies of the A ⁇ 1-6 peptide, respectively.
  • the synthesis (as shown in Figure 2J), the sequence of the synthesized gene fragment is shown in SEQ ID NO:277.
  • Pprotein-10copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop2S149-10copy-A ⁇ 1-6-lo op3G169 respectively, loop1, loop2, loop3 gene fragment of DNA encoding 10 copies of A ⁇ 1-6 peptide
  • the synthesis (as shown in Figure 2K), the sequence of the synthesized gene fragment is shown in SEQ ID NO:278.
  • the first protocol for the first embodiment is the gene fragment of the P particle circular structure of the chimeric human A ⁇ 1-m gene located between the mKpnI restriction site and the SalI restriction site, that is, chimerism only on loop1 N1 copies of the A ⁇ 1-m gene, the P protein prepared by this method is abbreviated as P protein-N1copy-A ⁇ 1-m-loop1;
  • the second scheme for the second embodiment is the gene fragment of the P particle loop structure of the chimeric human A ⁇ 1-m gene located between the SalI cleavage site and the mEagI cleavage site, ie (1) only in loop2
  • the N2 copies of the A ⁇ 1-m gene are chimeric
  • the P protein prepared by this method is abbreviated as P protein-N2copy-A ⁇ 1-m-loop2; (2) only the N3 copies of the A ⁇ 1-m gene are chimeric on loop3.
  • the prepared P protein is abbreviated as P protein-N3copy-A ⁇ 1-m-loop3; (3) chimeric N2 copies of A ⁇ 1-m gene are ligated on loop2 and N3 copies of A ⁇ 1-m gene are chimeric on loop3, which is prepared by the method
  • the P protein is abbreviated as P protein-N2copy-A ⁇ 1-m-loop2-N3copy-A ⁇ 1-m-loop3.
  • the mKpnI cleavage site obtained by mutation and the SalI cleavage site carried by the sequence are used for Example 2
  • the obtained pET26b-P protein-mEagI-mKpnI plasmid vector was digested with SalI/KpnI, and the original loop1 region was excised, and the plasmid was recovered as a vector.
  • the recombinant loop1 DNA fragment containing the multiple copies of the human A ⁇ 1-m sequence synthesized in Example 3 was subjected to SalI/KpnI double digestion to obtain a DNA fragment of interest.
  • the enzyme-cleaved DNA fragment was ligated to a restriction vector to obtain a PET26b plasmid capable of expressing a recombinant P protein having an A ⁇ 1-m immunogen.
  • Final sequencing confirms that the sequence is correct and the correct plasmid is obtained.
  • Example 3 the DNA fragment 10copy-A ⁇ 1-6-loop1G72 synthesized in Example 3 was subjected to double digestion with SalI/KpnI in the same manner to obtain a target gene fragment.
  • the digested vector was mixed with the target fragment, 0.75 ⁇ l of T4 ligase (purchased from Takara) was added, and 1.5 ⁇ l of ligase buffer (purchased from Takara) was added, and the mixture was ligated overnight at 16 ° C to obtain expression of PP-10copy.
  • the pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2 was digested with SalI/EagI by excision of the mEagI cleavage site and the sequence of the SalI cleavage site derived from the mutation, and the original loop2 was excised. And the loop3 region, the plasmid was recovered as a vector.
  • the recombinant loop2 and loop3 DNA fragments containing the multiple copies of the human A ⁇ 1-m sequence synthesized in Example 3 were subjected to SalI/EagI double digestion to obtain the desired DNA fragment.
  • the enzyme-cleaved DNA fragment was ligated to a restriction vector to obtain a PET26b plasmid capable of expressing a recombinant P protein having an A ⁇ 1-m immunogen.
  • Final sequencing confirms that the sequence is correct and the correct plasmid is obtained.
  • DNA fragment 1copy-A ⁇ 1-6-loop2S149 synthesized in Example 3 was subjected to double digestion with SalI/KpnI in the same manner to obtain a target gene fragment.
  • the digested vector was mixed with the target fragment, 0.75 ⁇ l of T4 ligase (purchased from Takara) was added, and 1.5 ⁇ l of ligase buffer (purchased from Takara) was added, and the mixture was ligated overnight at 16 ° C to obtain expression PP-1copy.
  • the plasmid vector of the - ⁇ 1-6-loop2S149 protein was correctly sequenced after sequencing, as shown in Fig. 1E.
  • a plasmid vector expressing the PP-10copy-A ⁇ 1-6-loop2S149 protein was constructed in the same manner as in 4.2.1.
  • the plasmid is shown in Figure 1F.
  • a plasmid vector expressing the PP-20copy-A ⁇ 1-6-loop3G169 protein was constructed in the same manner as in 4.2.1.
  • the plasmid is shown in Figure 1G.
  • a plasmid vector expressing the PP-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 protein was constructed in the same manner as in 4.2.1.
  • the plasmid is shown in Figure 1I.
  • the pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2 was digested with mKpnI/mEagI, and the original loop1 was excised. In the loop2 and loop3 regions, the plasmid was recovered as a vector.
  • the recombinant loop1, loop2 and loop3 DNA fragments containing the multiple copies of the human A ⁇ 1-m sequence synthesized in Example 3 were subjected to double digestion with mKpnI/mEagI to obtain a DNA fragment of interest.
  • the enzyme-cleaved DNA fragment was ligated to a restriction vector to obtain a PET26b plasmid capable of expressing a recombinant P protein having an A ⁇ 1-m immunogen.
  • Final sequencing confirms that the sequence is correct and the correct plasmid is obtained.
  • Example 3 the DNA fragment 3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169 synthesized in Example 3 was digested with EagI/KpnI in the same manner to obtain the target gene fragment. .
  • the digested vector was mixed with the target fragment, 0.75 ⁇ l of T4 ligase (purchased from Takara) was added, and 1.5 ⁇ l of ligase buffer (purchased from Takara) was added, and the mixture was ligated overnight at 16 ° C to obtain expression PP-1copy.
  • the plasmid vector of the - ⁇ 1-6-loop2S149 protein was correctly sequenced after sequencing, as shown in Fig. 1L.
  • a plasmid vector expressing the 10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 protein was constructed using the same method as in 4.3.1. The plasmid is shown in Figure 1H.
  • a plasmid vector expressing the PP-1copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop3G169 protein was constructed in the same manner as in 4.3.1. The plasmid is shown in Figure 1J.
  • a plasmid vector expressing the PP-1copy-A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1copy-A ⁇ 1-6-loop3G169 protein was constructed in the same manner as in 4.3.1. The plasmid is shown in Figure 1K.
  • a plasmid vector expressing the PP-10copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop2S14910copy-A ⁇ 1-6-loop3G169 protein was constructed in the same manner as in 4.3.1. The plasmid is shown in Figure 1M.
  • 1 ⁇ l of the recombinant plasmid prepared in the above examples was added to 100 ⁇ l of Escherichia coli BL21 competent cells (purchased from TransGen Co., Ltd.), ice-bathed for 30 min, and then heat-shocked for 90 s in a 42 ° C water bath for 2 min. 600 ⁇ l of LB medium was added to the mixture at 180 rpm/min, and cultured at 37 ° C for 1 h. The mixture was uniformly coated on LB solid medium containing kanamycin (15 ⁇ g/ml) resistance, and cultured at 37 ° C for 24 hours to obtain a strain capable of stably expressing the recombinant protein.
  • the colonies that picked up the growth were inoculated in 20 ml of LB medium, cultured at 37 ° C, 220 rpm, and when the OD value of the culture mixture reached 1.0, isopropyl thiogalactoside (IPTG, final concentration 0.33 mmol/L) was used.
  • IPTG isopropyl thiogalactoside
  • Induction induction at 16 ° C, 220 rpm overnight.
  • the bacterial solution was centrifuged at 4000 rpm for 20 min, the supernatant was discarded, the cell pellet was resuspended in PBS, centrifuged again at 4000 rpm for 20 min, and the supernatant was discarded to obtain a precipitate of the cells containing the protein of interest.
  • the crude protein extract was purified using a cation exchange column (available from GE Corporation).
  • the specific scheme is as follows: firstly, the exchange column is washed with ultrapure water, the volume is about 100 ml, and then the exchange column is equilibrated with a PB solution having a pH of 5.0, the flow rate is 2 ml/min, and then the crude liquid extract of 20 ml is at a flow rate of 1 ml/min. After adding the exchange column, after the sample is completely hung, the exchange column is washed with a PB solution having a pH of 7.0 to remove the heteroprotein, and then eluted with a PB solution containing a concentration of 1 mol/L sodium chloride to collect the peak protein.
  • Target protein is
  • the protein was further purified by a hydrophobic chromatography column (purchased from GE).
  • the specific operation method was as follows: first, the column was rinsed with ultrapure water, and then the hydrophobic column was washed with a pH of 7.0 PB solution at a flow rate of 2 ml/min. . After the column is well balanced, the protein sample is injected. After the sample is completely entered into the column, the gradient elution is carried out with PB of pH 7.0 and 1 mol/l sodium chloride solution for 2 hours, and the concentration of sodium chloride is 1 mol/l. The peak protein was harvested by dropping to 0.1 mol/l.
  • the size of 10 P-particle protein monomers was identified by reducing SDS-PAGE.
  • Figure 4. The upper panel of AJ shows that the size of PP-10copy-A ⁇ 1-6-loop1G72 protein is 45KD; the size of PP-1copy-A ⁇ 1-6-loop2S149 protein is 37KD; PP-10copy-A ⁇ 1-6-loopS149 protein The size of the PP-20copy-A ⁇ 1-6-loop3G169 protein is 55KD; the size of the PP-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 protein is 66KD; PP-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 protein size is 38KD; PP-1copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop3G169 protein size is 47KD; PP-1copy- The size of A ⁇ 1-6-loop1G72-1copy-A ⁇ 1-6-loop2S149-1copy
  • the 24-mer form of the P protein particles was further separated and purified by using Superdex 200 molecular sieve (purchased from GE).
  • the procedure was as follows: the column was rinsed with ultrapure water at a flow rate of 1 ml/min, and the volume of the column was washed and then used. Approximately 120 ml of pH 5 PB buffer was again rinsed with the column, then 2 ml of the protein extract was added to the column, washed with PB buffer at a flow rate of 1 ml/min, and the peak protein was harvested to obtain a P particle 24-mer form protein. The multimeric structure of the three proteins was detected by non-denaturing polyacrylamide gel electrophoresis.
  • the 10 protein bands were all above 225KDa. It can be seen that the recombinant P protein can self-assemble into 24 P protein particles in the form of a polymer, and after purification of the protein, remain in the form of its multimer.
  • the inventors further analyzed the particle size and morphology of the ten protein multimers.
  • the particle size was measured using a nanoparticle size analyzer (purchased from Malvern) according to the manufacturer's instructions.
  • the analysis results show that there are particles having an average diameter of about 20 nm in the above 10 kinds of recombinant P particle solutions, as shown in the lower diagram of FIG.
  • the particle size of the PP-10copy-A ⁇ 1-6-loop1G72 protein particles is 27.88 nm;
  • the particle size of PP-1copy-A ⁇ 1-6-loop2S149 protein particles is 17.44 nm; the particle size of PP-10copy-A ⁇ 1-6-loop2S149 protein particles is 27.64 nm; the particles of PP-20copy-A ⁇ 1-6-loop3G169 protein particles are 32.55nm;
  • PP-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149 protein particles have a particle size of 35.88 nm;
  • PP-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169 protein particles The diameter of the peptide is 17.02 nm; the particle size of the PP-1copy-A ⁇ 1-6-loop1G72-10copy-A ⁇ 1-6-loop3G169 protein particle is 28.92 nm; PP-1copy-A ⁇ 1-6-loop1
  • the results of electron microscopy showed that the morphology of the recombinant P protein was approximately spherical and was in the form of multimer.
  • 10 protein multimers mainly include particles of about 20 nm. Therefore, the above 10 recombinant P proteins can self-assemble in vitro to form a P particle 24mer.
  • the protein size and P particle size of the 127 P-particle proteins of the present invention are shown in Table 3.
  • the PP-10copy-A ⁇ 1-6-loop2S149 protein vaccine was selected and the female C57BL/6 mice (purchased from Beijing Huakangkang Biotechnology Co., Ltd.) for 6-8 weeks were used for immunization and immunization.
  • the doses were 12.5 ⁇ g, 25 ⁇ g, and 50 ⁇ g/head, supplemented to 100 ⁇ L/cell with sterile PBS, and 6 mice per group were used.
  • the immunization route was subcutaneous injection, and the immunological adjuvant was an aluminum adjuvant (purchased from Brenntag Biosector) and a CpG adjuvant (purchased from Takara Co., Ltd.) capable of specifically inducing humoral immunity to the body, and the nucleotide sequence thereof was as follows: TGTCGTCGTCGTTTGTCGTTTGTCGTT.
  • the immunological adjuvant was an aluminum adjuvant (purchased from Brenntag Biosector) and a CpG adjuvant (purchased from Takara Co., Ltd.) capable of specifically inducing humoral immunity to the body
  • the nucleotide sequence thereof was as follows: TGTCGTCGTCGTTTGTCGTTTGTCGTT.
  • mice were bled for blood on the day before immunization.
  • the blood samples were placed at 37 ° C for 2 h, then placed at 4 ° C for 1 h, centrifuged at 3000 rpm, and the supernatant serum was taken and stored for later use.
  • a ⁇ 1-42 purchased from Shanghai Jill Biochemical Co., Ltd.
  • as an antigen prepared into a 1 mg/ml solution with sterile PBS, diluted to 1 ng/ ⁇ l with antigen coating solution, coated 96-well plates, 100 ⁇ l per well, overnight at 4 °C.
  • PBST pH 7.4, 0.01 mol/L PBS, containing 0.05% Tween-20
  • a blocking solution pH 7.4, 0.01 mol/L PBS, 20% newborn calf serum
  • the cells were washed three times with PBST, and antisera of different dilution gradients (1:200, 1:800, 1:3200, 1:12800, 1:51200, 1:204800) were added, 100 ⁇ l per well, and incubated at 37 ° C for 2 hours.
  • a monoclonal antibody (from the elispot kit, purchased from BD) using a cytokine interferon gamma was coated in a 96-well plate at a concentration of 5 ⁇ g/ml, 50 ul per well, and coated at 4 ° C overnight. After the coated antibody was discarded and washed once with 10% fetal bovine serum in complete medium, 200 ⁇ l of the complete medium was added to each well, and the medium was discarded after blocking at 37 ° C for 1 hour. The rats were sacrificed by pulling the neck, and the spleen cells were taken to prepare a cell suspension having a cell concentration of 107 cells/ml. The cell suspension was added to a well-coated 96-well plate at 100 ul per well.
  • the 96-well plate was washed, and horseradish-peroxidized biotin secondary antibody (from elispot kit, purchased from BD) was added to 50 ul per well, cultured at room temperature for 2 h, washed four times with PBST, and washed twice with PBS, and added per well. 50 ⁇ l of Elispot coloring solution (AEC substrate), reacted at room temperature for 5 to 60 minutes, discarded the staining solution, washed with distilled water, and dried overnight, and the number of activated cells in the sample was calculated by a microscope.
  • horseradish-peroxidized biotin secondary antibody from elispot kit, purchased from BD
  • the experimental results are shown in Fig. 5B.
  • the A ⁇ 42 group in the T cell reaction positive control group showed more spots, which proved that it had a strong T cell response.
  • the 25 ⁇ g protein + aluminum adjuvant group also showed more spots, which proved that the aluminum adjuvant stimulated the body to produce a certain T cell response.
  • 25 ⁇ g The PP-10copy-A ⁇ 1-6-loop2-S149+CpG adjuvant group and the 50 ⁇ g group had no or fewer positive spots, demonstrating that no significant T cell response occurred in vivo, and as described in 3.1.1,
  • This immunization strategy is capable of stimulating mice to produce the highest titers of specific antibodies against A[beta]42.
  • 25 ⁇ g of PP-10copy-A ⁇ 1-6-loop2S149+CpG adjuvant was selected as the optimal immunization strategy.
  • the applicant first selected three representative recombinant proteins, using a protein vaccine dose of 25 ⁇ g/only, using CpG as an adjuvant strategy, using the nasal cavity.
  • Two immunological methods were used to compare the immune effects of three recombinant P-particle protein vaccines in female C57BL/6 mice at 6-8 weeks to compare nasal and subcutaneous immune effects, and to confirm the difference in immune effects of different proteins. Similar to the method of 3.1.1, the immunization was performed once every 2 weeks, and the immunization was performed 4 times. Before each immunization, the mice were subjected to tail-cutting and blood sampling, and the serum was subjected to ELISA. A total of 7 experimental groups and 3 control groups, the immunogen and immune mode of each group are shown in Table 5.
  • the mouse serum was the primary antibody after immunization
  • the HRP goat anti-mouse antibody was the secondary antibody
  • TMB was the substrate
  • the sulfuric acid was The solution was stopped, the reaction was terminated, and after the reaction was terminated, the absorbance was measured at 450 nm using a microplate reader, and the amount of antibody in the serum was compared by the magnitude of the absorbance.
  • PBS as a negative control to commercial antibody 6e10 (purchased from Covance) as a positive control
  • the serum of mice immunized with three proteins was used as an experimental group. As shown in Fig.
  • Applicants determined the immunization dose of the protein vaccine and the immunoassay, and the immunoassay of three representative recombinant proteins, determined that the dosage was 25 ⁇ g/mouse, and the immunological method was subcutaneous injection, and 127 candidate vaccines were determined at 6- Immune effect in 8-week female C57BL/6 mice.
  • the immunization procedure and method are as described in 3.2, and the results of the comparison of the immune effects are shown in Table 6.
  • PP-10copy-A ⁇ 1-6-loop1G72 PP-1copy-A ⁇ 1-6-loop2S149, PP-10copy-A ⁇ 1-6-loop2S149, PP-10copy-A ⁇ 1-6-loop3G169, PP-20copy-A ⁇ 1-6- loop3G169, PP-3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149, PP-10copy-A ⁇ 1-15-loop1G72-10copy-A ⁇ 1-6-loop2S149, PP-20copy-A ⁇ 1-15-loop1G72-40copy- A ⁇ 1-6-loop2S149, PP-3copy-A ⁇ 1-12-loop2S149-3copy-A ⁇ 1-6-loop3G169, PP-1copy-A ⁇ 1-6-loop2S149-10copy-A ⁇ 1-6-loop3G169, PP-20copy-A ⁇ 1-12- loop2S149-10copy-A ⁇ 1-6-loop3G169, PP-3copy-A ⁇ 1-6-loop1
  • the concentration of the induced antibody can be calculated, wherein PP-3copy-A ⁇ 1-6-loop1G72-3copy-A ⁇ 1-6-loop2S149-3copy-A ⁇ 1-6-loop3G169 after the fourth immunization
  • the concentration of the induced antibody was about 245.12 ⁇ g/ml. Therefore, the present invention has a good immune effect, and the concentration of the anti-A ⁇ 1-42 antibody in the serum of the mouse after immunization is high, and has a very good therapeutic effect, and is a very promising protein vaccine for treating AD.

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Abstract

提供了一种由嵌合Aβ1-m肽(m为6-15的整数)的诺如病毒衣壳P蛋白形成的重组P颗粒,所述重组P颗粒形成了一种有序和重复的抗原阵列。还提供了编码其的核苷酸序列、包含其的药物组合物及其用于制备治疗或预防阿尔茨海默症的药物的用途。还提供了制备所述重组P颗粒的方法。

Description

一种嵌合诺如病毒P颗粒及其制备和应用 技术领域
本发明涉及分子生物学和免疫学领域。具体地说,本发明涉及由嵌合Aβ1-m肽的诺如病毒衣壳P蛋白形成的重组P颗粒,其中所述重组P颗粒形成了一种有序且重复的抗原阵列,其中m为选自6-15的整数。
背景技术
阿尔茨海默症(Alzheimer’s disease,AD)又称老年痴呆病,是一种渐行性的神经退行性疾病,其主要临床特征是逐渐出现记忆力减退、认知功能障碍和行为异常,最终丧失独立生活能力,发病后10~20年因并发症死亡。目前这种最为常见的神经退行性疾病尚不能治愈,也没有有效的延缓手段,严重危害老年人的身心健康和生活质量。
研究者们普遍认为大脑组织中的淀粉样-β蛋白(Amyloid-βprotein,简称Aβ)是导致神经元损伤及认知功能障碍的主要致病物质。Aβ来源于其前体物质β-淀粉样前体蛋白(β-Amyloid precursor protein,简称βAPP)。βAPP是一种跨膜蛋白,在体内各种组织中广泛存在,在脑组织中的表达量最高。βAPP在淀粉样剪切途径中,能够被切割成为长度为38至43个氨基酸不等的Aβ蛋白,其中Aβ1-42蛋白是形成淀粉样沉积的主要类型。
目前,以刺激机体产生Aβ抗体从而清除大脑内Aβ1-42沉积为目的的Aβ主动免疫疫苗,是治疗阿尔茨海默症的重要方法。Aβ1-42多肽疫苗作为AD疫苗的先驱,在AD转基因鼠模型中表现出了良好的延缓记忆减退的治疗效果。然而,在Aβ1-42多肽疫苗的临床实验中,6%的AD患者出现了脑膜炎的副反应,且患者产生的Aβ1-42抗体水平较低。研究表明Aβ1-42多肽疫苗虽然可以引起抗体反应,但是由于其携带了大量T细胞表位,会使接种患者的脑神经细胞产生自身免疫性T细胞反应从而引发脑膜炎。相比于Aβ1-42,只包含Aβ多肽N端前15个氨基酸的多肽Aβ1-15,只含有B细胞表位而不含有T细胞表位,并且该多肽类疫苗能够刺激人体产生针对Aβ1-42蛋白的特异性免疫反应,并在人体试验中表现出良好的安全性。因此,Aβ1-15成为了一种非常有研究潜力的抗原肽。但Aβ1-15肽作为一种半抗原,其免疫效果以及诱导产生抗体的持续性不是很好。因此,亟需一种安全性较高且具有良好免疫效果的阿尔兹海默症的疫苗。
在现有技术中,通常优选分别合成病毒样颗粒和Aβ多肽,然后将两者偶联 以获得高免疫原性的疫苗。然而,通过偶联等方法得到的疫苗,难以对插入表位的数量进行控制,并且也难以对疫苗进行有效的纯化。
发明内容
本发明人发现,对于Aβ1-m(m为选自6-15的整数)肽而言,诺如病毒的衣壳P蛋白是非常理想的抗原展示平台。将不同拷贝数,不同长度的Aβ1-m(m为选自6-15的整数)肽编码序列插入到编码诺如病毒衣壳P蛋白的DNA序列的环状结构区域(loop)中时,Aβ1-m能够被展示于重组P蛋白形成的重组P颗粒的最表面,从而有利于最大程度地刺激机体产生针对Aβ1-42蛋白的特异性免疫反应。本发明的重组P颗粒制备方法简单,插入表位数量可控,纯化过程简单,制造成本低,安全性好,免疫原性高且诱导产生的抗体滴度高。
本发明利用基因工程手段,将多个拷贝的编码抗原肽Aβ1-m(m为选自6-15的整数)的DNA插入到编码P蛋白的loop1、loop2和/或loop3的DNA中。通过重组蛋白的体外表达和自我组装,形成能够充分展示Aβ1-m(m为选自6-15的整数)抗原表位的重组P颗粒,从而有利于增强疫苗免疫的有效性、持续性和安全性。
第一方面,本发明提供了一种由嵌合Aβ1-m肽的诺如病毒P蛋白形成的重组P颗粒,其中m为选自6-15的整数。在本发明的实施方案中,m可以为6、7、8、9、10、11、12、13、14或15。所述重组P颗粒形成了一种有序且重复的抗原阵列。其中至少一个所述Aβ1-m肽的氨基酸序列嵌入到所述诺如病毒衣壳P蛋白的loop1、loop2和/或loop3中。
在本发明的优选实施方案中,诺如病毒衣壳P蛋白的氨基酸序列如SEQ ID NO:1所示。在重组P颗粒中,N1个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第70位-第74位氨基酸,即I70、A71、G72、T73和Q74;N2个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第148位-第151位氨基酸,即,T148、S149、N150和D151;N3个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第168位-第171位氨基酸,即,D168、G169、S170和T171;其中N1、N2和N3为各自独立地选自0-40的整数,并且N1+N2+N3≥1。具体地,N1、N2和N3各自独立地选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39和40,并且N1+N2+N3≥1。
“Aβ1-m肽序列嵌入到一个氨基酸位点之后”意指所述Aβ1-m肽序列的N端直接与该氨基酸残基的C端通过肽键相连或通过氨基酸接头与该氨基酸相连。在优选的实施方案中,所述氨基酸接头为(Gly)n,优选n为1-10,更优选n=3。
插入至SEQ ID NO:1中的多个连续Aβ1-m肽序列之间可以直接相连也可通过氨基酸接头相连。在优选的实施方案中,所述氨基酸接头为(Gly)n,优选n为1-10,更优选n=3。
在本发明中,Aβ1-m肽序列意指完整Aβ1-42蛋白N端的前m个氨基酸组成的多肽,其中m为选自6-15的整数。例如,本发明中的Aβ1-m肽可以是完整Aβ1-42蛋白N端的1-6位,1-7位,1-8位,1-9位,1-10位,1-11位,1-12位,1-13位,1-14位或1-15位氨基酸组成的多肽。
本发明的Aβ1-m肽序列可以是人、小鼠、灵长类、兔、光滑爪蟾、大鼠、豚鼠的Aβ1-m肽。人、小鼠、灵长类、兔、光滑爪蟾、大鼠、豚鼠的Aβ1-15肽的氨基酸序列分别为SEQ ID NO:2-SEQ ID NO:8所示。根据这些序列,本领域技术人员可以容易地确定本发明的Aβ1-m肽(m为选自6-15的整数)。优选地,所述Aβ1-m肽为人Aβ1-m肽。
在本发明的优选实施方案中,所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的氨基酸序列为SEQ ID NO:15-141。更优选地,所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的氨基酸序列选自SEQ ID NO:24、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:57、SEQ ID NO:73、SEQ ID NO:92、SEQ ID NO:117、SEQ ID NO:132、SEQ ID NO:133和SEQ ID NO:134。
第二方面,本发明还提供了编码形成第一方面中的重组P颗粒的嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核苷酸序列,其中m为选自6-15的整数。在本发明的实施方案中,m可以为6、7、8、9、10、11、12、13、14或15。所述核苷酸序列包含编码诺如病毒衣壳P蛋白的核苷酸序列和至少一个拷贝的编码Aβ1-m肽的核苷酸序列,其中所述至少一个拷贝的编码Aβ1-m肽的核苷酸序列插入至编码诺如病毒衣壳P蛋白环状区域的核苷酸序列中,其中所述插入的编码Aβ1-m肽的核苷酸序列不会引起诺如病毒P蛋白的移码突变。
在具体的实施方案中,所述至少一个拷贝的编码Aβ1-m肽的核苷酸序列可插入至编码诺如病毒衣壳P蛋白同一环状区域的核苷酸序列中或插入至编码诺如病毒衣壳P蛋白不同的环状区域的核苷酸序列中。例如,当诺如病毒衣壳P蛋白的核苷酸序列为SEQ ID NO:9时,Loop1对应第208-222位核苷酸,Loop2对应第442-453位核苷酸,Loop3对应第502–513位核苷酸。
在本发明的具体实施方案中,插入Loop1、Loop2和Loop3的编码Aβ1-m肽的核苷酸序列的数目分别为N1、N2和N3;N1、N2和N3各自独立地选自0-40的整数,并且N1+N2+N3≥1。具体地,N1、N2和N3各自独立地选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39和40,并且N1+N2+N3≥1。
在本发明的具体实施方案中,所述编码Aβ1-m肽的核苷酸序列的5’端核苷酸通过3’,5’-磷酸二酯键直接与编码诺如病毒衣壳P蛋白环状区域的核苷酸相连或者通过DNA接头与编码诺如病毒衣壳P蛋白环状区域的核苷酸相连。所述插入无论是直接插入还是通过DNA接头插入,均不会在核苷酸序列的翻译中引起移码。在优选的实施方案中,所述DNA接头为(GGA)n或(GGG)n或(GGC)n,优选n为1-10,更优选n=3。
在本发明的具体实施方案中,插入至编码诺如病毒衣壳P蛋白的核苷酸序列中的多个连续的编码Aβ1-m肽的核苷酸序列之间通过3’,5’-磷酸二酯键直接相连或者通过DNA接头相连。所述插入无论是直接插入还是通过DNA接头插入,均不会在核苷酸序列的翻译中引起移码。在优选的实施方案中,所述DNA接头为(GGA)n或(GGG)n或(GGC)n,优选n为1-10,更优选n=3。
优选地,所述Aβ1-m肽核苷酸序列为编码人、小鼠、灵长类、兔、光滑爪蟾、大鼠、豚鼠的Aβ1-m肽的核苷酸序列。更优选地,所述Aβ1-m肽核苷酸序列为编码人Aβ1-m肽的核苷酸序列。
在本发明的优选实施方案中,所述编码嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核苷酸序列为SEQ ID NO:142-268。更优选地,所述编码嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核苷酸序列选自:SEQ ID NO:151、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:184、SEQ ID NO:200、SEQ ID NO:219、SEQ ID NO:244、SEQ ID NO:259、SEQ ID NO:260和SEQ ID NO:261。
第三方面,本发明还提供了一种可用于预防或治疗阿尔兹海默症的药物组合物,其包含本发明第一方面的重组P颗粒和可药用载体。所述药物组合物优选用于哺乳动物,更优选用于人。优选地,所述药物组合物是疫苗组合物,更优选地,所述疫苗组合物还包含佐剂。在本发明的一个具体实施方案中,所述佐剂是CpG。在本发明的另一个具体实施方案中,所述佐剂是铝佐剂。所述疫苗组合物优选通过皮下或鼻腔途径免疫,更优选通过皮下途径免疫。
第四方面,本发明还提供了本发明第一方面的重组p颗粒用于制备用于治疗 或预防阿尔兹海默症的药物中的应用。优选地,所述药物是疫苗。所述药物优选用于哺乳动物,更优选用于人。
第五方面,本发明还提供了一种用于制备本发明第一方面的重组P颗粒的方法,所述方法包括以下步骤:
i)获得含有编码嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核酸的表达载体,其中m为6-15的整数;
ii)将所述表达载体转入受体细胞;
iii)表达所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白并在自组装成重组P颗粒,
优选地,所述方法还包括分离和纯化步骤。
在一个具体的实施方案中,可以使用阳离子交换色谱法和/或疏水层析色谱法进行纯化。
附图说明
图1示出了各重组pET26b质粒构建体的图谱。
A为已经构建好的pET26b-Pprotein质粒示意图;
B为带有突变得到的mEagI酶切位点的pET26b-Pprotein-mEagI质粒示意图;
C为带有突变得到的mEagI和mKpnI酶切位点的pET26b-Pprotein-mEagI&mKpnI质粒示意图;
D为pET26b-Pprotein-10copy-Aβ1-6-loop1G72质粒示意图,在mKpnI酶切位点和SalI酶切位点间搭载目的基因片段P protein-10copy-Aβ1-6-loop1G72;
E为pET26b-P protein-1copy-Aβ1-6-loop2S149质粒示意图,在mEagI酶切位点和SalI酶切位点间搭载目的基因片段P protein-1copy-Aβ1-6-loop2S149;
F为pET26b-P protein-10copy-Aβ1-6-loop2S149质粒示意图,在mEagI酶切位点和SalI酶切位点间搭载目的基因片段P protein-10copy-Aβ1-6-loop2S149;
G为pET26b-P protein-20copy-Aβ1-6-loop3G169质粒示意图,在mEagI酶切位点和SalI酶切位点间搭载目的基因片段P protein-20copy-Aβ1-6-loop3G169;
H为pET26b-P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149质粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片Pprotein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149;
I为pET26b-P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169质 粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片段Pprotein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169;
J为pET26b-P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169质粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片段P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169;
K为pET26b-Pprotein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169质粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片段Pprotein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169。
L为pET26b-Pprotein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169质粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片段Pprotein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169。
M为pET26b-P protein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169质粒示意图,在mEagI酶切位点和mKpnI酶切位点间搭载目的基因片Pprotein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169。
图2示出了目的基因片段结构示意图。
A为P蛋白三个环状结构—loop1、loop2、loop3位置示意图以及定点突变位点和P蛋白基因自带酶切位点;
B为loop1上,位点G72后嵌合有十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-10copy-Aβ1-6-loop1G72;
C为loop2上,位点S149后嵌合有一个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-1copy-Aβ1-6-loop2S149;
D为loop2上,位点S149后嵌合有十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-10copy-Aβ1-6-loop2S149;
E为loop3上,位点G169后嵌合有二十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-20copy-Aβ1-6-loop3G169;
F为loop1上,位点G72后嵌合有十个拷贝的Aβ1-15基因,loop2上,位点S149后嵌合有十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149;
G为loop2上,位点S149后嵌合有三个拷贝的Aβ1-12基因,loop3上,位 点G169后嵌合有三个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169;
H为loop1上,位点G72后嵌合有一个拷贝的Aβ1-6基因,loop3上,位点G169后嵌合有十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169;
I为loop1上,位点G72后嵌合有一个拷贝的Aβ1-6基因,loop2上,位点S149后嵌合有一个拷贝的Aβ1-6基因,loop3上,位点G169后嵌合有一个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169;
J为loop1上,位点G72后嵌合有三个拷贝的Aβ1-6基因,loop2上,位点S149后嵌合有三个拷贝的Aβ1-6基因,loop3上,位点G169后嵌合有三个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169;
K为loop1上,位点G72后嵌合有十个拷贝的Aβ1-6基因,loop2上,位点S149后嵌合有十个拷贝的Aβ1-6基因,loop3上,位点G169后嵌合有十个拷贝的Aβ1-6基因的P颗粒目的基因片段示意图,简称P protein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169。
图3示出了10种重组P颗粒示意图。A为P protein-10copy-Aβ1-6-loop1G72示意图;B为P protein-1copy-Aβ1-6-loop2S149示意图;C为P protein-10copy-Aβ1-6-loop2S149示意图;D为P protein-20copy-Aβ1-6-loop3G169示意图;E为P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149示意图;F为P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169示意图;G为P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169示意图;H为P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169示意图;I为P protein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169示意图;J为P protein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169示意图。
图4示出了重组P颗粒纯化与性质的表征。A为PP-10copy-Aβ1-6-loop1G72蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;B为PP-1copy-Aβ1-6-loop2S149蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;C为PP-10copy-Aβ1-6-loop2S149蛋白SDS-PAGE和非变性蛋白电泳图谱, 以及电镜照片D为PP-20copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;E为PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;F为PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;G为PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;H为PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;I为PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片;J为PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169蛋白SDS-PAGE和非变性蛋白电泳图谱,以及电镜照片。
图5示出了PP-10copy-Aβ1-6-loop2S149蛋白疫苗在C57BL/6J小鼠模型中的最佳免疫剂量和最适免疫佐剂的确定。A为以不同剂量和不同免疫佐剂刺激的PP-10copy-Aβ1-6-loop2S149蛋白疫苗免疫后,小鼠免疫血清抗Aβ42抗体的检测。B为以不同形式的PP-10copy-Aβ1-6-loop2S149蛋白疫苗免疫后,小鼠脾淋巴细胞产生T细胞反应情况检测。
图6示出了三种不同形式的蛋白疫苗免疫小鼠后,小鼠免疫血清抗Aβ42抗体ELISA检测以及不同免疫组别Aβ42抗体水平的比较。A为PBS皮下免疫组四次免疫结果统计图;B为PBS鼻腔免疫组四次免疫结果统计图;C为CpG皮下免疫组四次免疫结果统计图;D为CpG鼻腔免疫组四次免疫结果统计图。E为PP-1copy-Aβ1-6-loop2S149以CpG为佐剂皮下免疫组四次免疫结果统计图;F为PP-1copy-Aβ1-6-loop2S149以CpG为佐剂鼻腔免疫组四次免疫结果统计图;G为PP-10copy-Aβ1-6-loop2S149以CpG为佐剂皮下免疫组四次免疫结果统计图;H为PP-10copy-Aβ1-6-loop2S149以CpG为佐剂鼻腔免疫组四次免疫结果统计图;I为PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169为佐剂皮下免疫组四次免疫结果统计图;J为PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169为佐剂鼻腔免疫组四次免疫结果统计图;K为各组第四次免疫后结果对比图,PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169以CpG为佐剂皮下免疫组免疫效果最佳。
序列表说明
SEQ ID NO:1为诺如病毒P蛋白的氨基酸序列。
SEQ ID NO:2-8分别为人、小鼠、灵长类、兔、光滑爪蟾、大鼠、豚鼠的Aβ1-15肽的氨基酸序列。
SEQ ID NO:9为编码诺如病毒P蛋白的核苷酸序列。
SEQ ID NO:10为编码人Aβ1-15肽的核苷酸序列。
SEQ ID NO:11为获得pET26b-P protein-mEagI质粒过程中所使用的定点突变正向引物。
SEQ ID NO:12为获得pET26b-P protein-mEagI质粒过程中所使用的定点突变反向引物。
SEQ ID NO:13为获得pET26b-P protein-mEagI&mKpnI质粒过程中所使用的定点突变正向引物。
SEQ ID NO:14为获得pET26b-P protein-mEagI&mKpnI质粒过程中所使用的定点突变反向引物。
SEQ ID NO:15-SEQ ID NO:141为编码本发明实施例中制备的嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核酸序列。
SEQ ID NO:142-SEQ ID NO:151为构建表达优选的10种重组P蛋白的重组质粒的过程中,合成的目的DNA片段的核苷酸序列,分别对应10copy-Aβ1-6-loop1G72、1copy-Aβ1-6-loop2S149、10copy-Aβ1-6-loop2S149、20copy-Aβ1-6-loop3G169、3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169、10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149、1copy-Aβ1-6-loop1-G72-10copy-Aβ1-6-loop3G169、1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169、3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169、10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169。
具体实施方式
定义:
除了另有说明以外,本发明中所有科技术语均使用本领域普通技术人员所通常理解的含义。
有序且重复的抗原阵列:是指多个Aβ1-m肽(m为选自6-15的整数)重复并且有序地排列在诺如病毒重组P颗粒的表面。
P蛋白(Pprotein):是指诺如病毒的衣壳蛋白中的P蛋白,其能够在体外自主装成P颗粒。用于本文时,在基因水平上使用Pprotein,意指编码P蛋白的基因片段、核苷酸序列、质粒等;在蛋白质水平上使用Pprotein,意指P蛋白单体或二聚体。在附图中,蛋白示意图均为Pprotein,例如图1、2和3中所示的编码P蛋白的质粒,以及图4中变性电泳中所示的P蛋白单体。
P颗粒(P particle,简称PP):是指诺如病毒中由P蛋白在体外自组装成的蛋白颗粒,以24聚体形式最为常见。用于本文时,仅在蛋白质水平上使用P particle(PP),意指多聚体(例如24聚体等)的形式,包括各种应用于性质检测的蛋白和用于免疫的蛋白,如图4中非变性电泳中所示的多聚体形式。
Pprotein-N1copy-Aβ1-m-loop1Ak1-N2copy-Aβ1-m-loop2Ak2-N3copy-Aβ1-m-loop3Ak3:意指在P蛋白的loop1环中第K1位氨基酸后嵌合有N1个拷贝的Aβ1-m,在loop2环中第K2位氨基酸后嵌合有N2个拷贝的Aβ1-m,并且loop3环中第K3位氨基酸后嵌合有N3个拷贝的Aβ1-m,并且m为各自独立地选自1-40的整数。除非另有说明,Aβ1-m与P蛋白之间通过具有三个甘氨酸的多肽接头连接。例如,Pprotein-1copy-Aβ1-6-loop2T148-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop2N150:意指在P蛋白的loop2环中的第148位的氨基酸后,以及第149位的氨基酸后,以及第150位的氨基酸后分别嵌合有1个拷贝的Aβ1-6,并且所有Aβ1-6与P蛋白之间通过具有3个由GGA编码的甘氨酸的氨基酸接头相连。
P protein-10copy-Aβ1-6-loop2S149(GGA)0:意指在P蛋白的loop2环中的第149位的氨基酸后嵌合有10个拷贝的Aβ1-6,并且Aβ1-6与P蛋白之间不具有接头,且各个Aβ1-6之间不具有氨基酸接头,即Aβ1-6与P蛋白之间及各个Aβ1-6之间通过肽键直接相连。
P protein-10copy-Aβ1-6-loop2S149(GGC)3:意指在P蛋白的loop2环中的第149位的氨基酸后嵌合有10个拷贝的Aβ1-6,并且Aβ1-6与P蛋白之间通过具有3个由GGC编码的甘氨酸的氨基酸接头相连,且各个Aβ1-6之间通过具有3个GGC编码的甘氨酸的氨基酸接头相连。
下面以具体实施例对本发明的技术方案做进一步的说明;但本发明并不限于这些实施例。除非另有说明,本文使用的制剂和设备均为普通市售品。
本发明提供了127种在P蛋白的loop1,loop2和/或loop3区域嵌合有多个拷贝的Aβ1-m肽(m为选自6-15的整数)的重组P颗粒,形成所述127种重组P颗粒的重组P蛋白如表1所示。
表1.形成本发明的127种重组P颗粒的重组P蛋白的氨基酸序列和编码其的核苷酸序列
Figure PCTCN2016087643-appb-000001
Figure PCTCN2016087643-appb-000002
Figure PCTCN2016087643-appb-000003
Figure PCTCN2016087643-appb-000004
Figure PCTCN2016087643-appb-000005
以下各表中的组号的重组P蛋白或重组P颗粒分别对应表1中相应组号的重组P蛋白。
本发明还提供了制备所述重组P蛋白颗粒的方法,包括以下步骤:
1.人工合成DNA片段,其包含编码嵌合有多拷贝Aβ1-m肽的P蛋白loop1、loop2和/或loop3结构域的DNA片段;
2.构建pET26b-P protein质粒(如图1A所示);
3.采用点突变法,在不改变氨基酸序列的前提下,在P蛋白的loop1前和loop3后进行定点突变,获得新的酶切位点mKpnI和mEagI(如图1B所示);
4.利用编码P蛋白的核酸上自带的酶切位点SalI以及所获得的酶切位点mKpnI和mEagI,根据不同的构建需求,用步骤1中获得的合成的编码嵌合有多拷贝Aβ1-m肽的P蛋白loop1、loop2和/或loop3结构域的DNA片段对多个野生型环状结构DNA进行整体替换,构建多种分别搭载多拷贝Aβ1-m肽的重组P蛋白表达质粒(如图1D-1M所示);
5.将步骤4中获得的表达质粒转入大肠杆菌内,稳定表达嵌合有Aβ1-m免疫原的P蛋白,并自组装形成24聚体形式的P颗粒。
此外,本发明还进行了以下分析验证实验:
1.通过粒径测定和电镜分析对优选的十种蛋白疫苗进行了性质表征(如图4所示)。
2.利用PP-10copy-Aβ1-6-loop2S149蛋白疫苗在C57BL/6J小鼠模型中进行了利用不同免疫剂量和不同免疫佐剂的实验,结果表明,以CpG为免疫佐剂时,25μg蛋白疫苗可刺激小鼠产生最高滴度的针对于Aβ1-42的特异性抗体,同时该蛋白疫苗不会引起小鼠产生针对于Aβ1-42的T细胞反应(如图5所示)。
3.以PP-1copy-Aβ1-6-loop2S149、PP-10copy-Aβ1-6-loop2S149、PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169三种蛋白疫苗为免疫原,以CpG为免疫佐剂,在小鼠模型中比较三种蛋白疫苗的免疫效果,同时分析皮下和鼻腔两种免疫方式对于蛋白疫苗免疫效果的影响(结果如图6所示)。实验结果表明,相比于鼻腔免疫,皮下免疫方式更利于蛋白疫苗诱导抗体的产生。
4.用127种候选蛋白进行免疫,比较多种蛋白的免疫效果,筛选出最优的候选疫苗。结果表明:相对于PBS对照组,多种疫苗均可以刺激小鼠产生针对于 Aβ1-42的特异性抗体;其中10种蛋白PP-10copy-Aβ1-6-loopG72、PP-1copy-Aβ1-6-loop2S149、PP-10copy-Aβ1-6-loop2S149、PP-20copy-Aβ1-6-loop3G169、PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149、PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169、PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169、PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169、PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169、PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169的免疫效果最佳,其刺激产生的Aβ42抗体浓度较高。
实施例1.pET26b-P protein质粒的构建
取4μg pET26载体质粒(购自Novagen公司),加入Nde I酶和Xho I酶(购自Takara公司)各1μl,并加入5μl酶切缓冲液(购自Takara公司),最后向体系中加入无菌水使终体积为50μl,37℃,酶切5小时,并对产物进行琼脂糖凝胶电泳,使用回收柱(购自invetrogen)进行回收,得到具有双酶切粘性末端的质粒载体。
通过基因合成的方法,合成如SEQ ID NO:9所示的P蛋白核苷酸序列,利用上述相同的双酶切方法,对合成的基因片段进行Nde I/Xho I双酶切,并对产物进行琼脂糖凝胶电泳,使用回收柱进行回收,得到具有双酶切粘性末端的目的基因片段。
取上述双酶切后的载体片段和目的片段(摩尔比为1:3,总体积15μl),混合,加入0.75μl的T4连接酶(购自Takara公司),以及1.5μl连接酶缓冲液(购自Takara公司),16℃连接过夜,得到具有能够表达P蛋白颗粒的pET26b-P protein质粒,如图1A所示。
实施例2.带有酶切位点mKpnI和mEagI的pET26b-P protein质粒的构建
定点突变方法如下:
通过定点突变的方法,利用实施例1已经构建的pET26b-P protein质粒,将
5’CCGCCG3’突变成5’CGGCCG3’以获得含有mEagI酶切位点的pET26b-P protein-mEagI质粒,并且不改变氨基酸序列。具体实施方法如下:利用一对包含突变位点的完全互补的双向引物:
SEQ ID NO:11(正向):
Figure PCTCN2016087643-appb-000006
SEQ ID NO:12(反向):
Figure PCTCN2016087643-appb-000007
对整个质粒进行PCR反应,其PCR反应体系为KOD-Plus DNA聚合酶体系(购自TOYOBO公司),反应体系总体积为50μl(缓冲液5μl,dNTP 0.2mM,硫酸镁1mM,上下游引物各0.3μM,模板DNA 50ng,KOD酶1μl,加水补充至终体积50μl)按照反应体系说明书进行PCR,得到20μlPCR产物,向产物中加入1μl DpnI酶(购自NEB公司),37℃酶切1h,取10μl酶切产物加入Tran1-Blue感受态细胞中(购自北京全式金公司),冰上放置30min后42℃热击45s,冰上放置2min,而后将其加入600μl无抗性的液体LB培养基中,37℃200rpm复苏1h。将菌液平铺于含有卡那霉素(15μg/ml)的LB固体培养板上,37℃倒置培养过夜。得到突变质粒的克隆,测序验证序列正确。pET26b-P protein-mEagI质粒如图1B所示。
2.利用上一步骤构建的pET26b-P protein-mEagI重组质粒(如图1B所示),将5’GGCACA3’突变成5’GGTACC3’以获得既含有mEagI酶切位点又含有mKpnI酶切位点的pET26b-P protein-mEagI&mKpnI质粒,并且不改变氨基酸序列,其具体实施方法如下:利用一对包含突变位点的完全互补的双向引物:
SEQ ID NO:13(正向):
Figure PCTCN2016087643-appb-000008
SEQ ID NO:14(反向):
5’GGTGAGAGCTGGGTGGTACCGAGCAGGACACC3’,利用与上述相同的构建方法,得到pET26b-P protein-mEagI&mKpnI质粒,测序验证序列正确。
pET26b-P protein-mEagI&mKpnI质粒如图1C所示。
实施例3.嵌合有人Aβ1-m基因的P颗粒环状结构目的基因片段的合成
此步骤共有3种不同的合成方案:
第一种为嵌合位于mKpnI酶切位点和SalI酶切位点间的人Aβ1-m基因的P蛋白环状结构目的基因片段,即仅在loop1上嵌合N1个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N 1copy-Aβ1-m-loop 1;
第二种为嵌合位于SalI酶切位点和mEagI酶切位点间的人Aβ1-m基因的P蛋白环状结构目的基因片段,即(1)仅在loop2上嵌合N2个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N2copy-Aβ1-m-loop2;(2)仅在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P  protein-N3copy-Aβ1-m-loop3;(3)在loop2上嵌合N2个拷贝的Aβ1-m基因并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3。其中m为各自独立地选自1~40的整数。
第三种为位于mKpnI酶切位点和mEagI酶切位点间的嵌合人Aβ1-m基因的P蛋白环状结构目的基因片段,即(1)在loop1上嵌合N1个拷贝的Aβ1-m基因,并且在loop2上嵌合N2个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2;(2)在loop1上嵌合N1个拷贝的Aβ1-m基因,并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3;(3)在loop1上嵌合N 1个拷贝的Aβ1-m基因,并且在loop2上嵌合N2个拷贝的Aβ1-m基因,并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3。其中m为各自独立地选自1~40的整数。
以下举例说明上述三种合成方案。
3.1方案一:
3.1.1
P protein-10copy-Aβ1-6-loop1G72的嵌合有编码10个拷贝的Aβ1-6肽的DNA的loop1基因片段的合成(如图2B所示),合成的基因片段序列如SEQ ID NO:269所示。
3.2方案二:
3.2.1
P protein-1copy-Aβ1-6-loop2S149的嵌合有编码1个拷贝的Aβ1-6肽的DNA的loop2基因片段的合成(如图2C所示),合成的基因片段序列如SEQ ID NO:270所示。
3.2.2
P protein-10copy-Aβ1-6-loop2S149的嵌合有编码10个拷贝的Aβ1-6肽的DNA的loop2基因片段的合成(如图2D所示),合成的基因片段序列如SEQ ID NO:271所示。
3.2.3
P protein-20copy-Aβ1-6-loop3G169的嵌合有编码20个拷贝的Aβ1-6肽的DNA的loop3基因片段的合成(如图2E所示),合成的基因片段序列如SEQ ID  NO:272所示。
3.2.4
P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169的嵌合有编码3个拷贝的Aβ1-12肽的DNA的loop2和编码3个拷贝的Aβ1-6肽的DNA的loop3的基因片段的合成(如图2G所示),合成的基因片段序列如SEQ ID NO:273所示。
3.3方案三:
3.3.1
P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149的分别嵌合有编码10个拷贝的Aβ1-15肽的DNA的loop1和编码10个拷贝的Aβ1-6肽的DNA的loop2的基因片段(如图2F所示),合成的基因片段序列如SEQ ID NO:274所示。
3.3.2
P protein-1copy-Aβ1-6-loop1-G72-10copy-Aβ1-6-loop3G169的分别嵌合有编码1个拷贝的Aβ1-6肽的DNA的loop1和编码10个拷贝的Aβ1-6肽的DNA的loop3的基因片段的合成(如图2H所示),合成的基因片段序列如SEQ ID NO:275所示。
3.3.3
P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169的分别嵌合有编码1个拷贝的Aβ1-6肽的DNA的loop123基因片段的合成(如图2I所示),合成的基因片段序列如SEQ ID NO:276所示。
3.3.4
P protein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169的分别嵌合有编码3个拷贝的Aβ1-6肽的DNA的loop1、loop2、loop3基因片段的合成(如图2J所示),合成的基因片段序列如SEQ ID NO:277所示。
3.3.5
Pprotein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-lo op3G169的分别嵌合有编码10个拷贝的Aβ1-6肽的DNA的loop1、loop2、loop3基因片段的合成(如图2K所示),合成的基因片段序列如SEQ ID NO:278所示。
此外,除了上述10种重组P蛋白之外,还有其他117种重组P蛋白的合成方法分别使用上述三种方案,具体如表2所示:
表2.本发明的127种重组P蛋白的合成方法和127种表达重组P蛋白的质 粒的构建方法
Figure PCTCN2016087643-appb-000009
Figure PCTCN2016087643-appb-000010
Figure PCTCN2016087643-appb-000011
实施例4.稳定表达嵌合有Aβ1-m免疫原的P蛋白的pET26b载体的构建
与实施例3相对应,此步骤共有3种不同的合成方案:
第一种针对实施例3的方案一,为位于mKpnI酶切位点和SalI酶切位点间的嵌合人Aβ1-m基因的P颗粒环状结构目的基因片段,即仅在loop1上嵌合N1个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1;
第二种针对实施例3的方案二,为位于SalI酶切位点和mEagI酶切位点间的嵌合人Aβ1-m基因的P颗粒环状结构目的基因片段,即(1)仅在loop2上嵌合N2个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N2copy-Aβ1-m-loop2;(2)仅在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N3copy-Aβ1-m-loop3;(3)在loop2上嵌合N2个拷贝的Aβ1-m基因并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3。
第三种针对实施例3的合成嵌合有Aβ1-m免疫原的P蛋白的方案三,
为位于mKpnI酶切位点和mEagI酶切位点间的嵌合人Aβ1-m基因的P颗粒环状结构目的基因片段,(1)在loop1上嵌合N1个拷贝的Aβ1-m基因,并且在loop2上嵌合N2个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2;(2)在loop1上嵌合N1个拷贝的Aβ1-m基因,并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3;(3)在loop1上嵌合N1个拷贝的Aβ1-m基因,并且在loop2上嵌合N2个拷贝的Aβ1-m基因,并且在loop3上嵌合N3个拷贝的Aβ1-m基因,此方法制备的P蛋白简称P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3。
以下举例说明上述三种合成方案。
4.1方案一:
利用突变而得的mKpnI酶切位点和序列自带的SalI酶切位点,对实施例2 获得的pET26b-P protein-mEagI-mKpnI质粒载体进行SalI/KpnI双酶切,切除原有的loop1区域,回收质粒作为载体。
同时对实施例3中合成的含有多个拷贝的人Aβ1-m序列的重组loop1DNA片段进行SalI/KpnI双酶切,得到目的DNA片段。
将所述酶切目的DNA片段连接到酶切载体上,得到能够表达嵌合有Aβ1-m免疫原的重组P蛋白的PET26b质粒。最后测序验证序列是否正确,得到正确的质粒。
4.1.1表达PP-10copy-Aβ1-6-loop1G72蛋白的质粒载体的构建
取4μg实施例2中获得的pET26b-P protein-mEagI-mKpnI质粒载体,加入Sal I酶和Kpn I酶(购自Takara公司)各1μl,并加入5μl酶切缓冲液(购自Takara公司),最后向体系中加入无菌水使终体积为50μl,37℃,酶切5小时,并对酶切后对产物进行琼脂糖凝胶电泳,并用胶回收试剂盒(购自天根生化技术有限公司)对产物进行回收,得到具有粘性末端的质粒载体。
同时,对实施例3中合成的DNA片段10copy-Aβ1-6-loop1G72,以同样的方法进行SalI/KpnI双酶切,得到目的基因片段。
将酶切后的载体与目的片段混合,加入T4连接酶(购自Takara公司)0.75μl,并加入1.5μl连接酶缓冲液(购自Takara公司),16℃,连接过夜,得到表达PP-10copy-Aβ1-6-loop1G72蛋白的质粒载体,测序后质粒正确,如图1D所示。
4.2方案二:
利用突变而得的mEagI酶切位点和序列自带的SalI酶切位点,对实施例2获得的pET26b-P protein-mEagI-mKpnI质粒载体进行SalI/EagI双酶切,切除原有的loop2和loop3区域,回收质粒作为载体。
同时对实施例3中合成的含有多个拷贝的人Aβ1-m序列的重组loop2和loop3DNA片段进行SalI/EagI双酶切,得到目的DNA片段。
将所述酶切目的DNA片段连接到酶切载体上,得到能够表达嵌合有Aβ1-m免疫原的重组P蛋白的PET26b质粒。最后测序验证序列是否正确,得到正确的质粒。
4.2.1表达PP-1copy-Aβ1-6-loop2S149蛋白的质粒载体的构建
取4μg实施例2中获得的pET26b-P protein-mEagI-mKpnI质粒载体,加入Sal I酶和EagI酶(购自Takara公司)各1μl,并加入适宜的酶切缓冲液(购自Takara公司),最后向体系中加入无菌水使终体积为50μl,37℃,酶切5小 时,并对酶切后对产物进行琼脂糖凝胶电泳,并用胶回收试剂盒(购自天根生化技术有限公司)对产物进行回收,得到具有粘性末端的质粒载体。
同时对实施例3中合成的DNA片段1copy-Aβ1-6-loop2S149,以同样的方法进行SalI/KpnI双酶切,得到目的基因片段。
将酶切后的载体与目的片段混合,加入T4连接酶(购自Takara公司)0.75μl,并加入1.5μl连接酶缓冲液(购自Takara公司),16℃,连接过夜,得到表达PP-1copy-Aβ1-6-loop2S149蛋白的质粒载体,测序后质粒正确,如图1E所示。
4.2.2表达PP-10copy-Aβ1-6-loop2S149蛋白的质粒载体的构建
使用与4.2.1相同的方法,构建表达PP-10copy-Aβ1-6-loop2S149蛋白的质粒载体。质粒如图1F所示。
4.2.3表达PP-20copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
使用与4.2.1相同的方法,构建表达PP-20copy-Aβ1-6-loop3G169蛋白的质粒载体。质粒如图1G所示。
4.2.4表达PP-3copy-Aβ1-12-loopS149-3copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
使用与4.2.1相同的方法,构建表达PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169蛋白的质粒载体。质粒如图1I所示。
4.3方案三:
利用突变而得的mEagI酶切位点和突变而得的mKpnI酶切位点,对实施例2获得的pET26b-P protein-mEagI-mKpnI质粒载体进行mKpnI/mEagI双酶切,切除原有的loop1、loop2和loop3区域,回收质粒作为载体。
同时对实施例3中合成的含有多个拷贝的人Aβ1-m序列的重组loop1,loop2和loop3DNA片段进行mKpnI/mEagI双酶切,得到目的DNA片段。
将所述酶切目的DNA片段连接到酶切载体上,得到能够表达嵌合有Aβ1-m免疫原的重组P蛋白的PET26b质粒。最后测序验证序列是否正确,得到正确的质粒。
4.3.1表达PP-3copy-Aβ1-6-loop1-G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
取4μg实施例2中获得的pET26b-P protein-mEagI-mKpnI质粒载体,加入Kpn I酶和EagI酶(购自Takara公司)各1μl,并加入适宜的酶切缓冲液(购自Takara公司),最后向体系中加入无菌水使终体积为50μl,37℃,酶切 5小时,对产物进行琼脂糖凝胶电泳,并用胶回收试剂盒(购自天根生化技术有限公司)对酶切后产物进行回收,得到具有粘性末端的质粒载体。
同时对实施例3中合成的DNA片段3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169,以同样的方法进行EagI/KpnI双酶切,得到目的基因片段。
将酶切后的载体与目的片段混合,加入T4连接酶(购自Takara公司)0.75μl,并加入1.5μl连接酶缓冲液(购自Takara公司),16℃,连接过夜,得到表达PP-1copy-Aβ1-6-loop2S149蛋白的质粒载体,测序后质粒正确,如图1L所示。
4.3.2表达PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149蛋白的质粒载体的构建
使用与4.3.1相同的方法,构建表达10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149蛋白的质粒载体。质粒如图1H所示。
4.3.3表达PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
使用与4.3.1相同的方法,构建表达PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169蛋白的质粒载体。质粒如图1J所示。
4.3.4表达PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
使用与4.3.1相同的方法,构建表达PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169蛋白的质粒载体。质粒如图1K所示。
4.3.5表达PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169蛋白的质粒载体的构建
使用与4.3.1相同的方法,构建表达PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S14910copy-Aβ1-6-loop3G169蛋白的质粒载体。质粒如图1M所示。
此外,除了上述10种重组P蛋白之外,还有其他117种表达重组P蛋白的质粒的构建方法分别使用上述三种方案,具体如表2所示,获得的重组质粒未示出。
实施例5嵌合有人Aβ1-m免疫原的P蛋白的表达和纯化
5.1重组P颗粒蛋白的表达
分别取上述实施例中制成的重组质粒1μl加入100μl大肠杆菌BL21感受态细胞(购自TransGen公司)中,冰浴30min,然后在42℃水浴中,热激90s后,冰浴2min。向混合物中加入LB培养基600μl,180rpm/min,37℃培养1h。将混合物均匀涂布在含有卡那霉素(15μg/ml)抗性的LB固体培养基上,37℃培养24小时,即可获得可以稳定表达重组蛋白的菌株。挑取生长的菌落接种在20ml LB培养基中,37℃,220rpm培养,并在培养混合物的OD值达到1.0时,用异丙基硫代半乳糖苷(IPTG,终浓度0.33mmol/L)进行诱导,16℃,220rpm诱导过夜。诱导完成后,将菌液以4000rpm离心20min,弃上清,用PBS重悬菌体沉淀,再次以4000rpm离心20min,弃上清,得到含有目的蛋白的菌体的沉淀。
5.2重组P颗粒蛋白的提取以及纯化
向5.1获得的菌体沉淀中加入20ml蛋白缓冲液(PH=8.0,含50mM Tris,300Mm KCl)重悬,在冰上将菌体超声30min破碎菌体,将混合物以12000rpm,4℃离心30min,取上清。将上清过0.45μm滤膜后得到蛋白的粗提液。
重组P颗粒蛋白的结构如图3A-J所示。
利用阳离子交换柱(购自GE公司)对蛋白质粗提液进行纯化。其具体方案如下:首先用超纯水润洗交换柱,体积约为100ml,然后用pH为5.0的PB溶液平衡交换柱,流速2ml/min,然后将20ml蛋白粗提液以1ml/min的流速加入交换柱,待样品完全挂柱后,用pH为7.0的PB溶液冲洗交换柱,以除去杂蛋白,然后用含有浓度为1mol/L氯化钠的PB溶液进行洗脱,收集峰值蛋白,得到目的蛋白。
继续用疏水层析柱(购自GE公司)对蛋白进行进一步纯化,具体操作方法如下:首先用超纯水润洗柱子,然后用pH为7.0的PB溶液润洗疏水柱,流速为2ml/min。柱子平衡好之后,注入蛋白样品,待样品完全进入柱子后,用pH为7.0的PB与1mol/l氯化钠溶液进行梯度洗脱,洗脱时长为2小时,氯化钠浓度从1mol/l下降到0.1mol/l,收获峰值蛋白。
通过还原性SDS-PAGE鉴定10种P颗粒蛋白单体的大小。图4.A-J的上部图分别示出PP-10copy-Aβ1-6-loop1G72蛋白的大小为45KD;PP-1copy-Aβ1-6-loop2S149蛋白的大小为37KD;PP-10copy-Aβ1-6-loopS149蛋白的大小为45KD;PP-20copy-Aβ1-6-loop3G169蛋白的大小为55KD;PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149蛋白的大小为66KD; PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169蛋白的大小为38KD;PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169蛋白的大小为47KD;PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169蛋白的大小为3KD;PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169蛋白的大小为45KD;PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169蛋白的大小为66KD。
然后利用Superdex 200分子筛(购自GE公司)进一步分离纯化P蛋白颗粒的24聚体形式,其操作步骤如下:用超纯水润洗柱子,流速1ml/min,润洗一个柱体积,然后用体积约120ml的pH5的PB缓冲液再次润洗柱子,然后将2ml蛋白提取液加入柱子中,用PB缓冲液以1ml/min的流速冲洗,收获峰值蛋白,得到P颗粒24聚体形式蛋白。并用非变性聚丙烯酰胺凝胶电泳检测三种蛋白的多聚体结构,如图4A-J的中间图所示,10种蛋白条带均在225KDa之上,可知重组P蛋白可以自我组装成24聚体形式的P蛋白颗粒,并且蛋白纯化后,依旧保持了其多聚体的形式。
实施例6嵌合有人Aβ1-m免疫原的P蛋白颗粒的性质表征
发明人进一步分析了10种蛋白多聚体的粒径大小和形态。粒径利用纳米粒径仪(购自马尔文公司)依据生产商的使用说明进行检测。分析结果表明在上述10种重组P颗粒溶液中存在平均直径在20nm左右的颗粒,如图4A-J的下部图所示,PP-10copy-Aβ1-6-loop1G72蛋白颗粒的粒径为27.88nm;PP-1copy-Aβ1-6-loop2S149蛋白颗粒的粒径为17.44nm;PP-10copy-Aβ1-6-loop2S149蛋白颗粒的粒径为27.64nm;PP-20copy-Aβ1-6-loop3G169蛋白颗粒的粒为32.55nm;PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149蛋白颗粒的粒径为35.88nm;PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169蛋白颗粒的粒径为17.02nm;PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169蛋白颗粒的粒径为28.92nm;PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169蛋白颗粒的粒径为18.14nm;PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169蛋白颗粒的粒径为25.56nm;PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169 蛋白颗粒的粒径为36.09nm。同时,电镜测试结果表明,重组P蛋白的形态为近似于球形的颗粒,并为多聚体形式。根据标尺可知,10种蛋白多聚体主要包括20nm左右的颗粒。因此,上述10种重组P蛋白在体外均能自组装,形成P颗粒24聚体。本发明的127种P颗粒蛋白的蛋白大小和P颗粒粒径如表3所示。
表3.本发明的127种不同形式的重组P颗粒蛋白大小及P颗粒粒径
Figure PCTCN2016087643-appb-000012
Figure PCTCN2016087643-appb-000013
3.重组P颗粒蛋白疫苗的免疫效果
3.1P颗粒蛋白疫苗免疫剂量和佐剂的确定
选取PP-10copy-Aβ1-6-loop2S149蛋白疫苗,在6-8周的雌性C57BL/6小鼠(购自北京华阜康生物科技股份有限公司)中进行免疫剂量和免疫佐剂的摸索,免疫剂量分别为12.5μg,25μg,50μg/只,用无菌PBS补至100μL/只,每组选用6只小鼠。免疫途径采用皮下注射方式,免疫佐剂为铝佐剂(购自Brenntag Biosector公司)和能够特异性激发机体产生体液免疫的CpG佐剂(购自Takara公司),其核苷酸序列如下:TGTCGTCGTCGTTTGTCGTTTGTCGTT。第1天,第15天和第29天共计免疫3次,每次免疫前一天取血,第三次免疫两周后处死小鼠,检测重组P颗粒蛋白疫苗诱导小鼠的体液免疫和细胞免疫的免疫应答。共分为7个实验组和3个对照组,每组的免疫剂量、佐剂和免疫抗原如表4所示。
表4.PP-10copy-Aβ1-6-loop2S149的免疫方案
Figure PCTCN2016087643-appb-000014
3.1.1体液免疫—ELISA检测实验
每次免疫前一天对小鼠割尾取血,血样37℃放置2h后,4℃放置1h,3000rpm离心,取上层血清,冻存待用。以Aβ1-42(购自上海吉尔生化公司)为抗原,用无菌PBS配制成1mg/ml溶液,用抗原包被液稀释至1ng/μl,包被 96孔板,每孔100μl,4℃包被过夜。每孔用300μlPBST(pH7.4,0.01mol/L PBS,含有0.05%Tween-20)洗涤三次后,加入封闭液(pH7.4,0.01mol/L PBS,20%的新生牛血清),37℃封闭2小时。用PBST洗涤三次,加入不同稀释梯度(1:200、1:800、1:3200、1:12800、1:51200、1:204800)的抗血清,每孔100μl,37℃,孵育2小时。用PBST洗涤三次,加入0.3μg/ml的HRP(辣根过氧化物酶)山羊抗小鼠二抗(购自北京鼎国昌盛生物技术有限责任公司),每孔100μl,37℃,孵育1小时。PBST洗涤三次,加入底物四甲基联苯胺(TMB)(购自天根生化科技有限公司),每孔100μl,避光显色25分钟,每孔加入2M硫酸50μl,终止反应,酶标仪(购自Bio-red公司)450nm处检测吸光度。
实验结果如图5.A所示,以CpG为免疫佐剂,并以25μg为剂量时,或者以50μg为剂量而不使用免疫佐剂时,PP-10copy-Aβ1-6-loop2-S149蛋白疫苗在所有所示稀释倍数时,都可刺激小鼠产生具有相对较高滴度的针对Aβ42的特异性抗体。
3.1.2细胞免疫-ELISPOT检测
利用细胞因子干扰素γ的单克隆抗体(来自elispot试剂盒,购自BD公司)包被96孔板,浓度5μg/ml,每孔50ul,加盖4℃包被过夜。弃去包被抗体,用10%胎牛血清的完全培养基洗涤一次后,在每孔加入该完全培养基200μl,37℃封闭1小时后弃去培养基。拉颈将实验鼠处死,取其脾细胞,制成细胞浓度为107个/ml细胞悬液,将细胞悬液加入包被好的96孔板中,每孔100ul。每孔加入1μg/ml的特异性抗原Aβ1-42抗原100μl,37℃,5%CO2培养箱中培养24小时,刺激、活化细胞。24小时后,用无菌水洗板两次,无菌PBST(pH7.4,0.01mol/L PBS,含有0.05%Tween-20)缓冲液洗涤6次,洗去细胞。每孔加入干扰素γ的抗体(来自elispot试剂盒,购自BD公司),50μl,抗体浓度为2μg/ml,室温孵育两小时。洗涤96孔板,加入辣根过氧化物标记的生物素二抗(来自elispot试剂盒,购自BD公司)每孔50ul,室温培养2h,PBST洗涤四次,PBS洗涤2次后,每孔加入50μlElispot显色液(AEC底物),避光室温反应5~60分钟,弃去染色液,用蒸馏水洗涤,过夜干燥后,利用显微镜计算出样品中被激活细胞的数目。
实验结果如图5B所示,T细胞反应阳性对照组Aβ42组有较多的斑点数出现,证明其有较强的T细胞反应。而25μg蛋白+铝佐剂组也有较多的斑点数出现,证明铝佐剂会刺激机体产生一定的T细胞反应。而25μg  PP-10copy-Aβ1-6-loop2-S149+CpG佐剂组和50μg组没有或有较少的阳性斑点数,证明其在体内无明显的T细胞反应发生,并且如3.1.1中所述,该免疫策略能够刺激小鼠产生最高滴度的针对于Aβ42的特异性抗体。而出于疫苗的安全性考虑,选择25μg PP-10copy-Aβ1-6-loop2S149+CpG佐剂作为最佳免疫策略。
3.2三种重组P颗粒蛋白疫苗的免疫效果比较
通过蛋白疫苗的免疫剂量和免疫佐剂的确定实验,申请人接下来首先挑选了三种较有代表性的重组蛋白,采用蛋白疫苗剂量为25μg/只,以CpG为佐剂的策略,利用鼻腔和皮下注射两种免疫方式来比较三种重组P颗粒蛋白疫苗在6-8周的雌性C57BL/6小鼠中的免疫效果,以对比鼻腔和皮下免疫效果,并确认不同蛋白的免疫效果差异。与3.1.1的方法相似,每2周免疫一次,共免疫4次,每次免疫前对小鼠进行割尾取血,对血清进行ELISA检测。共分为7个实验组和3个对照组,每组的免疫原和免疫方式如表5所示。
表5.三种代表性P颗粒蛋白的免疫方案
Figure PCTCN2016087643-appb-000015
应用3.1.1所述的方法,以Aβ1-42为抗原,包被于ELISA板上,以免疫后小鼠血清为一抗,HRP山羊抗小鼠抗体为二抗,TMB为底物,硫酸为终止液,进行反应,终止反应后,在450nm处,利用酶标仪对吸光度进行检测,通过吸光度的大小比较血清中抗体的含量。并以PBS为阴性对照,以商用抗体6e10 (购自Covance公司)为阳性对照,以三种蛋白免疫小鼠前后的血清为实验组进行实验,结果如图6所示,三种蛋白都可以刺激小鼠机体产生针对于Aβ42的特异性抗体。说明本发明疫苗具有良好的免疫原性。并用商用抗体6e10为阳性对照,绘制标准曲线,用于抗体浓度的计算,免疫效果比较结果如图6所示。三种蛋白的两种免疫方式都能够产生针对Aβ42的特异性抗体,其OD值都明显高于PBS组和CpG组,并随着免疫次数的增加,抗体浓度不断上升,在第四次免疫时,抗体水平能够达到最高值。通过对比可知,皮下免疫的效果要好于鼻腔免疫。
3.3多种重组P颗粒蛋白疫苗的免疫效果比较
申请人通过蛋白疫苗的免疫剂量和免疫佐剂的确定实验,以及三种代表性的重组蛋白的免疫试验,确定采用剂量为25μg/只,免疫方式为皮下注射,测定127种候选疫苗在6-8周的雌性C57BL/6小鼠中的免疫效果。免疫流程和方法如3.2中所述,免疫效果比较结果如表6所示。
表6.以127种不同形式的P颗粒作为免疫原的蛋白疫苗刺激小鼠产生的Aβ42抗体的浓度
Figure PCTCN2016087643-appb-000016
Figure PCTCN2016087643-appb-000017
Figure PCTCN2016087643-appb-000018
其中,PP-10copy-Aβ1-6-loop1G72、PP-1copy-Aβ1-6-loop2S149、PP-10copy-Aβ1-6-loop2S149、PP-10copy-Aβ1-6-loop3G169、PP-20copy-Aβ1-6-loop3G169、PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149、PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149、PP-20copy-Aβ1-15-loop1G72-40copy-Aβ1-6-loop2S149、PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169、PP-1copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169、PP-20copy-Aβ1-12-loop2S149-10copy-Aβ1-6-loop3G169、PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-12-loop3G169、PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169、PP-3copy-Aβ1-6-loop1G72-10copy-Aβ1-12-loop3G169、PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169、PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169、PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169。这17种P颗粒免疫效果较好,能够诱导产生高浓度的抗体。
根据阳性抗体6e10制作标准曲线,其线性拟合曲线为y=47.692x+0.2964,R2=0.99。根据该标准曲线,在线性范围内,可计算诱导产生抗体的浓度,其中PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169在第四次免疫后诱导产生的抗体浓度在245.12μg/ml左右。因此,本发明具有良好的免疫效果,免疫后小鼠血清中抗Aβ1-42抗体的浓度较高,具有非常好的治疗效果,是一种非常有潜力的治疗AD症的蛋白疫苗。

Claims (10)

  1. 一种重组P颗粒,其由嵌合Aβ1-m肽的诺如病毒衣壳P蛋白形成,其中m为选自6-15的整数,所述重组P颗粒形成了一种有序且重复的抗原阵列,至少一个所述Aβ1-m肽的氨基酸序列嵌入到所述诺如病毒衣壳P蛋白的loop1、loop2和/或loop3中,优选地,所述诺如病毒衣壳P蛋白的氨基酸序列为SEQ ID NO:1所示的序列。
  2. 权利要求1的重组P颗粒,其中N1个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第70位-第74位氨基酸,即I70、A71、G72、T73和Q74;N2个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第148位-第151位氨基酸,即T148、S149、N150和D151;N3个Aβ1-m肽序列嵌入到选自以下的一个或多个氨基酸位点之后:SEQ ID NO:1的第168位-第171位氨基酸,即D168、G169、S170和T171,其中N1、N2和N3各自独立地为选自0-40的整数,并且N1+N2+N3≥1。
  3. 权利要求1或2的重组P颗粒,其中嵌入至所述诺如病毒衣壳P蛋白中的多个连续Aβ1-m肽序列之间直接相连,或者通过多肽接头相连,其中所述多肽接头优选为(Gly)n,优选n=1-10,更优选n=3。
  4. 权利要求1或2的重组P颗粒,其中所述Aβ1-m肽与所述诺如病毒衣壳P蛋白直接相连,或者通过多肽接头相连,其中所述多肽接头优选为(Gly)n,优选n=1-10,更优选n=3。
  5. 权利要求1或2的重组P颗粒,其中所述Aβ1-m肽序列为包含在选自SEQ ID NO:2-8的序列中的氨基酸序列,优选地,所述Aβ1-m肽序列包含在SEQ ID NO:2中。
  6. 权利要求1的重组P颗粒,其中所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的氨基酸序列为SEQ ID NO:15-SEQ ID NO:141;优选地,所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的氨基酸序列为SEQ ID NO:24、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:73、SEQ ID NO:92、SEQ ID NO:117、SEQ ID NO:132、SEQ ID NO:133或SEQ ID NO:134。
  7. 一种核酸,其编码形成权利要求1-6任一项的重组P颗粒,优选地,所述核酸序列为SEQ ID NO:142-SEQ ID NO:268,更优选地,所述核苷酸序列为SEQ ID NO:151、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:184、SEQ ID NO:200、SEQ ID NO:219、SEQ ID NO:244、SEQ ID NO:259、SEQ ID  NO:260或SEQ ID NO:261。
  8. 一种用于预防或治疗阿尔兹海默病的药物组合物,其包含权利要求1-6任一项的重组P颗粒和可药用载体,所述药物组合物优选用于哺乳动物,更优选用于人;优选地,所述药物组合物是疫苗,更优选地,所述疫苗还包含佐剂,最优选地,所述疫苗还包含CpG佐剂或铝佐剂;优选地,所述疫苗优选通过皮下或鼻腔途径免疫,更优选通过皮下途径免疫。
  9. 权利要求1-6任一项的重组P颗粒用于制备用于治疗或预防阿尔兹海默病的药物中的用途,优选地,所述药物是疫苗。
  10. 一种用于制备权利要求1-6任一项的重组P颗粒的方法,所述方法包括以下步骤:
    i)获得含有编码嵌合Aβ1-m肽的诺如病毒衣壳P蛋白的核酸的表达载体,其中m为6-15的整数;
    ii)将所述表达载体转入受体细胞;
    iii)表达所述嵌合Aβ1-m肽的诺如病毒衣壳P蛋白并在自组装成重组P颗粒,
    优选地,所述方法还包括分离和纯化步骤,更优选地,所述纯化步骤包括使用阳离子交换色谱法和/或疏水层析色谱法进行纯化。
PCT/CN2016/087643 2015-07-15 2016-06-29 一种嵌合诺如病毒p颗粒及其制备和应用 WO2017008638A1 (zh)

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