WO2017032303A1 - 一种用于展示目的多肽的多肽载体及其用途 - Google Patents

一种用于展示目的多肽的多肽载体及其用途 Download PDF

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WO2017032303A1
WO2017032303A1 PCT/CN2016/096481 CN2016096481W WO2017032303A1 WO 2017032303 A1 WO2017032303 A1 WO 2017032303A1 CN 2016096481 W CN2016096481 W CN 2016096481W WO 2017032303 A1 WO2017032303 A1 WO 2017032303A1
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amino acid
protein
polypeptide
epitope
hbv
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PCT/CN2016/096481
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English (en)
French (fr)
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张天英
袁权
郭雪染
张莹
赵勤俭
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Application filed by 厦门大学, 厦门万泰沧海生物技术有限公司 filed Critical 厦门大学
Priority to JP2018510399A priority Critical patent/JP6863966B2/ja
Priority to CA2996497A priority patent/CA2996497A1/en
Priority to AU2016313660A priority patent/AU2016313660B2/en
Priority to BR112018003580-7A priority patent/BR112018003580A2/zh
Priority to EP16838561.5A priority patent/EP3342866B1/en
Priority to KR1020187008233A priority patent/KR102116320B1/ko
Priority to US15/754,766 priority patent/US10548973B2/en
Publication of WO2017032303A1 publication Critical patent/WO2017032303A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N2730/00—Reverse transcribing DNA viruses
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    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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    • C12N2730/10011—Hepadnaviridae
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2740/00—Reverse transcribing RNA viruses
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the invention relates to the field of genetic engineering vaccines, the field of molecular virology and the field of immunology.
  • the present invention particularly relates to the field of Hepatitis B virus (HBV) infection treatment.
  • the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a peptide carrier and for inserting a nucleotide sequence encoding a polypeptide of interest.
  • the nucleic acid molecule of the present invention after insertion of a nucleotide sequence encoding a polypeptide of interest and expressed as a recombinant protein, is capable of displaying the polypeptide of interest (eg, an epitope of interest or an epitope of interest in an antigen), And/or, the recombinant protein is capable of forming a virus-like particle and displaying the polypeptide of interest.
  • the invention relates to a recombinant protein comprising the polypeptide vector and a polypeptide of interest. Further, the invention relates to the use of said nucleic acid molecule and said recombinant protein.
  • the present invention particularly relates to a vaccine or pharmaceutical composition for preventing, alleviating or treating a disease associated with HBV infection or a disease associated with HBV infection, such as hepatitis B, comprising a polypeptide vector comprising the polypeptide of the present invention and an epitope derived from HBV. Recombinant protein.
  • Vaccines are an effective means of dealing with infectious diseases. Vaccines can be classified into preventive vaccines and therapeutic vaccines depending on the population. Prophylactic vaccines are mainly used to prevent viral infections, including attenuated vaccines, inactivated vaccines, and genetically engineered vaccines, which protect the body from infection by inducing neutralizing antibodies. Therapeutic vaccines are mainly used to treat diseases such as persistent viral infections and tumors. In these diseases, patients generally exhibit a state of immune tolerance to target antigens, and therefore, researchers have attempted to induce an effective immune response against a target antigen by various forms of vaccine. Therapeutic vaccines mainly include nucleic acid vaccines, viral vector vaccines, genetic engineering vaccines, etc., among which genetically engineered vaccines have obvious advantages.
  • the genetically engineered vaccines that have been marketed include hepatitis B virus vaccine, human papillomavirus (HPV) vaccine, and hepatitis E virus (Hepatitis E).
  • Virus, HEV Virus, HEV vaccines, all of which are in the form of virus-like particles (VLPs).
  • a virus-like particle refers to a hollow particle composed of one or more structural proteins of a certain virus, which does not contain viral nucleic acid and cannot be autonomously replicated, but is identical or similar in morphology to a true virion.
  • the virus-like particles have the following advantages: strong immunogenicity, good safety, inactivation, display of exogenous peptides and induction of specific immune responses against exogenous peptides, and therefore important applications in the field of vaccines. value.
  • HBV infection-related liver diseases At present, about 2 billion people worldwide have been infected with HBV, and about 350 million of them have chronic HBV infections. These people eventually die from HBV infection-related liver diseases by 15%-25%. More than 1 million people worldwide die from end-stage liver disease caused by hepatitis B every year. China is the hardest hit area of HBV infection, and there are currently about 93 million hepatitis B carriers. In recent years, with the continuous improvement of the case report system, the incidence of hepatitis B-related diseases, the mortality rate has not declined.
  • N-stage liver disease such as severe hepatitis (liver failure), cirrhosis and liver cancer
  • the best clinical endpoint is to make patients reach the serology of hepatitis B virus surface antigen.
  • Negative or serological conversion ie complete elimination of HBV.
  • the efficacy of existing drugs to achieve this goal is very limited.
  • the inventors of the present application are based on three bat-derived hepatitis B virus core antigen proteins (ie, the core antigen protein (RBHBcAg) of the roundleaf bat HBV (RBHBV); the bat hepatitis B virus (tent) -making bat HBV, TBHBV) core antigen protein (TBHBcAg); horshoe bat HBV (HBHBV) core antigen protein (HBHBcAg), developed a new class of peptides for the display of peptides of interest A peptide carrier.
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide vector.
  • nucleic acid molecules can be inserted for coding purposes
  • the produced recombinant protein is capable of efficiently forming a VLP, and is capable of displaying a polypeptide of interest inserted into the VLP surface, so that the polypeptide of interest can be effectively recognized by the body's immune system, and the body is induced to generate a specific immune response against the polypeptide of interest.
  • the polypeptide vector of the present invention can be used as a vaccine vector for displaying a polypeptide of interest, such as a peptide (for example, an epitope) from an antigen of interest; and a recombinant protein comprising the polypeptide vector of the present invention and a polypeptide of interest can be used as a vaccine It is used to induce the body to produce a specific immune response against the polypeptide of interest.
  • a polypeptide of interest such as a peptide (for example, an epitope) from an antigen of interest
  • a recombinant protein comprising the polypeptide vector of the present invention and a polypeptide of interest can be used as a vaccine It is used to induce the body to produce a specific immune response against the polypeptide of interest.
  • the inventors of the present application have surprisingly found that the C-terminus of the RBHBcAg protein, the TBHBcAg protein, and the HBHBcAg protein are both arginine-rich regions, which are not essential for VLP assembly. Therefore, the polypeptide carrier of the present invention may not contain the C-terminal region of the RBHBcAg protein, the TBHBcAg protein, and the HBHBcAg protein.
  • the RBHBcAg vector of the present invention may delete part or all of amino acids 145-189 of the RBHBcAg protein (for example, amino acids 150-189 of the RBHBcAg protein are deleted); the TBHBcAg vector of the present invention may be deleted from the 149th of the TBHBcAg protein. Part or all of the amino acid at position 188 (for example, deleting amino acids 154-188 of the TBHBcAg protein); the HBHBcAg vector of the present invention may delete part or all of amino acids 145 to 189 of the HBHBcAg protein (for example, the deletion of the HBHBcAg protein) 150-189 amino acids).
  • the polypeptide vector of the present invention is particularly suitable for use in displaying epitopes derived from human hepatitis B virus (e.g., epitopes in HBsAg from human HBV), capable of being induced in a subject
  • a particularly strong specific immune response against HBsAg is significantly superior to existing hepatitis B vaccines (eg, vaccines containing the same epitope constructed with human HBV HBcAg as a polypeptide vector).
  • the present invention also provides a polypeptide vector that is particularly suitable for use in displaying epitopes derived from human hepatitis B virus.
  • the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide vector or a variant thereof, the variant having at least 90% (eg, at least 91) The identity of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), or under stringent conditions or high stringency conditions, with the nucleoside Acid sequence hybridization, wherein the polypeptide carrier is selected from the group consisting of:
  • RBHBcAg vector which differs from the core antigen protein of hoof hemiver hepatitis B virus (RBHBcAg; for example, its amino acid sequence is shown in SEQ ID NO: 1) in that: (a) the N-terminus of the RBHBcAg protein is 78- One or more amino acid residues in the amino acid residue at position 83 (eg, 1, 2, 3, 4, 5 or 6 amino acid residues; for example, amino acid residues 78-83, The amino acid residues 78-82, amino acid residues 78-81, or amino acid residues 78-80 are deleted or replaced with a linker (eg, a flexible linker; eg, as SEQ ID NO: 43 The linker shown); and (b) optionally, the C-terminus of the RBHBcAg protein is deleted by 1-40 amino acid residues (eg, 1-5, 5-10, 10-15, 15-20) , 20-25, 25-30, 30-35, or 35-40 amino acid residues);
  • a linker
  • a TBHBcAg vector which differs from the core antigen protein of TB-hepatitis B virus (TBHBcAg; for example, its amino acid sequence is shown in SEQ ID NO: 2) in that: (a) the N-terminal 80th of the TBHBcAg protein One or more amino acid residues in the -84 amino acid residue (for example, 1, 2, 3, 4 or 5 amino acid residues; for example, amino acid residues 80-84, 80- The amino acid residue at position 83 or the amino acid residue at positions 80-82 is deleted or replaced with a linker (for example, a flexible linker; for example, a linker as shown in SEQ ID NO: 43); and (b) Optionally, the C-terminus of the TBHBcAg protein is deleted from 1 to 35 amino acid residues (eg, 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, Or 30-35 amino acid residues); or
  • HBHBcAg vector which differs from the core antigen protein of HBV Hepatitis B virus (HBHBcAg; for example, its amino acid sequence is shown in SEQ ID NO: 3) in that: (a) the N-terminal 78 of the HBHBcAg protein One or more amino acid residues in the -83 amino acid residue (eg, 1, 2, 3, 4, 5 or 6 amino acid residues; for example, amino acid residues 78-83, The amino acid residues 78-82, amino acid residues 78-81, or amino acid residues 78-80 are deleted or replaced with a linker (eg, a flexible linker; eg, as SEQ ID NO: The linker shown by 43); and (b) optionally, the C-terminus of the HBHBcAg protein is deleted by 1-40 amino acid residues (for example, 1-5, 5-10, 10-15, 15-20) , 20-25, 25-30, 30-35, or 35-40 ammonia Acid residue).
  • HBHBcAg core antigen protein
  • the variant has at least 90% identity to the nucleotide sequence encoding the polypeptide carrier, eg, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • the variant is capable of hybridizing to the nucleotide sequence encoding the polypeptide carrier under stringent conditions. In a preferred embodiment, the variant is capable of hybridizing to the nucleotide sequence encoding the polypeptide carrier under high stringency conditions.
  • the RBHBcAg protein is wild-type RBHBcAg.
  • the amino acid sequence of the RBHBcAg protein is set forth in SEQ ID NO: 1.
  • the RBHBcAg vector differs from the RBHBcAg protein by one or more contiguous amino acid residues in the amino acid residues 78-83 of the N-terminus of the RBHBcAg protein (eg, 1, 2, 3) , 4, 5 or 6 contiguous amino acid residues) were deleted or replaced with a linker.
  • the amino acid residue at position 79, the amino acid residue at position 80, the amino acid residue at position 81, the amino acid residue at position 82, or the amino acid residue at position 83 may be deleted or replaced with a linker.
  • the connector is for example a flexible connector.
  • Such flexible linkers are well known to those skilled in the art, for example, GGGGGSGGGGTGSEFGGGGSG
  • the RBHBcAg vector differs from the RBHBcAg protein in that: (1) one or more contiguous amino acid residues in the amino acid residues 78-83 of the N-terminus of the RBHBcAg protein (eg, 1, 2) , 3, 4, 5 or 6 contiguous amino acid residues) are deleted or replaced by a linker as defined above; and (2) RBHBcAg The C-terminus of the protein is deleted by 1-40 amino acid residues.
  • the C-terminus of the RBHBcAg protein is deleted 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, Or 35-40 amino acid residues; for example, 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are deleted 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues.
  • the TBHBcAg protein is wild-type TBHBcAg.
  • the amino acid sequence of the TBHBcAg protein is set forth in SEQ ID NO: 2.
  • the TBHBcAg vector differs from the TBHBcAg protein by one or more contiguous amino acid residues in the amino acid residues 80-84 of the N-terminus of the TBHBcAg protein (eg, 1, 2, 3) One, four, or five consecutive amino acid residues) were deleted or replaced with a linker.
  • amino acid residues 80-84 at the N-terminus of the TBHBcAg protein amino acid residues 80-83, amino acid residues 80-82, amino acid residues 80-81, amino acid residues 81-84 Base, amino acid residues 81-83, amino acid residues 81-82, amino acid residues 82-84, amino acid residues 82-83, amino acid residues 83-84, 80th
  • the amino acid residue, the 81st amino acid residue, the 82nd amino acid residue, the 83rd amino acid residue, or the 84th amino acid residue may be deleted or replaced with a linker.
  • the connector is for example a flexible connector.
  • Such flexible linkers are well known to those skilled in the art, for example, GGGGGSGGGGTGSEFGGGGSGGGGS (SEQ ID NO: 43).
  • the TBHBcAg vector differs from the TBHBcAg protein in that: (1) one or more contiguous amino acid residues in the amino acid residues 80-84 of the N-terminus of the TBHBcAg protein (eg, 1, 2) , 3, 4, or 5 contiguous amino acid residues) are deleted or replaced with a linker, as defined above; and (2) the C-terminus of the TBHBcAg protein is deleted by 1-35 amino acid residues.
  • the C-terminus of the TBHBcAg protein is deleted 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, or 30-35 Amino acid residues; for example, deleted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, twenty two, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acid residues.
  • the HBHBcAg protein is wild-type HBHBcAg.
  • the amino acid sequence of the HBHBcAg protein is set forth in SEQ ID NO:3.
  • the HBHBcAg vector differs from the HBHBcAg protein by one or more contiguous amino acid residues in the amino acid residues 78-83 of the N-terminus of the HBHBcAg protein (eg, 1, 2, 3) , 4, 5 or 6 contiguous amino acid residues) were deleted or replaced with a linker.
  • one or more contiguous amino acid residues in the amino acid residues 78-83 of the N-terminus of the HBHBcAg protein eg, 1, 2, 3) , 4, 5 or 6 contiguous amino acid residues
  • the amino acid residue at position 79, the amino acid residue at position 80, the amino acid residue at position 81, the amino acid residue at position 82, or the amino acid residue at position 83 may be deleted or replaced with a linker.
  • the connector is for example a flexible connector.
  • Such flexible linkers are well known to those skilled in the art, for example, GGGGGSGGG
  • the HBHBcAg vector differs from the HBHBcAg protein in that: (1) one or more contiguous amino acid residues in the amino acid residues 78-83 of the N-terminus of the HBHBcAg protein (eg, 1, 2) , 3, 4, 5 or 6 contiguous amino acid residues) are deleted or replaced by a linker as defined above; and (2) 1 to 40 amino acid residues are deleted at the C-terminus of the HBHBcAg protein .
  • the C-terminus of the HBHBcAg protein is deleted 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, Or 35-40 amino acid residues; for example, 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are deleted 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues.
  • restriction sites are particularly advantageous, as is known to those skilled in the art.
  • a restriction enzyme cleavage site is introduced at a position encoding the deleted one or more amino acid residues.
  • a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker and/or one or both ends thereof.
  • one or more restriction sites are introduced in the nucleic acid molecule of the invention and/or one or both of its ends.
  • restriction sites are known to those skilled in the art, including, but not limited to, restriction enzymes such as EcoR I, BamH I, Hind II, Hind III, Hpa I, Hpa II, Mbo I, Mbo II, and the like. Identification of the cleavage site.
  • amino acid sequence of the polypeptide vector is selected from the group consisting of SEQ ID NOS: 4-9.
  • the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 12-17.
  • the nucleic acid molecule is used to insert a nucleotide sequence encoding a polypeptide of interest.
  • the nucleotide sequence encoding the polypeptide of interest is inserted at a position encoding the deleted one or more amino acid residues, or inserted into a nucleotide sequence encoding the linker or one end thereof Or both ends.
  • the nucleotide sequence encoding the polypeptide of interest is inserted in-frame into the nucleotide sequence encoding the polypeptide vector.
  • the nucleotide sequence encoding the polypeptide of interest is inserted into a nucleotide sequence encoding a polypeptide vector using a restriction enzyme cleavage site.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a polypeptide of interest, wherein the polypeptide of interest is heterologous to the polypeptide carrier, and the polypeptide encoding the polypeptide of interest
  • the nucleotide sequence is inserted at a position encoding the deleted one or more amino acid residues, or inserted into a nucleotide sequence encoding the linker or one or both ends thereof.
  • the nucleotide sequence encoding the polypeptide of interest is inserted in-frame into the nucleotide sequence encoding the polypeptide vector.
  • the nucleotide sequence encoding the polypeptide of interest is inserted into a nucleotide sequence encoding a polypeptide vector using a restriction enzyme cleavage site.
  • the polypeptide of interest comprises or is an antigen or An epitope peptide having an epitope.
  • the polypeptide of interest is an epitope peptide, such as an epitope peptide comprising an epitope from HIV, PDL1 or HBV, particularly human HBV.
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a TBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is amino acids 361-375 of the GP120 protein.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acids 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOS: 20-22 and 60-62.
  • the nucleic acid molecule comprises or consists of a nucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-40 and 69-74 Sequence composition.
  • the invention relates to a vector comprising a nucleic acid molecule of the invention as defined above.
  • Vectors useful for insertion of a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors.
  • the vector of the invention may be a eukaryotic expression vector or a prokaryotic expression vector.
  • the vector of the invention is, for example, a plasmid, a cosmid, or a bacteriophage or the like.
  • the invention also relates to a host cell comprising the nucleic acid molecule or vector described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the invention features a method of displaying a polypeptide of interest, comprising:
  • nucleic acid molecule of the present invention inserting a nucleotide sequence encoding the polypeptide of interest into a nucleic acid molecule of the present invention (in particular, inserted into a nucleotide sequence encoding a polypeptide carrier), thereby obtaining a nucleic acid molecule encoding a recombinant protein;
  • the polypeptide of interest is heterologous to the polypeptide carrier.
  • the nucleotide sequence encoding the polypeptide of interest is inserted at a position encoding the deleted one or more amino acid residues, or inserted into a nucleoside encoding the linker In the acid sequence or at one or both ends.
  • the nucleotide sequence encoding the polypeptide of interest is inserted in-frame into the nucleotide sequence encoding the polypeptide vector.
  • the nucleotide sequence encoding the polypeptide of interest is inserted into a nucleotide sequence encoding a polypeptide vector using a restriction enzyme cleavage site.
  • the polypeptide of interest comprises either an antigen or an epitope peptide comprising an epitope.
  • the polypeptide of interest is a table A peptide, for example, an epitope peptide comprising an epitope from HIV, PDL1 or HBV (particularly human HBV).
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a TBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is amino acids 361-375 of the GP120 protein.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acids 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOS: 20-22 and 60-62.
  • the nucleic acid molecule encoding the recombinant protein comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-40 and 69-74.
  • the invention in another aspect, relates to a recombinant protein comprising a polypeptide vector and a polypeptide of interest, wherein the polypeptide vector is as defined above, and wherein the polypeptide of interest is inserted into the polypeptide vector.
  • the polypeptide of interest is inserted at the position of the deleted one or more amino acid residues, or inserted into the linker or at one or both ends thereof.
  • the polypeptide of interest comprises either an antigen or an epitope peptide comprising an epitope.
  • the polypeptide of interest is an epitope peptide, such as an epitope peptide comprising an epitope from HIV, PDL1 or HBV, particularly human HBV.
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a TBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HIV, PDL1 or HBV (particularly human HBV) (eg, HBsAg from human HBV) Epitope (eg linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is 361-375 of the GP120 protein Amino acid.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acid 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOS: 20-22 and 60-62.
  • the amino acid sequence of the polypeptide vector is selected from the group consisting of SEQ ID NOS: 4-9.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-40 and 69-74.
  • the invention features a virus-like particle comprising or consisting of a recombinant protein of the invention.
  • the invention relates to a pharmaceutical composition (eg, a vaccine) comprising a recombinant protein of the invention or a virus-like particle of the invention, and, optionally, one or more pharmaceutically acceptable carriers ( Vehicle) or excipient (eg adjuvant).
  • a pharmaceutical composition eg, a vaccine
  • the recombinant protein of the invention or the virus-like particle of the invention is present in the pharmaceutical composition in an effective amount.
  • the pharmaceutical composition of the present invention may comprise recombinant protein or virus-like particles in an amount effective to prevent or treat HBV infection or a disease associated with HBV infection (for example, hepatitis B).
  • the present invention relates to a method of preventing or treating a HBV infection or a disease associated with HBV infection (eg, hepatitis B), comprising administering a recombinant protein of the present invention to a subject in need thereof or A virus-like particle or pharmaceutical composition, wherein the polypeptide of interest comprises an epitope from HBV, particularly human HBV.
  • the polypeptide of interest is an epitope peptide comprising an epitope from HBV, particularly human HBV.
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg Linear epitope)).
  • the polypeptide vector is a TBHBcAg vector, and the epitope peptide comprises or is an epitope derived from HBV (particularly human HBV) (eg, an epitope (eg, a linear epitope) of HBsAg from human HBV).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg eg Linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOs: 22 and 60-62.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 35-40 and 69-74.
  • the recombinant protein or virus-like particle or pharmaceutical composition of the invention is administered in an amount effective to prevent or treat HBV infection or a disease associated with HBV infection (eg, hepatitis B).
  • HBV infection or a disease associated with HBV infection eg, hepatitis B.
  • the present invention relates to the use of the above recombinant protein or virus-like particle for the preparation of a medicament for preventing or treating a HBV infection or a disease associated with HBV infection (for example, hepatitis B), wherein said
  • the polypeptide of interest comprises an epitope derived from HBV, particularly human HBV.
  • the polypeptide of interest is an epitope peptide comprising an epitope from HBV, particularly human HBV.
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg Linear epitope)).
  • the polypeptide vector is a TBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg eg Linear gauge)).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg eg Linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOs: 22 and 60-62.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 35-40 and 69-74.
  • the present invention relates to the above recombinant protein or virus-like particle or pharmaceutical composition for preventing or treating a HBV infection or a disease associated with HBV infection (for example, hepatitis B), wherein the polypeptide of interest comprises Epitope of HBV (especially human HBV).
  • the polypeptide of interest is an epitope peptide comprising an epitope from HBV, particularly human HBV.
  • the polypeptide of interest comprises or is selected from the group consisting of HBsAg of human HBV or an epitope peptide comprising an epitope of HBsAg (eg, a linear epitope).
  • the polypeptide vector is an RBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg Linear epitope)).
  • the polypeptide vector is a TBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg eg Linear epitope)).
  • the polypeptide vector is a HBHBcAg vector and the epitope peptide comprises or is an epitope from HBV (particularly human HBV) (eg, an epitope from HBsAg of human HBV (eg eg Linear epitope)).
  • the polypeptide of interest comprises or is selected from the HBsAg protein of human HBV or an epitope peptide comprising an epitope of the HBsAg protein (eg, a linear epitope).
  • the HBV is selected from the group consisting of HBV genotypes A, B, C and D.
  • the epitope of the HBsAg protein is amino acids 113-135 of the HBsAg protein.
  • the amino acid sequence of the polypeptide of interest is selected from the group consisting of SEQ ID NOs: 22 and 60-62.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 35-40 and 69-74.
  • the present invention relates to a method of preventing or treating HIV infection or a disease associated with HIV infection (eg, AIDS), comprising administering a recombinant protein or virus sample of the present invention to a subject in need thereof
  • a particle or pharmaceutical composition wherein the polypeptide of interest comprises an epitope from HIV.
  • the polypeptide of interest is an epitope peptide comprising an epitope from HIV.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is amino acids 361-375 of the GP120 protein.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO: 20.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28.
  • the recombinant protein or virus-like particle or pharmaceutical composition of the invention is administered in an amount effective to prevent or treat HIV infection or a disease associated with HIV infection (e.g., AIDS).
  • the present invention relates to the use of the above recombinant protein or virus-like particle for the preparation of a medicament for preventing or treating HIV infection or a disease associated with HIV infection (for example, AIDS), wherein the polypeptide of interest Contains epitopes from HIV.
  • the polypeptide of interest is an epitope peptide comprising an epitope from HIV.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is a GP120 protein Amino acids 361-375.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO: 20.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28.
  • the present invention relates to the above recombinant protein or virus-like particle or pharmaceutical composition for preventing or treating HIV infection or a disease associated with HIV infection (for example, AIDS), wherein the polypeptide of interest comprises from HIV Epitope.
  • the polypeptide of interest is an epitope peptide comprising an epitope from HIV.
  • the polypeptide of interest comprises or is selected from the GP120 protein of HIV or an epitope peptide comprising an epitope of a GP120 protein (eg, a linear epitope).
  • the epitope peptide comprises or is amino acids 361-375 of the GP120 protein.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO: 20.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28.
  • the present invention relates to a method of preventing or treating cancer (eg, non-small cell lung cancer) comprising administering to a subject in need thereof a recombinant protein or virus-like particle or pharmaceutical composition of the present invention
  • cancer eg, non-small cell lung cancer
  • the polypeptide of interest comprises an epitope of a human PD-L1 protein.
  • the polypeptide of interest is an epitope peptide comprising an epitope of a human PD-L1 protein.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acid 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO:21.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-34.
  • the recombinant protein or virus-like particle or pharmaceutical composition of the invention is administered in an amount effective to prevent or treat cancer (eg, non-small cell lung cancer).
  • cancer eg, non-small cell lung cancer
  • the present invention relates to the use of the above recombinant protein or virus-like particle for the preparation of a medicament for preventing or treating cancer (for example, non-small cell lung cancer), wherein the polypeptide of interest comprises human PD-L1 The epitope of the protein.
  • the polypeptide of interest is an epitope peptide comprising an epitope of a human PD-L1 protein.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acid 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO:21.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-34.
  • the present invention relates to the above recombinant protein or virus-like particle or pharmaceutical composition for preventing or treating cancer (for example, non-small cell lung cancer), wherein the polypeptide of interest comprises an antigenic epitope of human PD-L1 protein Bit.
  • the polypeptide of interest is an epitope peptide comprising an epitope of a human PD-L1 protein.
  • the polypeptide of interest comprises or is selected from a human PD-L1 protein or an epitope peptide comprising an epitope (eg, a linear epitope) of a human PD-L1 protein.
  • the epitope peptide comprises or is amino acid 147-160 of the human PD-L1 protein.
  • the amino acid sequence of the polypeptide of interest is set forth in SEQ ID NO:21.
  • the recombinant protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-34.
  • the invention relates to a polynucleotide encoding the above recombinant protein and a vector comprising the polynucleotide.
  • Vectors useful for insertion of a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors.
  • the vector of the invention may be a eukaryotic expression vector or a prokaryotic expression vector.
  • the vector of the invention is, for example, a plasmid, a cosmid, or a bacteriophage or the like.
  • the invention also relates to a host fine comprising the above polynucleotide or vector Cell.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention may also be a cell line, such as a 293T cell.
  • the present invention is also a method of producing the above recombinant protein, which comprises culturing a host cell of the present invention under conditions suitable for expression of the recombinant protein, and recovering the recombinant protein.
  • RBHBcAg and "RBHBcAg protein” refer to a core antigenic protein from roundleaf bat HBV (RBHBV), It is well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: KC790373.1).
  • amino acid residues 78-83 at the N-terminus of the RBHBcAg protein means the amino acid residues 78-83 of the polypeptide represented by SEQ ID NO: 1.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as RBHBcAg proteins of different genotypes or subtypes may be naturally occurring or artificially introduced in the amino acid sequence of the RBHBcAg protein. ) without affecting its biological function.
  • RBHBcAg protein shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 1 as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the RBHBcAg protein, it includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment in its natural or artificial variant. For example, the expression “amino acid residues 78-83 of the N-terminus of the RBHBcAg protein” package The amino acid residues 78-83 of SEQ ID NO: 1, and the corresponding fragments thereof (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • wild-type RBHBcAg refers to a core antigenic protein that is naturally found in the hoofed hepatitis B virus.
  • RBHBcAg vector refers to a polypeptide vector derived from an RBHBcAg protein. As described in detail above, the RBHBcAg vector differs from the RBHBcAg protein in that: (a) one or more amino acid residues in the amino acid residues 78-83 of the N-terminus of the RBHBcAg protein are deleted or replaced with a linker. And (b) optionally, the C-terminus of the RBHBcAg protein is deleted by 1-40 amino acid residues.
  • TBHBcAg and "TBHBcAg protein" of the bat hepatitis B virus are referred to as tent-making bat HBV (TBHBV).
  • Core antigenic proteins which are well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: KC790378.1).
  • amino acid residues at positions N-80 of the TBHBcAg protein refers to amino acid residues 80-84 of the polypeptide represented by SEQ ID NO: 2.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as different genotypes or subtypes of TBHBcAg protein may be naturally occurring or artificially introduced in the amino acid sequence of the TBHBcAg protein. ) without affecting its biological function.
  • the term "TBHBcAg protein” shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 2, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the TBHBcAg protein, it includes not only the sequence fragment of SEQ ID NO: 2 but also the corresponding sequence fragment in its natural or artificial variant. For example, the expression “amino acid residues at positions N-80 of the TBHBcAg protein” includes amino acid residues 80-84 of SEQ ID NO: 2, and corresponding fragments thereof in variants (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned, the highest percentage is obtained.
  • number identity a segment located at an equivalent position in the compared sequence.
  • wild-type TBHBcAg refers to a core antigenic protein that is naturally found in the bat hepatitis B virus.
  • TBHBcAg vector refers to a polypeptide vector derived from a TBHBcAg protein. As described in detail above, the TBHBcAg vector differs from the TBHBcAg protein in that: (a) one or more amino acid residues in the amino acid residues 80-84 of the N-terminus of the TBHBcAg protein are deleted or replaced with a linker. And (b) optionally, the C-terminus of the TBHBcAg protein is deleted from 1 to 35 amino acid residues.
  • HBHBcAg and "HBHBcAg protein” refer to a core antigen from horseshoe bat HBV (HBHBV). Proteins, which are well known to those skilled in the art (see, for example, NCBI GENBANK database accession number: KC790377.1).
  • amino acid residue at positions N-78-83 of the HBHBcAg protein means the amino acid residues 78-83 of the polypeptide represented by SEQ ID NO: 3.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as HBHBcAg proteins of different genotypes or subtypes may be naturally occurring or artificially introduced in the amino acid sequence of the HBHBcAg protein. ) without affecting its biological function.
  • HBHBcAg protein shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 3, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the HBHBcAg protein, it includes not only the sequence fragment of SEQ ID NO: 3 but also the corresponding sequence fragment in its natural or artificial variant. For example, the expression “amino acid residues at positions N-78-83 of the HBHBcAg protein” includes amino acid residues 78-83 of SEQ ID NO: 3, and corresponding fragments thereof in variants (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • wild-type HBHBcAg refers to a core antigenic protein that is naturally found in the Helicoverpa armigera hepatitis B virus.
  • HBHBcAg vector refers to a polypeptide vector derived from a HBHBcAg protein. As described in detail above, the HBHBcAg vector differs from the HBHBcAg protein in that: (a) one or more amino acid residues in the amino acid residues 78-83 of the N-terminus of the HBHBcAg protein are deleted or replaced with a linker. And (b) optionally, the C-terminus of the HBHBcAg protein is deleted by 1-40 amino acid residues.
  • HBcAg of human HBV and “Hu-HBcAg” refer to the core antigenic protein of human hepatitis B virus, which is well known to those skilled in the art (see, for example, the NCBI GENBANK database accession number). :AAO63517.1). As used herein, when referring to the amino acid sequence of HBcAg of human HBV, it is described using the sequence shown in NCBI GENBANK database accession number: AAO63517.1.
  • HBsAg of human HBV and “Hu-HBsAg” refer to the surface antigen major protein of human hepatitis B virus, which is well known to those skilled in the art (see, for example, NCBI GENBANK database login) No.: AAF24729.1).
  • amino acid residues 113-135 of the HBsAg protein refers to amino acid residues 113 to 135 of the polypeptide represented by SEQ ID NO: 44.
  • mutations or mutations including but not limited to, substitutions, deletions and/or additions, such as HBsAg proteins of different genotypes or subtypes may be naturally occurring or artificially introduced in the amino acid sequence of the HBsAg protein.
  • the term "HBsAg protein” shall include all such sequences, including, for example, the sequence set forth in SEQ ID NO: 44, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the HBsAg protein, it includes not only the sequence fragment of SEQ ID NO: 44 but also the corresponding sequence fragment in its natural or artificial variant. For example, the expression “amino acid residues 113-135 of the HBsAg protein” includes the amino acid residues 113-135 of SEQ ID NO: 44, and the corresponding fragments thereof (natural or artificial).
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • the expression "the deletion of Y amino acid residues at the C-terminus of the X protein” means that the last Y amino acid residues at the C-terminus of the X protein are all removed.
  • the expression "the C-terminal deletion of 1 to 40 amino acid residues of the RBHBcAg protein” means that the last 1-40 amino acid residues at the C-terminus of the RBHBcAg protein are all removed.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • Align program DNAstar, Inc.
  • the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blos sum 62 matrix.
  • the PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the same percentage between the two amino acid sequences Sex.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include the replacement of amino acid residues with amino acid residues with similar side chains Substituent substitutions, for example, using residues that are physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.) Replacement.
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • hybridization refers to the anneal of two single-stranded nucleic acid molecules having complementary sequences to each other under conditions (suitable temperature and ionic strength, etc.) to form double-stranded nucleic acids on a base-pairing basis. the process of. Nucleic acid hybridization can be carried out between DNA-DNA, or between DNA-RNA or RNA-RNA, as long as there is a complementary sequence between them, base pairing can be performed.
  • nucleic acid hybridization see, for example, Henegariu O et al, (1999).
  • stringent conditions or high stringency conditions are usually used.
  • Stringent conditions and high stringency conditions are well known in the field of molecular biology.
  • stringent conditions may mean that hybridization is carried out in 6X sodium chloride/sodium citrate (SSC) at about 45 ° C, followed by 0.2-SSC/0.1% SDS at about 50-65 ° C. Wash one or more times.
  • High stringency conditions may mean that hybridization is carried out in 6 x SSC at about 45 °C followed by one or more washes in 0.1 x SSC/0.2% SDS at about 68 °C.
  • linker refers to a short peptide used to join two molecules, such as a protein.
  • linkers are well known to those skilled in the art and include, but are not limited to, flexible linkers such as (Gly) 4 , (Gly) 4 -Ser, ((Gly) 4 -Ser) 3 and the like.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • restriction site refers to a restriction site recognized by a restriction endonuclease.
  • restriction sites are known to those skilled in the art and include, but are not limited to, restriction enzymes such as EcoR I, BamH I, Hind II, Hind III, Hpa I, Hpa II, Mbo I, Mbo II and the like. Identification of the cleavage site.
  • epitope and epitopope refer to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • epitope of HBsAg refers to a site present in HBsAg that is capable of being specifically bound by an immunoglobulin or antibody.
  • the structure and function of human HBV HBsAg have been studied in depth.
  • many literatures have reported epitopes on HBsAg of human HBV. See, for example, WO 97/39029 A2; WO 85/04103 A1; Xiaoxing Qiu et al, The Journal of Immunology, 1996, Vol. 156, pp. 3350-3356; WO 2013/185558A1 and the like.
  • epitope peptide refers to a peptide segment on an antigen capable of forming an epitope/used as an epitope.
  • a single epitope peptide is capable of being specifically recognized/bound by an antibody directed against the epitope.
  • the epitope contained in the epitope peptide may be a linear epitope or a conformational epitope.
  • the epitope peptide comprises a linear epitope, it may comprise or be a contiguous stretch of amino acids (ie, peptide fragments) that constitute the epitope in the antigen.
  • the epitope peptide comprises a conformational epitope, it may comprise or be a contiguous stretch of amino acids (ie, peptide fragments) in the antigen spanning all of the amino acid residues involved in the conformational epitope.
  • the epitope peptide preferably has a length of no more than 500 amino acid residues, such as a length of no more than 400 amino acid residues, a length of no more than 300 amino acid residues, no more than 200 The length of an amino acid residue, not longer than 100 amino acid residues, not longer than 90 amino acid residues, not longer than 80 amino acid residues, not exceeding 70 amino acid residues, not exceeding 60 The length of the amino acid residue, the length of no more than 50 amino acid residues, the length of no more than 40 amino acid residues, the length of no more than 30 amino acid residues, or the length of no more than 25 amino acid residues.
  • polypeptide carrier refers to a carrier protein that can serve as a carrier for an epitope peptide, ie, it can be inserted into a epitope at a specific position (eg, inside the protein, N-terminus or C-terminus)
  • the peptide is such that the epitope peptide can be presented such that the epitope peptide can be recognized by the antibody or immune system.
  • carrier proteins including For example, HPV L1 protein (the epitope peptide can be inserted between amino acids 130-131 of the protein or between amino acids 426-427, see Slippitzky, K. et al.
  • the present invention provides a new class of polypeptide vectors for use in displaying polypeptides of interest, and is particularly suitable for use in displaying epitopes containing human hepatitis B virus (eg, epitopes in HBsAg from human HBV).
  • An epitope peptide e.g., epitopes in HBsAg from human HBV.
  • An epitope peptide e.g., epitopes in HBsAg from human HBV.
  • a linker e.g., a flexible or rigid linker
  • recombinant protein merely means that the described protein is not naturally occurring and is not intended to define the manner in which the protein is produced/obtained.
  • the recombinant protein of the present invention can be produced by any known method including, but not limited to, genetic engineering methods and synthetic methods.
  • virus-like particle refers to a hollow particle composed of one or more structural proteins of a virus that does not contain viral nucleic acid and is not capable of autonomous replication, but is morphologically and structurally The real virions are the same or similar.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers. .
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator when When it is delivered to the body together with the antigen, it can enhance the body's immune response to the antigen or alter the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector in which E. coli (strain) is derived from commercially available strains, for example but not Limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the term "subject" refers to a mammal, such as a primate mammal, such as a human.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, HBV infection or a disease associated with HBV infection
  • an amount sufficient to prevent, prevent, or delay the onset of a disease eg, HBV infection or a disease associated with HBV infection
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • the present invention provides a novel polypeptide vector which has broad versatility and can be used for efficiently displaying various polypeptides of interest (for example, antigen epitope/antigenic peptide) and inducing the host immune system to produce a purpose. Specific immune response of the polypeptide.
  • polypeptides of interest for example, antigen epitope/antigenic peptide
  • polypeptides of interest include, but are not limited to, epitopes/antigenic peptides derived from HIV (eg, epitopes/antigenic peptides of HIV-derived GP120 proteins; For example, a polypeptide comprising amino acids 361 to 375 of the GP120 protein) derived from an epitope/antigenic peptide of a human PD-L1 protein (for example, a polypeptide comprising amino acids 147 to 160 of the human PD-L1 protein), and An epitope/antigenic peptide derived from human HBV (for example, an epitope/antigenic peptide of a HBsAg protein derived from human HBV; for example, a polypeptide comprising amino acids 113-135 of the HBsAg protein).
  • HIV epitopes/antigenic peptides of HIV-derived GP120 proteins
  • a polypeptide comprising amino acids 361 to 375 of the GP120 protein derived from an epitope/antigenic
  • the polypeptide vector of the present invention is particularly suitable for displaying an epitope derived from human hepatitis B virus (e.g., an epitope in HBsAg derived from human HBV), and is capable of inducing a particularly strong specific immunity against HBsAg in a subject.
  • the response is significantly superior to existing hepatitis B vaccines (eg, vaccines containing the same epitope constructed with HBcAg of human HBV as a polypeptide vector).
  • Figure 1 is a schematic diagram showing a cloning scheme for inserting a polypeptide of interest (target antigen peptide) into the RBHBcAg vector, TBHBcAg vector and HBHBcAg vector of the present invention to construct a recombinant protein.
  • a polypeptide of interest target antigen peptide
  • Figure 2 shows the results of SDS-PAGE of the 18 recombinant proteins constructed in Example 2, and the transmission electron microscopic observation results of the virus-like particles formed from the recombinant protein.
  • Figure 3 is a graph showing the change in antibody titer against the target polypeptide in the recombinant mouse against serum in the serum of BALB/C mice after immunization of BALB/C mice with the virus-like particles formed by the 18 recombinant proteins constructed in Example 2.
  • Vertical axis antibody titer (log 10); horizontal axis: time (week).
  • Figure 3A The polypeptide of interest used is SEQ ID NO: 20, and the titer of the anti-GP120 antibody is detected;
  • Figure 3B the polypeptide of interest used is SEQ ID NO: 21, and the titer of the anti-PD-L1 antibody is detected;
  • Figure 3C The polypeptide of interest used is SEQ ID NO: 22 and the titer of the anti-HBsAg antibody is detected.
  • Figure 4 shows the level of HBsAg in serum of mice after treatment of HBV transgenic male (Figure 4A) and female ( Figure 4B) mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22). The change.
  • Vertical axis level of HBsAg (IU/ml); horizontal axis: time (week). Arrows indicate the time point at which the mice were administered virus-like particles.
  • Figure 5 shows the level of HBV DNA in mouse serum as a function of time after treatment of HBV transgenic male mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22).
  • Vertical axis level of HBV DNA (Log10 IU/ml); horizontal axis: time (week). Arrows indicate the time point at which the mice were administered virus-like particles.
  • Figure 6 shows the dilution of anti-HBsAg antibodies in mouse serum after treatment of HBV transgenic male ( Figure 6A) and female ( Figure 6B) mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22). The degree of change over time.
  • Vertical axis titer of anti-HBsAg antibody; horizontal axis: time (week).
  • Fig. 7 shows the results of transmission electron microscopic observation of virus-like particles formed from the six recombinant proteins constructed in Example 5.
  • Figure 8 shows antibody titers against the corresponding target polypeptide (SEQ ID NO: 60, 22, 61, 62) in mouse sera after immunization of BALB/C mice with virus-like particles of 8 recombinant proteins for 3 weeks.
  • the epitope peptide of HBsAg protein from HBV genotype A (SEQ ID NO: 60) was used to detect antibody titers of mouse sera immunized with RBHBcAg149-SEQ60 and TBHBcAg153-SEQ60; with HBV genotype B
  • the epitope peptide of HBsAg protein (SEQ ID NO: SEQ22) to detect antibody titer of mouse sera immunized with RBHccAg149-SEQ22 and TBHBcAg153-SEQ22
  • epitope peptide with HBsAg protein from HBV genotype C (SEQ ID NO : 61) to detect antibody titers of mouse sera immunized with RBHBcA
  • Figure 9 shows that treatment of HBV transgenic males (Figure 9A) and female ( Figure 9B) mice with virus-like particles formed from four recombinant proteins (SEQ ID NO: 36, 69, 70, 71) in mouse serum
  • the level of HBsAg changes with time, wherein the vertical axis: the level of HBsAg (IU/ml); the horizontal axis: time (week).
  • Table 1 Sequence information of SEQ ID NO: 1-44
  • the RBHBcAg189 vector differs from the RBHBcAg protein (SEQ ID NO: 1) in that the amino acid residues 78-81 of the RBHBcAg protein are replaced with the linker shown in SEQ ID NO: 43; the amino acid sequence of the RBHBcAg189 vector is as SEQ ID NO: 4, the nucleotide sequence is shown in SEQ ID NO: 12;
  • TBHBcAg188 vector differs from TBHBcAg protein (SEQ ID NO: 2) in that amino acid residues 80-83 of RBHBcAg protein are replaced by SEQ ID NO: 43 The linker shown; the amino acid sequence of the TBHBcAg188 vector is shown in SEQ ID NO: 6, and the nucleotide sequence is shown in SEQ ID NO: 14.
  • the HBHBcAg189 vector differs from the HBHBcAg protein (SEQ ID NO: 3) in that the amino acid residues 78-81 of the RBHBcAg protein are replaced with a linker as shown in SEQ ID NO: 43; the amino acid sequence of the HBHBcAg189 vector is as SEQ ID NO: 8, the nucleotide sequence is shown in SEQ ID NO: 16.
  • the HBcAg183 vector was designed and used as a control.
  • the HBcAg183 vector differs from the HBcAg protein of human HBV in that the amino acid residues 79-81 of the HBcAg protein of human HBV are replaced with a linker as shown in SEQ ID NO: 43; the amino acid sequence of the HBcAg183 vector is SEQ ID NO: As shown in FIG. 10, the nucleotide sequence is shown in SEQ ID NO: 18.
  • nucleotide sequences of the above four vectors were all entrusted to Biotech Engineering Shanghai (Stock) Co., Ltd. for whole gene synthesis.
  • the full-length genes of the above four vectors and the truncated bodies thereof were amplified by PCR using the primers in Table 2.
  • a total of 8 PCR products were obtained, namely: the gene encoding RBHBcAg189 vector (SEQ ID NO: 12; the encoded amino acid sequence is SEQ ID NO: 4), the gene encoding RBHBcAg149 vector (SEQ ID NO: 13; The amino acid sequence is SEQ ID NO: 5), the gene encoding the TBHBcAg188 vector (SEQ ID NO: 14; the encoded amino acid sequence is SEQ ID NO: 6), the gene encoding the TBHBcAg153 vector (SEQ ID NO: 15; The amino acid sequence is SEQ ID NO: 7), the gene encoding the HBHBcAg189 vector (SEQ ID NO: 16; the encoded amino acid sequence is SEQ ID NO: 8), the gene encoding the HBHBcAg149 vector (SEQ ID NO: 17; The amino acid sequence is SEQ ID NO: 9), the gene encoding the HBcAg183 vector (SEQ ID NO: 18; the encoded amino acid sequence is SEQ ID NO:
  • the pTO-T7 vector was used with NdeI and HindIII (Luo Wenxin, Zhang Jun, Yang Haijie, et al. Construction and preliminary application of a prokaryotic expression vector with enhancer [J]. Chinese Journal of Biotechnology, 2000, 16(5): 578-581) Double enzyme digestion to obtain a linearized vector.
  • the obtained 8 PCR products were ligated with a linearized vector and transformed into DH5a competent bacteria using the Gibson assembly cloning method (New England Biolabs (UK) Ltd). The transformed bacteria were plated, then monoclonal colonies were picked, plasmids were extracted and sequenced. As verified by sequencing, 8 plasmids containing the nucleotide sequence encoding the polypeptide vector were obtained.
  • the three polypeptides of interest are: the polypeptide HIV-GP120-aa361-375 (ie, amino acids 361-375 derived from the HIV GP120 protein, the amino acid sequence of which is shown in SEQ ID NO: 20); the polypeptide hPDL1-aa147-160 (ie, amino acids 147-160 derived from human PD-L1 protein, the amino acid sequence of which is shown in SEQ ID NO: 21); polypeptide HBsAg-aa113-135 (ie, derived from human HBV surface antigen major protein (HBsAg) Amino acids 113-135, the amino acid sequence of which is set forth in SEQ ID NO:22).
  • the polypeptide HIV-GP120-aa361-375 ie, amino acids 361-375 derived from the HIV GP120 protein, the amino acid sequence of which is shown in SEQ ID NO: 20
  • the polypeptide hPDL1-aa147-160 ie, amino acids 147-160 derived from human PD
  • the sense and antisense coding sequences of the three polypeptides of interest (as shown in Table 3) were directly synthesized and annealed to obtain a gene fragment encoding the polypeptide of interest having a sticky end.
  • the six plasmids (RBHBcAg189, RBHBcAg149, TBHBcAg188, TBHBcAg153, HBHBcAg189, and HBHBcAg149) obtained in Example 1 were digested with BamHI and EcoRI, respectively, to obtain six linearized vectors.
  • the 18 expression plasmids constructed in the previous step were used to express and purify the recombinant protein encoded by the expression plasmid by the same method.
  • the expression and purification process of these recombinant proteins is exemplified below by taking RBHBcAg149-SEQX (SEQX, SEQ20, SEQ21 or SEQ22) as an example.
  • Figure 2 shows the results of SDS-PAGE of the 18 recombinant proteins constructed, and the transmission electron microscopic observation results of the virus-like particles formed from the recombinant protein.
  • the results showed that the 18 recombinant proteins obtained were all more than 85% pure and were able to assemble into virus-like particles of about 30 nm in diameter.
  • virus-like particles formed from the recombinant proteins of the present invention. These virus-like particles are capable of inducing the body to produce antibodies that specifically bind to the target antigen.
  • the immunization protocol is as follows: the immune adjuvant is aluminum hydroxide adjuvant; the immunization dose is 3 ug/dose; the immunization method is intramuscular injection of the lateral thigh of the hind limb; the immunization procedure is booster immunization after the first immunization + 2 weeks, and the total immunization is performed twice.
  • the coated antigen of the reaction plate is a target antigen corresponding to the above three polypeptides of interest, namely: gp120 protein of HIV-1 (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number 11233-V08H), human PD- L1 protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), human hepatitis B virus surface antigen main protein (HBsAg, purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) recombinantly expressed by CHO cells.
  • gp120 protein of HIV-1 purchasedd from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., catalog number 11233-V08H
  • human PD- L1 protein purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • human hepatitis B virus surface antigen main protein HBsAg, purchased from Beijing Wantai Biopharmaceutical Co., Ltd.
  • the three recombinant proteins were diluted with 50 mM CB buffer (NaHCO 3 /Na 2 CO 3 buffer, final concentration 50 mM, pH 9.6) at pH 9.6 to a final concentration of 2 ⁇ g/mL to obtain a coating solution. . 100 ⁇ L of the coating solution was added to each well of a 96-well microtiter plate, and coated at 2-8 ° C for 16-24 hours, and then coated at 37 ° C for 2 hours.
  • 50 mM CB buffer NaHCO 3 /Na 2 CO 3 buffer, final concentration 50 mM, pH 9.6
  • 100 ⁇ L of the coating solution was added to each well of a 96-well microtiter plate, and coated at 2-8 ° C for 16-24 hours, and then coated at 37 ° C for 2 hours.
  • PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20); then 200 ⁇ L of blocking solution (20% Na 2 HPO 4 containing 20% calf serum and 1% casein) was added to each well. /NaH 2 PO 4 buffer solution, pH 7.4), and blocked at 37 ° C for 2 hours. Discard the blocking solution.
  • the plate was then dried and placed in an aluminum foil pouch and stored at 2-8 ° C until use.
  • Sample dilution The mouse serum was diluted to 1:100, 1:500, 1:2500, 1:12500, 1:62500, 1:312500, 1:1562500 with 7 dilutions in PBS containing 20% newborn calf serum. gradient.
  • ELISA assay 100 ⁇ L of the diluted serum sample was added to each well of the coated plate and incubated at 37 ° C for 30 minutes. The plate was then washed 5 times with PBST wash (20 mM PB 7.4, 150 mM NaCl, 0.1% Tween 20). After washing, 100 ⁇ L of GAM-HRP reaction solution was added to each well of the plate, and incubated at 37 ° C for 30 minutes. The plate was then washed 5 times with PBST wash (20 mM PB 7.4, 150 mM NaCl, 0.1% Tween 20).
  • TMB color developer supplied by Beijing Wantai Biopharmaceutical Co., Ltd.
  • stop solution provided by Beijing Wantai Biopharmaceutical Co., Ltd.
  • Calculation of antibody titer take a sample well with a reading value between 0.2 and 2.0, perform a regression curve on the dilution factor and the reading value of the sample, and calculate a dilution factor of the sample when the reading value is equal to 2 times the background value, and The sample dilution factor was used as the titer of the specific antibody in the serum.
  • Figure 3 shows the antibody titer against target antigen in mouse serum after immunization of BALB/C mice with virus-like particles formed by 18 recombinant proteins.
  • Figure 3A The polypeptide of interest used is SEQ ID NO: 20, and the titer of the anti-GP120 antibody is detected;
  • Figure 3B the polypeptide of interest used is SEQ ID NO: 21, and the titer of the anti-PD-L1 antibody is detected;
  • Figure 3C The polypeptide of interest used is SEQ ID NO: 22 and the titer of the anti-HBsAg antibody is detected.
  • the results showed that the virus-like particles formed by the 18 recombinant proteins had good immunogenicity and were able to induce high titers of antibodies that specifically bind to the target antigen.
  • Example 4 Evaluation of anti-HBV therapeutic effect of virus-like particles displaying HBsAg epitope (SEQ ID NO: 22)
  • HBcAg183-SEQ22 the amino acid sequence of which is set forth in SEQ ID NO: 41; and HBcAg149-SEQ22, the amino acid sequence of which is set forth in SEQ ID NO: 42 Show
  • virus-like particles formed from these two recombinant proteins ie, HBcAg183-SEQ22, the amino acid sequence of which is set forth in SEQ ID NO: 41; and HBcAg149-SEQ22, the amino acid sequence of which is set forth in SEQ ID NO: 42 Show
  • the HBV transgenic mouse model was used to evaluate the anti-HBV therapeutic effects of the five virus-like particles displaying the HBsAg epitope (SEQ ID NO: 22) prepared in Example 2 and the two virus-like particles prepared in the present example.
  • the immunization protocol is as follows: the immunological adjuvant is aluminum hydroxide adjuvant; the immunization dose is 12 ⁇ g/dose; the immunization method is intramuscular injection of the lateral thigh of the hind limb; the immunization procedure is immunization once every 0, 2, 3, 4, 5, and 6 weeks. A total of 6 immunizations.
  • the detection of the titer of Ant i-HBsAg in serum was performed as described in Example 3.2, and the virological indicators in the serum of the mice, i.e., the levels of HBV DNA and HBsAg, were measured.
  • Figure 4 shows the level of HBsAg in serum of mice after treatment of HBV transgenic male ( Figure 4A) and female ( Figure 4B) mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22).
  • Figure 5 shows the level of HBV DNA in mouse serum as a function of time after treatment of HBV transgenic male mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22).
  • Figure 6 shows the dilution of anti-HBsAg antibodies in mouse serum after treatment of HBV transgenic male (Figure 6A) and female ( Figure 6B) mice with different virus-like particles displaying the same epitope peptide (SEQ ID NO: 22). The degree of change over time.
  • the six polypeptide vectors constructed based on the bat hepatitis B virus core protein can be used to efficiently display epitope peptides of HBsAg from human HBV (for example, HBsAg-aa113-135), which is capable of forming VLPs and inducing the body to produce high titers of ant i-HBsAg antibodies in the host, thereby inhibiting the levels of HBV DNA and HBsAg in mice (ie, HBV DNA and HBsAg) Decreased significantly).
  • virus-like particles based on the polypeptide carriers of the present invention e.g., RBHBcAg149 and TBHBcAg153
  • virus-like particles constructed based on human HBV-based HBcAg. have a particularly significant anti-HBV therapeutic effect, which is superior to virus-like particles constructed based on human HBV-based HBcAg. .
  • the polypeptide carrier of the present invention is capable of forming a VLP, is suitable for displaying various polypeptides of interest, and is capable of inducing the body to produce high titers of antibodies against the polypeptide of interest;
  • the present invention The polypeptide vector is particularly suitable for displaying an epitope of human HBV (for example, an epitope of HBsAg of human HBV), which is capable of inducing the body to produce high titers of antibodies against HBsAg, and to eliminate or inhibit the levels of HBV DNA and HBsAg in vivo, And its effect is superior to the polypeptide carrier constructed based on human HBV-based HBcAg.
  • the recombinant proteins of the present invention displaying epitopes of human HBV have the potential to treat HBV infection and are particularly suitable for inducing effective, specific, therapeutic anti-HBV immunity.
  • Example 5 Preparation and evaluation of virus-like particles displaying HBsAg epitopes of HBV from different genotypes
  • the HBsAg epitope (SEQ ID NO: 22) used in Examples 2-4 was derived from HBV genotype B.
  • HBV genotype B The HBsAg epitope (SEQ ID NO: 22) used in Examples 2-4 was derived from HBV genotype B.
  • RBHBcAg149 and TBHBcAg153 were constructed from different genotypes (genotypes A, C, and D) using RBHBcAg149 and TBHBcAg153 as exemplary polypeptide vectors.
  • Recombinant proteins of the HBsAg epitope and evaluated the ability of the constructed recombinant protein to assemble into virus-like particles, as well as the immunogenicity of the resulting virus-like particles and the therapeutic effect of anti-HBV infection.
  • the polypeptide of interest in addition to the HBsAg epitope (from HBV genotype B, SEQ ID NO: 22) used in Examples 2-4, the polypeptide of interest also includes HBsAg from HBV genotypes A, C and D.
  • Protein epitopes amino acids 113-135), which are named: HBsAg-aa113-135-A, HBsAg-aa113-135-C and HBsAg-aa113-135-D, respectively, and their sequences (SEQ ID NO: 60-62) is shown in Table 4.
  • Table 4 Sequence of amino acids 113-135 of the HBsAg protein from HBV genotypes A, C and D
  • the sense and antisense coding sequences of the three polypeptides of interest (as shown in Table 5) were directly synthesized and annealed to obtain a gene fragment encoding the polypeptide of interest having a sticky end.
  • Table 5 Justice and antisense coding sequences for three polypeptides of interest
  • the three gene fragments encoding the polypeptide of interest prepared as described above were ligated into the linearized vectors RBHBcAg149 and TBHBcAg153, respectively, as described in Example 2, thereby obtaining an expression plasmid encoding the recombinant protein (6 kinds: RBHBcAg149) - SEQ60, RBHBcAg149-SEQ61, RBHBcAg149-SEQ62, TBHBcAg153-SEQ60, TBHBcAg153-SEQ61, TBHBcAg153-SEQ62).
  • the amino acid sequences of the recombinant proteins encoded by these expression plasmids are shown in Table 6.
  • the recombinant protein encoded by the expression plasmid was expressed and purified using the six expression plasmids constructed in the previous step as described in Example 2. Subsequently, the state of the VLP formed by these recombinant proteins was observed using a transmission electron microscope.
  • Figure 7 shows the results of transmission electron microscopy of virus-like particles formed from the six recombinant proteins constructed. The results showed that the six recombinant proteins obtained were able to assemble into virus-like particles of about 30 nm in diameter. These results indicate that the polypeptide vector constructed by the present invention has broad versatility, can be used to display epitope peptides of HBV from various genotypes (for example, aa113-135 of HBsAg protein), and is capable of forming a good VLP.
  • Figure 8 shows that BALB/C mice were immunized with virus-like particles formed from 8 recombinant proteins.
  • antibody titers against the corresponding target polypeptide SEQ ID NO: 60, 22, 61, 62
  • SEQ ID NO: 60 the epitope peptide of HBsAg protein from HBV genotype A
  • SEQ ID NO: 60 To detect antibody titer of mouse sera immunized with RBHBcAg149-SEQ60 and TBHBcAg153-SEQ60; use epitope peptide (SEQ ID NO: SEQ22) of HBsAg protein from HBV genotype B to detect RBHBcAg149-SEQ22 and TBHBcAg153
  • the anti-HBV therapeutic effect of the virus-like particles formed by the four recombinant proteins was evaluated using the method described in Example 4. The experimental results are shown in Fig. 9.
  • Figure 9 shows virus-like particles formed using the four recombinant proteins (RBHBcAg149-SEQ22, RBHBcAg149-SEQ60, RBHBcAg149-SEQ61, RBHBcAg149-SEQ62; sequences SEQ ID NO: 36, 69, 70, 71, respectively) constructed as above.
  • the results showed that in the mice immunized with VLP, the level of HBsAg in the serum of the mice showed a significant decrease after immunization.
  • polypeptide vectors of the present invention e.g., RBHBcAg149 and TBHBcAg153
  • RBHBcAg149 and TBHBcAg153 can be used to efficiently display epitope peptides of HBsAg of human HBV from different genotypes (e.g., genotypes A, B, C, and D) (e.g., HBsAg- Aa113-135).
  • the recombinant protein constructed based on the polypeptide carrier of the present invention and the epitope peptide of HBsAg is capable of forming a VLP, and is capable of inducing a body to produce a high titer of anti-HBsAg antibody in the host, and thereby The level of HBsAg in mice was inhibited (i.e., the level of HBsAg was significantly decreased).
  • a recombinant protein containing the polypeptide carrier of the present invention and an epitope peptide of HBsAg can be used for the prevention and treatment of infection of various genotypes of HBV, and thus can be used for development of new anti-HBV vaccines and drugs.

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Abstract

提供一种核酸分子，其包含编码多肽载体的核苷酸序列。还提供包含该多肽载体和目的多肽的重组蛋白以及该核酸分子和该重组蛋白的用途。还提供可用于预防、减轻或治疗HBV感染或与HBV感染相关的疾病（例如乙肝）的疫苗或药物组合物，其包含该多肽载体和来自HBV表位的重组蛋白。

Description

一种用于展示目的多肽的多肽载体及其用途 技术领域
本发明涉及基因工程疫苗领域、分子病毒学领域和免疫学领域。此外，本发明还特别涉及乙型肝炎病毒(Hepatitis B virus,HBV)感染治疗领域。具体而言，本发明涉及一种核酸分子，其包含编码多肽载体(peptide carrier)的核苷酸序列，且用于插入编码目的多肽的核苷酸序列。特别地，本发明的所述核酸分子在插入编码目的多肽的核苷酸序列且表达为重组蛋白后，所述多肽载体能够展示所述目的多肽(例如目的抗原或抗原中的目的表位)，和/或，所述重组蛋白能够形成病毒样颗粒并展示所述目的多肽。此外，本发明还涉及包含所述多肽载体以及目的多肽的重组蛋白。进一步，本发明还涉及，所述核酸分子和所述重组蛋白的用途。此外，本发明还特别涉及，可用于预防、减轻或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)的疫苗或药物组合物，其包含含有本发明的多肽载体和来自HBV的表位的重组蛋白。
背景技术
疫苗是应对传染病的有效手段。根据适用人群的不同，疫苗可分为预防性疫苗和治疗性疫苗。预防性疫苗主要应用于预防病毒感染，包括减毒疫苗、灭活疫苗、基因工程疫苗，其主要通过诱导机体产生中和抗体来保护机体不被病毒感染。治疗性疫苗主要应用于治疗持续性病毒感染以及肿瘤等疾病。在这些疾病中，患者对于靶抗原通常表现为免疫耐受的状态，因此，研发人员尝试通过多种形式的疫苗来诱导机体产生针对靶抗原的有效免疫应答。治疗性疫苗主要包括核酸疫苗、病毒载体疫苗、基因工程疫苗等，其中基因工程疫苗具有明显的优势。
已上市的基因工程疫苗包括乙型肝炎病毒疫苗、人乳头瘤病毒(Human Papillomavirus，HPV)疫苗、戊型肝炎病毒(Hepatitis E virus，HEV)疫苗，它们均为病毒样颗粒(virus-like particles,VLPs)的形式。病毒样颗粒是指，由某种病毒的一个或多个结构蛋白构成的空心颗粒，其不包含病毒核酸，不能进行自主复制，但在形态上与真正的病毒粒子相同或相似。病毒样颗粒具有以下优点：免疫原性强、安全性好、不易失活、可展示外源肽段并诱导机体产生针对外源肽段的特异性免疫应答，因此，在疫苗领域具有重要的应用价值。
目前，全球约有20亿人感染过HBV，其中约有3.5亿的慢性HBV感染者，这些感染者最终死于HBV感染相关肝脏疾病的风险可达15％-25％。全球每年超过100万人死于乙肝导致的终末期肝病。我国是HBV感染的重灾区，目前约有9300万乙肝携带者。近年来，随着病例报告系统不断健全，乙肝相关疾病的发生率，病死率不降反升。
当前针对慢性HBV感染的治疗药物主要可分为两类，即干扰素类(Interferon)和核苷/核苷酸类似物(NAs)。慢性HBV感染的治疗的最终目标是，阻止重症肝炎(肝衰竭)、肝硬化和肝癌等终末期肝脏疾病的发生；其临床最佳治疗终点是，使患者达到乙型肝炎病毒表面抗原的血清学阴转或血清学转换，即完全清除HBV。但现有药物实现该目标的疗效十分有限。因此，针对慢性HBV感染者开发新的、能更有效地清除病毒、尤其是能有效清除HBsAg或大幅降低HBsAg水平的创新型治疗药物和方法是迫切而必要的，并且，研发治疗性疫苗是具有一定潜力的策略。
发明内容
本申请发明人基于三种蝙蝠来源的乙型肝炎病毒核心抗原蛋白(即，蹄蝠乙型肝炎病毒(roundleaf bat HBV，RBHBV)的核心抗原蛋白(RBHBcAg)；筑帐蝠乙型肝炎病毒(tent-making bat HBV，TBHBV)的核心抗原蛋白(TBHBcAg)；菊头蝠乙型肝炎病毒(horseshoe bat HBV，HBHBV)的核心抗原蛋白(HBHBcAg))，开发了一类新的用于展示目的多肽的多肽载体(peptide carrier)。由此，本发明提供了一种核酸分子，其含有编码多肽载体的核苷酸序列。此类核酸分子能够插入编码目的 多肽的核苷酸序列，并表达为含有多肽载体和目的多肽(例如目的抗原，或抗原中的目的表位)的重组蛋白。进一步，所产生的重组蛋白能够有效形成VLP，并能够在VLP表面展示插入其中的目的多肽，从而所述目的多肽能够有效地被机体的免疫系统识别，诱发机体产生针对目的多肽的特异性免疫应答。因此，本发明的多肽载体可用作疫苗载体，用于展示目的多肽，例如来自目的抗原的肽段(例如表位)；并且，包含本发明的多肽载体和目的多肽的重组蛋白可用作疫苗，用于诱发机体产生针对目的多肽的特异性免疫应答。
此外，本申请的发明人还出人意料地发现，RBHBcAg蛋白、TBHBcAg蛋白和HBHBcAg蛋白的C端均为富含精氨酸的区域，其对于VLP组装不是必须的。因此，本发明的多肽载体可以不包含RBHBcAg蛋白、TBHBcAg蛋白和HBHBcAg蛋白的C端区域。例如，本发明的RBHBcAg载体可以缺失RBHBcAg蛋白的第145-189位氨基酸的部分或全部(例如，缺失RBHBcAg蛋白的第150-189位氨基酸)；本发明的TBHBcAg载体可以缺失TBHBcAg蛋白的第149-188位氨基酸的部分或全部(例如，缺失TBHBcAg蛋白的第154-188位氨基酸)；本发明的HBHBcAg载体可以缺失HBHBcAg蛋白的第145-189位氨基酸的部分或全部(例如，缺失HBHBcAg蛋白的第150-189位氨基酸)。
此外，本申请的发明人还出人意料地发现，本发明的多肽载体特别适合用于展示来自人乙肝病毒的抗原表位(例如来自人HBV的HBsAg中的表位)，能够在受试者中诱发特别强的清除HBsAg的特异性免疫应答，其功效显著优于现有的乙肝疫苗(例如，以人HBV的HBcAg为多肽载体构建的、含有相同表位的疫苗)。因此，本发明还提供了一种特别适合用于展示来自人乙肝病毒的抗原表位的多肽载体。
因此，在一个方面，本发明提供了一种核酸分子，其包含编码多肽载体的核苷酸序列或者其变体，所述变体与所述核苷酸序列具有至少90％(例如，至少91％、92％、93％、94％、95％、96％、97％、98％、或99％)的同一性，或者在严紧条件或者高严紧条件下，能够与所述核苷 酸序列杂交，其中，所述多肽载体选自：
(1)RBHBcAg载体，其与蹄蝠乙型肝炎病毒的核心抗原蛋白(RBHBcAg；例如，其氨基酸序列如SEQ ID NO:1所示)的区别在于：(a)RBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个，5个或6个氨基酸残基；例如，第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、或第78-80位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，RBHBcAg蛋白的C端缺失1-40个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨基酸残基)；
(2)TBHBcAg载体，其与筑帐蝠乙型肝炎病毒的核心抗原蛋白(TBHBcAg；例如，其氨基酸序列如SEQ ID NO:2所示)的区别在于：(a)TBHBcAg蛋白的N端第80-84位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个或5个氨基酸残基；例如，第80-84位氨基酸残基、第80-83位氨基酸残基、或第80-82位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，TBHBcAg蛋白的C端缺失1-35个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，或30-35个氨基酸残基)；或
(3)HBHBcAg载体，其与菊头蝠乙型肝炎病毒的核心抗原蛋白(HBHBcAg；例如，其氨基酸序列如SEQ ID NO:3所示)的区别在于：(a)HBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个，5个或6个氨基酸残基；例如，第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、或第78-80位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，HBHBcAg蛋白的C端缺失1-40个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨 基酸残基)。
在一个优选的实施方案中，所述变体与所述编码多肽载体的核苷酸序列具有至少90％的同一性，例如，至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
在一个优选的实施方案中，所述变体能够在严紧条件下与所述编码多肽载体的核苷酸序列杂交。在一个优选的实施方案中，所述变体能够在高严紧条件下与所述编码多肽载体的核苷酸序列杂交。
在一个优选的实施方案中，RBHBcAg蛋白是野生型RBHBcAg。在一个优选的实施方案中，RBHBcAg蛋白的氨基酸序列如SEQ ID NO:1所示。
在一个优选的实施方案中，RBHBcAg载体与RBHBcAg蛋白的区别在于，RBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，5个或6个连续氨基酸残基)被缺失或被置换为连接体。例如，RBHBcAg蛋白的N端第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、第78-80位氨基酸残基、第78-79位氨基酸残基、第79-83位氨基酸残基、第79-82位氨基酸残基、第79-81位氨基酸残基、第79-80位氨基酸残基、第80-83位氨基酸残基、第80-82位氨基酸残基、第80-81位氨基酸残基、第81-83位氨基酸残基、第81-82位氨基酸残基、第82-83位氨基酸残基、第78位氨基酸残基、第79位氨基酸残基、第80位氨基酸残基、第81位氨基酸残基、第82位氨基酸残基、或第83位氨基酸残基可被缺失或被置换为连接体。在一个优选的实施方案中，所述连接体例如为柔性连接体。此类柔性连接体是本领域技术人员公知的，例如，GGGGGSGGGGTGSEFGGGGSGGGGS(SEQ ID NO:43)。
在一个优选的实施方案中，RBHBcAg载体与RBHBcAg蛋白的区别在于：(1)RBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，5个或6个连续氨基酸残基)被缺失或被置换为连接体，如上文所定义的；和(2)RBHBcAg 蛋白的C端缺失1-40个氨基酸残基。在一个优选的实施方案中，RBHBcAg蛋白的C端被缺失1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨基酸残基；例如，被缺失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、或40个氨基酸残基。
在一个优选的实施方案中，TBHBcAg蛋白是野生型TBHBcAg。在一个优选的实施方案中，TBHBcAg蛋白的氨基酸序列如SEQ ID NO:2所示。
在一个优选的实施方案中，TBHBcAg载体与TBHBcAg蛋白的区别在于，TBHBcAg蛋白的N端第80-84位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，或5个连续氨基酸残基)被缺失或被置换为连接体。例如，TBHBcAg蛋白的N端第80-84位氨基酸残基、第80-83位氨基酸残基、第80-82位氨基酸残基、第80-81位氨基酸残基、第81-84位氨基酸残基、第81-83位氨基酸残基、第81-82位氨基酸残基、第82-84位氨基酸残基、第82-83位氨基酸残基、第83-84位氨基酸残基、第80位氨基酸残基、第81位氨基酸残基、第82位氨基酸残基、第83位氨基酸残基、或第84位氨基酸残基可被缺失或被置换为连接体。在一个优选的实施方案中，所述连接体例如为柔性连接体。此类柔性连接体是本领域技术人员公知的，例如，GGGGGSGGGGTGSEFGGGGSGGGGS(SEQ ID NO:43)。
在一个优选的实施方案中，TBHBcAg载体与TBHBcAg蛋白的区别在于：(1)TBHBcAg蛋白的N端第80-84位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，或5个连续氨基酸残基)被缺失或被置换为连接体，如上文所定义的；和(2)TBHBcAg蛋白的C端缺失1-35个氨基酸残基。在一个优选的实施方案中，TBHBcAg蛋白的C端被缺失1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，或30-35个氨基酸残基；例如，被缺失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、或35个氨基酸残基。
在一个优选的实施方案中，HBHBcAg蛋白是野生型HBHBcAg。在一个优选的实施方案中，HBHBcAg蛋白的氨基酸序列如SEQ ID NO:3所示。
在一个优选的实施方案中，HBHBcAg载体与HBHBcAg蛋白的区别在于，HBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，5个或6个连续氨基酸残基)被缺失或被置换为连接体。例如，HBHBcAg蛋白的N端第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、第78-80位氨基酸残基、第78-79位氨基酸残基、第79-83位氨基酸残基、第79-82位氨基酸残基、第79-81位氨基酸残基、第79-80位氨基酸残基、第80-83位氨基酸残基、第80-82位氨基酸残基、第80-81位氨基酸残基、第81-83位氨基酸残基、第81-82位氨基酸残基、第82-83位氨基酸残基、第78位氨基酸残基、第79位氨基酸残基、第80位氨基酸残基、第81位氨基酸残基、第82位氨基酸残基、或第83位氨基酸残基可被缺失或被置换为连接体。在一个优选的实施方案中，所述连接体例如为柔性连接体。此类柔性连接体是本领域技术人员公知的，例如，GGGGGSGGGGTGSEFGGGGSGGGGS(SEQ ID NO:43)。
在一个优选的实施方案中，HBHBcAg载体与HBHBcAg蛋白的区别在于：(1)HBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个连续氨基酸残基(例如，1个，2个，3个，4个，5个或6个连续氨基酸残基)被缺失或被置换为连接体，如上文所定义的；和(2)HBHBcAg蛋白的C端缺失1-40个氨基酸残基。在一个优选的实施方案中，HBHBcAg蛋白的C端被缺失1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨基酸残基；例如，被缺失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、或40个氨基酸残基。
如本领域技术人员已知的，限制性酶切位点的引入是特别有利的。因此，在一个优选的实施方案中，在本发明的核酸分子中，在编码所述被缺失的一个或多个氨基酸残基的位置处引入限制性酶切位点。在一个优选的实施方案中，在本发明的核酸分子中，在编码所述连接体的核苷酸序列中和/或其一端或两端引入限制性酶切位点。在一个优选的实施方案中，在本发明的核酸分子中和/或其一端或两端引入一个或多个限制性酶切位点。各种限制性酶切位点是本领域技术人员已知的，其包括但不限于，EcoR I、BamH I、HindⅡ、HindⅢ，Hpa I、Hpa Ⅱ，Mbo I、Mbo Ⅱ等限制性内切酶识别的酶切位点。
在一个优选的实施方案中，所述多肽载体的氨基酸序列选自SEQ ID NO:4-9。
在一个优选的实施方案中，所述核酸分子包含选自SEQ ID NO:12-17的核苷酸序列。
在一个优选的实施方案中，所述核酸分子用于插入编码目的多肽的核苷酸序列。例如，所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端。在一个优选的实施方案中，以符合读框的方式将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。在一个优选的实施方案中，利用限制性酶切位点，将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。
在一个优选的实施方案中，所述核酸分子还包含，编码目的多肽的核苷酸序列，其中，所述目的多肽相对于所述多肽载体而言是异源的，并且所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端。在一个优选的实施方案中，以符合读框的方式将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。在一个优选的实施方案中，利用限制性酶切位点，将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。
在一个优选的实施方案中，所述目的多肽包含或者是抗原或者含 有抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽是表位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的第361-375位氨基酸。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白的第147-160位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62。在一个优选的实施方案中，所述核酸分子包含或者由编码选自SEQ ID NO:23-40和69-74的氨基酸序列的核苷酸 序列组成。
在另一个方面，本发明涉及，含有如上所定义的本发明核酸分子的载体(vector)。
可用于插入目的多核苷酸(例如，本发明的核酸分子)的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个优选的实施方案中，本发明的载体可以是真核表达载体或原核表达载体。在一个优选的实施方案中，本发明的载体是例如质粒，粘粒，或噬菌体等。
在另一个方面，本发明还涉及包含上述核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明涉及一种展示目的多肽的方法，其包括:
(1)将编码所述目的多肽的核苷酸序列插入至本发明的核酸分子中(特别是，插入至编码多肽载体的核苷酸序列中)，从而获得编码重组蛋白的核酸分子；和
(2)将步骤(1)中的编码重组蛋白的核酸分子表达为重组蛋白。
在一个优选的实施方案中，所述目的多肽相对于所述多肽载体而言是异源的。在一个优选的实施方案中，所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端。在一个优选的实施方案中，以符合读框的方式将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。在一个优选的实施方案中，利用限制性酶切位点，将编码目的多肽的核苷酸序列插入编码多肽载体的核苷酸序列中。
在一个优选的实施方案中，所述目的多肽包含或者是抗原或者含有抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽是表 位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的第361-375位氨基酸。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白的第147-160位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62。在一个优选的实施方案中，所述编码重组蛋白的核酸分子包含或者由编码选自SEQ ID NO:23-40和69-74的氨基酸序列的核苷酸序列组成。
在另一个方面，本发明涉及一种重组蛋白，其包含多肽载体和目的多肽，其中，所述多肽载体如上文所定义，并且，所述目的多肽被插入至所述多肽载体中。
在一个优选的实施方案中，所述目的多肽被插入至所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至所述连接体中或其一端或两端。
在一个优选的实施方案中，所述目的多肽包含或者是抗原或者含有抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽是表位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HIV、PDL1或HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的第361-375 位氨基酸。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白第147-160位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62。在一个优选的实施方案中，所述多肽载体的氨基酸序列选自SEQ ID NO:4-9。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:23-40和69-74的氨基酸序列组成。
在另一个方面，本发明涉及一种病毒样颗粒，其包含或者由本发明的重组蛋白组成。
在另一个方面，本发明涉及一种药物组合物(例如疫苗)，其包含本发明的重组蛋白或本发明的病毒样颗粒，和任选地，一种或多种药学上可接受的载体(vehicle)或赋形剂(例如佐剂)。在一个优选的实施方案中，本发明的重组蛋白或本发明的病毒样颗粒以有效量存在于药物组合物中。例如，本发明的药物组合物可包含有效预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)的量的重组蛋白或病毒样颗粒。
在另一个方面，本发明涉及一种预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)的方法，其包括，给有此需要的受试者施用本发明的重组蛋白或病毒样颗粒或药物组合物，其中所述目的多肽包含来自HBV(特别是人HBV)的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中， 所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
在一个优选的实施方案中，以有效预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)的量施用本发明的重组蛋白或病毒样颗粒或药物组合物。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)，其中所述目的多肽包含来自HBV(特别是人HBV)的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性 表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒或药物组合物，其用于预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)，其中所述目的多肽包含来自HBV(特别是人HBV)的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg或含有HBsAg的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述多肽载体为RBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为TBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。在一个优选的实施方案中，所述多肽载体为HBHBcAg载体，且所述表位肽包含或者是来自HBV(特别是人HBV)的抗原表位(例如，来自人HBV的HBsAg的表位(例如线性表位))。
在一个优选的实施方案中，所述目的多肽包含或者选自人HBV的HBsAg蛋白或含有HBsAg蛋白的表位(例如线性表位)的表位肽。在一 个优选的实施方案中，所述HBV选自HBV基因型A、B、C和D。在一个优选的实施方案中，所述HBsAg蛋白的表位为HBsAg蛋白的第113-135位氨基酸。
在一个优选的实施方案中，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
在另一个方面，本发明涉及一种预防或治疗HIV感染或与HIV感染相关的疾病(例如，AIDS)的方法，其包括，给有此需要的受试者施用本发明的重组蛋白或病毒样颗粒或药物组合物，其中所述目的多肽包含来自HIV的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HIV的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的第361-375位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:20所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:23-28的氨基酸序列组成。
在一个优选的实施方案中，以有效预防或治疗HIV感染或与HIV感染相关的疾病(例如，AIDS)的量施用本发明的重组蛋白或病毒样颗粒或药物组合物。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗HIV感染或与HIV感染相关的疾病(例如，AIDS)，其中所述目的多肽包含来自HIV的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HIV的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的 第361-375位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:20所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:23-28的氨基酸序列组成。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒或药物组合物，其用于预防或治疗HIV感染或与HIV感染相关的疾病(例如，AIDS)，其中所述目的多肽包含来自HIV的抗原表位。在一个优选的实施方案中，所述目的多肽是包含来自HIV的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自HIV的GP120蛋白或含有GP120蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是GP120蛋白的第361-375位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:20所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:23-28的氨基酸序列组成。
在另一个方面，本发明涉及一种预防或治疗癌症(例如，非小细胞肺癌)的方法，其包括，给有此需要的受试者施用本发明的重组蛋白或病毒样颗粒或药物组合物，其中所述目的多肽包含人PD-L1蛋白的抗原表位。在一个优选的实施方案中，所述目的多肽是包含人PD-L1蛋白的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白第147-160位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:21所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:29-34的氨基酸序列组成。
在一个优选的实施方案中，以有效预防或治疗癌症(例如，非小细胞肺癌)的量施用本发明的重组蛋白或病毒样颗粒或药物组合物。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗癌症(例如，非小细胞肺癌)，其中所述目的多肽包含人PD-L1蛋白的抗原表位。在一个优选的实施方案中，所述目的多肽是包含人PD-L1蛋白的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白第147-160位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:21所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:29-34的氨基酸序列组成。
在另一个方面，本发明涉及上述重组蛋白或病毒样颗粒或药物组合物，其用于预防或治疗癌症(例如，非小细胞肺癌)，其中所述目的多肽包含人PD-L1蛋白的抗原表位。在一个优选的实施方案中，所述目的多肽是包含人PD-L1蛋白的抗原表位的表位肽。在一个优选的实施方案中，所述目的多肽包含或者选自人PD-L1蛋白或含有人PD-L1蛋白的表位(例如线性表位)的表位肽。
在一个优选的实施方案中，所述表位肽包含或者是人PD-L1蛋白第147-160位氨基酸。在一个优选的实施方案中，所述目的多肽的氨基酸序列如SEQ ID NO:21所示。在一个优选的实施方案中，所述重组蛋白包含或者由选自SEQ ID NO:29-34的氨基酸序列组成。
在另一个方面，本发明涉及编码上述重组蛋白的多核苷酸以及含有该多核苷酸的载体。
可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体。在一个优选的实施方案中，本发明的载体可以是真核表达载体或原核表达载体。在一个优选的实施方案中，本发明的载体是例如质粒，粘粒，或噬菌体等。
在另一个方面，本发明还涉及包含上述多核苷酸或载体的宿主细 胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的宿主细胞还可以是细胞系，例如293T细胞。
在另一个方面，本发明还涉及制备上述重组蛋白的方法，其包括，在适合表达所述重组蛋白的条件下培养本发明的宿主细胞，和，回收所述重组蛋白。
本发明中相关术语的说明及解释
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“蹄蝠乙型肝炎病毒的核心抗原蛋白(RBHBcAg)”和“RBHBcAg蛋白”是指，来自蹄蝠乙型肝炎病毒(roundleaf bat HBV，RBHBV)的核心抗原蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：KC790373.1)。
如本文中所使用的，当提及RBHBcAg蛋白的氨基酸序列时，其使用SEQ ID NO：1所示的序列来进行描述。例如，表述“RBHBcAg蛋白的N端第78-83位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第78-83位氨基酸残基。然而，本领域技术人员理解，在RBHBcAg蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同基因型或基因亚型的RBHBcAg蛋白)，而不影响其生物学功能。因此，在本发明中，术语“RBHBcAg蛋白”应包括所有此类序列，包括例如SEQ ID NO：1所示的序列以及其天然或人工的变体。并且，当描述RBHBcAg蛋白的序列片段时，其不仅包括SEQ ID NO：1的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“RBHBcAg蛋白的N端第78-83位氨基酸残基”包 括，SEQ ID NO：1的第78-83位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“野生型RBHBcAg”是指，天然存在于蹄蝠乙型肝炎病毒中的核心抗原蛋白。
如本文中所使用的，术语“RBHBcAg载体”是指，衍生自RBHBcAg蛋白的多肽载体。如上文所详细描述的，RBHBcAg载体与RBHBcAg蛋白存在着如下差异：(a)RBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基被缺失或被置换为连接体；以及(b)任选地，RBHBcAg蛋白的C端被缺失1-40个氨基酸残基。
如本文中所使用的，术语“筑帐蝠乙型肝炎病毒的核心抗原蛋白(TBHBcAg)”和“TBHBcAg蛋白”是指，来自筑帐蝠乙型肝炎病毒(tent-making bat HBV，TBHBV)的核心抗原蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：KC790378.1)。
如本文中所使用的，当提及TBHBcAg蛋白的氨基酸序列时，其使用SEQ ID NO：2所示的序列来进行描述。例如，表述“TBHBcAg蛋白的N端第80-84位氨基酸残基”是指，SEQ ID NO：2所示的多肽的第80-84位氨基酸残基。然而，本领域技术人员理解，在TBHBcAg蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同基因型或基因亚型的TBHBcAg蛋白)，而不影响其生物学功能。因此，在本发明中，术语“TBHBcAg蛋白”应包括所有此类序列，包括例如SEQ ID NO：2所示的序列以及其天然或人工的变体。并且，当描述TBHBcAg蛋白的序列片段时，其不仅包括SEQ ID NO：2的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“TBHBcAg蛋白的N端第80-84位氨基酸残基”包括，SEQ ID NO：2的第80-84位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分 数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“野生型TBHBcAg”是指，天然存在于筑帐蝠乙型肝炎病毒中的核心抗原蛋白。
如本文中所使用的，术语“TBHBcAg载体”是指，衍生自TBHBcAg蛋白的多肽载体。如上文所详细描述的，TBHBcAg载体与TBHBcAg蛋白存在着如下差异：(a)TBHBcAg蛋白的N端第80-84位氨基酸残基中的一个或多个氨基酸残基被缺失或被置换为连接体；以及(b)任选地，TBHBcAg蛋白的C端被缺失1-35个氨基酸残基。
如本文中所使用的，术语“菊头蝠乙型肝炎病毒的核心抗原蛋白(HBHBcAg)”和“HBHBcAg蛋白”是指，来自菊头蝠乙型肝炎病毒(horseshoe bat HBV，HBHBV)的核心抗原蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：KC790377.1)。
如本文中所使用的，当提及HBHBcAg蛋白的氨基酸序列时，其使用SEQ ID NO：3所示的序列来进行描述。例如，表述“HBHBcAg蛋白的N端第78-83位氨基酸残基”是指，SEQ ID NO：3所示的多肽的第78-83位氨基酸残基。然而，本领域技术人员理解，在HBHBcAg蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同基因型或基因亚型的HBHBcAg蛋白)，而不影响其生物学功能。因此，在本发明中，术语“HBHBcAg蛋白”应包括所有此类序列，包括例如SEQ ID NO：3所示的序列以及其天然或人工的变体。并且，当描述HBHBcAg蛋白的序列片段时，其不仅包括SEQ ID NO：3的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“HBHBcAg蛋白的N端第78-83位氨基酸残基”包括，SEQ ID NO：3的第78-83位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“野生型HBHBcAg”是指，天然存在于菊头蝠乙型肝炎病毒中的核心抗原蛋白。
如本文中所使用的，术语“HBHBcAg载体”是指，衍生自HBHBcAg蛋白的多肽载体。如上文所详细描述的，HBHBcAg载体与HBHBcAg蛋白存在着如下差异：(a)HBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基被缺失或被置换为连接体；以及(b)任选地，HBHBcAg蛋白的C端被缺失1-40个氨基酸残基。
如本文中所使用的，术语“人HBV的HBcAg”和“Hu-HBcAg”是指，人乙型肝炎病毒的核心抗原蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：AAO63517.1)。如本文中所使用的，当提及人HBV的HBcAg的氨基酸序列时，其使用NCBI GENBANK数据库登录号：AAO63517.1所示的序列来进行描述。
如本文中所使用的，术语“人HBV的HBsAg”和“Hu-HBsAg”是指，人乙型肝炎病毒的表面抗原主蛋白，其是本领域技术人员公知的(参见，例如NCBI GENBANK数据库登录号：AAF24729.1)。
如本文中所使用的，当提及人HBV的HBsAg的氨基酸序列时，其使用SEQ ID NO：44所示的序列(即，NCBI GENBANK数据库登录号：AAF24729.1)来进行描述。例如，表述“HBsAg蛋白的第113-135位氨基酸残基”是指，SEQ ID NO：44所示的多肽的第113-135位氨基酸残基。然而，本领域技术人员理解，在HBsAg蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同基因型或基因亚型的HBsAg蛋白)，而不影响其生物学功能。因此，在本发明中，术语“HBsAg蛋白”应包括所有此类序列，包括例如SEQ ID NO：44所示的序列以及其天然或人工的变体。并且，当描述HBsAg蛋白的序列片段时，其不仅包括SEQ ID NO：44的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“HBsAg蛋白的第113-135位氨基酸残基”包括，SEQ ID NO：44的第113-135位氨基酸残基，以及其变体(天然或人工)中的相应片段。根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，表述“X蛋白的C端缺失Y个氨基酸残基”是指，X蛋白的C端最后Y个氨基酸残基全部被去除。例如，表述“RBHBcAg蛋白的C端缺失1-40个氨基酸残基”是指，RBHBcAg蛋白的C端最后1-40个氨基酸残基全部被去除。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Mi l ler(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blos sum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残 基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“杂交”是指，相互间具有互补序列的两个单链核酸分子在一定条件下(适宜的温度及离子强度等)按碱基互补配对原则退火形成双链核酸的过程。核酸杂交可以在DNA-DNA之间，也可在DNA-RNA或RNA-RNA之间进行，只要它们之间存在互补序列，可以进行碱基配对。关于核酸杂交的进一步详细描述，可参见例如，Henegariu O等人，(1999).“Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling”,Nature Biotechnology 18:345-348；Ezaki T等人，1989.Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains.Int.J.of Systemic Bacteriology 29(3):224-229；和Herrington C等人，1998.PCR 3:PCR in situ hybridization:a practical  approach,Volume 3.Oxford:Oxford University Press。
为了保证核酸杂交的特异性，通常使用严紧条件或者高严紧条件。严紧条件和高严紧条件在分子生物学领域中是熟知的。例如，严紧条件可以是指，在6×氯化钠/柠檬酸钠(SSC)中、于约45℃下进行杂交，随后在0.2×SSC/0.1％SDS中、于约50－65℃下进行一次或多次洗涤。高严紧条件可以是指，在6×SSC中、于约45℃下进行杂交，随后在0.1×SSC/0.2％SDS中、于约68℃下进行一次或多次洗涤。关于本领域技术人员已知的其它严紧条件或高严紧条件，可参见例如Ausubel,F.M.等编,1989,Current Protocols in Molecular Biology,第1卷,Green Publishing Associates,Inc.,和John Wiley&Sons,Inc.,纽约，第6.3.1-6.3.6和2.10.3页。
如本文中所使用的，术语“连接体”是指，用于连接两个分子(例如蛋白)的短肽。此类连接体是本领域技术人员公知的，包括但不限于柔性连接体，例如(Gly)4，(Gly)4-Ser，((Gly)4-Ser)3等等。
在本发明中，术语“多肽”和“蛋白质”具有相同的含义，可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“限制性酶切位点”是指，限制性内切酶识别的酶切位点。此类限制性酶切位点是本领域技术人员已知的，其包括但不限于，EcoR I、BamH I、HindⅡ、HindⅢ，Hpa I、Hpa Ⅱ，Mbo I、Mbo Ⅱ等限制性内切酶识别的酶切位点。
如本文中所使用的，术语“抗原表位”和“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷， G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“HBsAg的表位”是指，存在于HBsAg中的、能够被免疫球蛋白或抗体特异性结合的部位。已对人HBV的HBsAg的结构和功能进行了深入的研究。并且，已有许多文献报道了人HBV的HBsAg上的表位。参见例如，WO 97/39029A2；WO 85/04103A1；Xiaoxing Qiu等，The Journal of Immunology，1996，第156卷，第3350-3356页；WO 2013/185558A1等。
如本文中所使用的，术语“表位肽”是指，抗原上能够形成表位/用作表位的肽段。在一些情况下，单独的表位肽即能够被针对所述表位的抗体特异性识别/结合。在另一些情况下，可能需要将表位肽与多肽载体融合，以便表位肽能够被特异性抗体识别。表位肽所包含的表位可以是线性表位，也可以是构象表位。当表位肽包含线性表位时，其可以包含或者是抗原中构成该表位的连续氨基酸区段(即，肽片段)。当表位肽包含构象表位时，其可以包含或者是抗原中横跨该构象表位所涉及的所有氨基酸残基的连续氨基酸区段(即，肽片段)。在本发明的某些实施方案中，表位肽优选地具有不超过500个氨基酸残基的长度，例如不超过400个氨基酸残基的长度，不超过300个氨基酸残基的长度，不超过200个氨基酸残基的长度，不超过100个氨基酸残基的长度，不超过90个氨基酸残基的长度，不超过80个氨基酸残基的长度，不超过70个氨基酸残基的长度，不超过60个氨基酸残基的长度，不超过50个氨基酸残基的长度，不超过40个氨基酸残基的长度，不超过30个氨基酸残基的长度，或不超过25个氨基酸残基的长度。
如本文中所使用的，术语“多肽载体”是指这样的载体蛋白，其可以充当表位肽的载体，即，其可以在特定位置处(例如蛋白内部，N端或C端)插入表位肽，以便该表位肽能够呈现出来，从而该表位肽能够被抗体或免疫系统识别。之前的文献已报道了此类载体蛋白，包 括例如，HPV L1蛋白(可以将表位肽插入在所述蛋白的第130-131位氨基酸之间或在第426-427位氨基酸之间，参见Slupetzky,K.等Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops[J].J Gen Virol,2001,82:2799-2804；Varsani,A.等Chimeric human papillomavirus type 16(HPV-16)L1particles presenting the common neutralizing epitope for the L2minor capsid protein of HPV-6and HPV-16[J].J Virol,2003,77:8386-8393)，CRM197蛋白(可以将表位肽连接至该蛋白或其片段的N末端或C末端)等。如上文所论述的，本发明提供了一类新的多肽载体，其用于展示目的多肽，并且特别适合用于展示含有来自人乙肝病毒的抗原表位(例如来自人HBV的HBsAg中的表位)的表位肽。在本发明的实施方案中，可以在表位肽与多肽载体之间使用连接体(例如柔性或刚性连接体)，以促进二者各自的折叠。
如本文中所使用的，术语“重组蛋白”仅表示，所描述的蛋白不是天然存在的，而不意欲限定该蛋白的产生/获得方式。本发明的重组蛋白可以通过任何已知的方法来产生，包括但不限于，基因工程方法和人工合成方法。
如本文中所使用的，术语“病毒样颗粒”是指，由某种病毒的一个或多个结构蛋白构成的空心颗粒，其不包含病毒核酸，不能进行自主复制，但在形态和结构上与真正的病毒粒子相同或相似。
如本文中所使用的，术语“药学可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当 其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体(vector)组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698,ER2566,BL21(DE3),B834(DE3),BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如HBV感染或与HBV感染相关的疾病)有效量是指，足以预防，阻止，或延迟疾病(例如HBV感染或与HBV感染相关的疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果：
(1)本发明提供了一种新的多肽载体，其具有广泛的通用性，能够用于高效展示各种目的多肽(例如，抗原表位/抗原肽段)，并诱发宿主免疫系统产生针对目的多肽的特异性免疫应答。此类目的多肽(例如，抗原表位/抗原肽段)包括但不限于，来源于HIV的抗原表位/抗原肽段(例如，来源于HIV的GP120蛋白的抗原表位/抗原肽段；例如含有GP120蛋白的第361-375位氨基酸的多肽)，来源于人PD-L1蛋白的抗原表位/抗原肽段(例如，含有人PD-L1蛋白第147-160位氨基酸的多肽)，和来源于人HBV的抗原表位/抗原肽段(例如，来源于人HBV的HBsAg蛋白的抗原表位/抗原肽段；例如含有HBsAg蛋白的第113-135位氨基酸的多肽)。
(2)本发明的多肽载体特别适合用于展示来自人乙肝病毒的抗原表位(例如来自人HBV的HBsAg中的表位)，能够在受试者中诱发特别强的清除HBsAg的特异性免疫应答，其功效显著优于现有的乙肝疫苗(例如，以人HBV的HBcAg为多肽载体构建的、含有相同表位的疫苗)。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而 不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1展示了，将目的多肽(靶抗原肽段)插入本发明的RBHBcAg载体、TBHBcAg载体和HBHBcAg载体以构建重组蛋白的克隆方案的示意图。
图2展示了，实施例2中所构建的18种重组蛋白的SDS-PAGE结果，以及由所述重组蛋白形成的病毒样颗粒的透射电镜观察结果。
图3展示了，用实施例2中构建的18种重组蛋白形成的病毒样颗粒免疫BALB/C小鼠后，小鼠血清中抗重组蛋白中的目的多肽的抗体滴度随时间的变化情况。纵轴：抗体滴度(log10)；横轴：时间(周)。图3A：所使用的目的多肽为SEQ ID NO:20，且检测抗GP120抗体的滴度；图3B：所使用的目的多肽为SEQ ID NO:21，且检测抗PD-L1抗体的滴度；图3C：所使用的目的多肽为SEQ ID NO:22，且检测抗HBsAg抗体的滴度。
图4展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性(图4A)和雌性(图4B)小鼠后，小鼠血清中HBsAg的水平随时间的变化情况。纵轴：HBsAg的水平(IU/ml)；横轴：时间(周)。箭头指示，给小鼠施用病毒样颗粒的时间点。
图5展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性小鼠后，小鼠血清中HBV DNA的水平随时间的变化情况。纵轴：HBV DNA的水平(Log10IU/ml)；横轴：时间(周)。箭头指示，给小鼠施用病毒样颗粒的时间点。
图6展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性(图6A)和雌性(图6B)小鼠后，小鼠血清中抗HBsAg抗体的滴度随时间的变化情况。纵轴：抗HBsAg抗体的滴度；横轴：时间(周)。
图7显示了，由实施例5中构建的6种重组蛋白形成的病毒样颗粒的透射电镜观察结果。
图8展示了，用8种重组蛋白形成的病毒样颗粒免疫BALB/C小鼠3周后，小鼠血清中抗相应靶多肽(SEQ ID NO：60，22，61，62)的抗体滴度；其中，用来自HBV基因型A的HBsAg蛋白的表位肽(SEQ ID NO：60)来检测用RBHBcAg149-SEQ60和TBHBcAg153-SEQ60免疫的小鼠血清的抗体滴度；用来自HBV基因型B的HBsAg蛋白的表位肽(SEQ ID NO:SEQ22)来检测用RBHBcAg149-SEQ22和TBHBcAg153-SEQ22免疫的小鼠血清的抗体滴度；用来自HBV基因型C的HBsAg蛋白的表位肽(SEQ ID NO:61)来检测用RBHBcAg149-SEQ61和TBHBcAg153-SEQ61免疫的小鼠血清的抗体滴度；并且，使用来自HBV基因型D的HBsAg蛋白的表位肽(SEQ ID NO:62)来检测用RBHBcAg149-SEQ62和TBHBcAg153-SEQ62免疫的小鼠血清的抗体滴度。结果显示，8种重组蛋白形成的病毒样颗粒均具有良好的免疫原性，能够诱导小鼠产生高滴度的特异性结合靶抗原的抗体。
图9展示了，用4种重组蛋白(SEQ ID NO：36，69，70，71)形成的病毒样颗粒治疗HBV转基因雄性(图9A)和雌性(图9B)小鼠后，小鼠血清中HBsAg的水平随时间的变化情况，其中，纵轴：HBsAg的水平(IU/ml)；横轴：时间(周)。
序列信息
本发明涉及的部分序列(SEQ ID NO:1-44)的信息提供于下面的表1中。
表1：SEQ ID NO:1-44的序列信息
Figure PCTCN2016096481-appb-000001
Figure PCTCN2016096481-appb-000002
Figure PCTCN2016096481-appb-000003
Figure PCTCN2016096481-appb-000004
Figure PCTCN2016096481-appb-000005
Figure PCTCN2016096481-appb-000006
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.编码多肽载体的质粒的构建
在本实施例中，我们构建了编码多肽载体的质粒。
1.1编码多肽载体的核苷酸序列的制备
基于三种蝙蝠来源的乙型肝炎病毒核心抗原蛋白(即，RBHBcAg蛋白，TBHBcAg蛋白，HBHBcAg蛋白)，设计了下述多肽载体：
RBHBcAg189载体，其与RBHBcAg蛋白(SEQ ID NO:1)的区别在于，RBHBcAg蛋白的第78-81位氨基酸残基被替换为如SEQ ID NO:43所示的连接体；RBHBcAg189载体的氨基酸序列如SEQ ID NO:4所示，核苷酸序列如SEQ ID NO:12所示；
TBHBcAg188载体，其与TBHBcAg蛋白(SEQ ID NO:2)的区别在于，RBHBcAg蛋白的第80-83位氨基酸残基被替换为如SEQ ID NO:43所 示的连接体；TBHBcAg188载体的氨基酸序列如SEQ ID NO:6所示，核苷酸序列如SEQ ID NO:14所示；
HBHBcAg189载体，其与HBHBcAg蛋白(SEQ ID NO:3)的区别在于，RBHBcAg蛋白的第78-81位氨基酸残基被替换为如SEQ ID NO:43所示的连接体；HBHBcAg189载体的氨基酸序列如SEQ ID NO:8所示，核苷酸序列如SEQ ID NO:16所示。
另外，还基于人HBV的HBcAg蛋白，设计了HBcAg183载体，用作对照。HBcAg183载体与人HBV的HBcAg蛋白的区别在于，人HBV的HBcAg蛋白的第79-81位氨基酸残基被替换为如SEQ ID NO:43所示的连接体；HBcAg183载体的氨基酸序列如SEQ ID NO:10所示，核苷酸序列如SEQ ID NO:18所示。
上述四种载体的核苷酸序列均委托生工生物工程上海(股份)有限公司进行全基因合成。
1.2编码多肽载体的质粒的制备
以上述合成的核苷酸序列为模板，使用表2中的引物，通过PCR分别扩增上述4种载体的全长基因以及其截短体(即，C端截短的基因片段)。一共获得8种PCR产物，分别是：编码RBHBcAg189载体的基因(SEQ ID NO:12；其编码的氨基酸序列为SEQ ID NO:4)、编码RBHBcAg149载体的基因(SEQ ID NO:13；其编码的氨基酸序列为SEQ ID NO:5)、编码TBHBcAg188载体的基因(SEQ ID NO:14；其编码的氨基酸序列为SEQ ID NO:6)、编码TBHBcAg153载体的基因(SEQ ID NO:15；其编码的氨基酸序列为SEQ ID NO:7)、编码HBHBcAg189载体的基因(SEQ ID NO:16；其编码的氨基酸序列为SEQ ID NO:8)、编码HBHBcAg149载体的基因(SEQ ID NO:17；其编码的氨基酸序列为SEQ ID NO:9)、编码HBcAg183载体的基因(SEQ ID NO:18；其编码的氨基酸序列为SEQ ID NO:10)、和编码HBcAg149载体的基因(SEQ ID NO:19；其编码的氨基酸序列为SEQ ID NO:11)。
用NdeI和HindIII将pTO-T7载体(罗文新,张军,杨海杰,等. 一种带增强子的原核高效表达载体的构建及初步应用[J].生物工程学报,2000,16(5):578-581)双酶切，获得线性化载体。利用Gibson assembly克隆方法(New England Biolabs(UK)Ltd)，将获得的8种PCR产物与线性化载体连接，并转化入DH5a感受态细菌中。将经转化的细菌涂板培养，然后挑取单克隆菌落，提取质粒并进行测序。经测序验证，获得8种含有编码多肽载体的核苷酸序列的质粒。
上述PCR过程涉及的引物示于表2中。
表2：引物序列
Figure PCTCN2016096481-appb-000007
实施例2.重组蛋白的制备
在本实施例中，我们将编码目的多肽的核苷酸序列插入实施例1构建的质粒中，并获得了含有目的多肽和多肽载体的重组蛋白。将目的多肽(靶抗原肽段)插入本发明的RBHBcAg载体、TBHBcAg载体和HBHBcAg载体以构建重组蛋白的克隆方案的示意图如图1所示。
2.1编码含有目的多肽和多肽载体的重组蛋白的表达质粒的构建
本实施例利用3种目的多肽来验证本发明的多肽载体用于展示肽 段的通用性。这3种目的多肽为：多肽HIV-GP120-aa361-375(即，来源于HIV GP120蛋白的第361-375位氨基酸，其氨基酸序列如SEQ ID NO:20所示)；多肽hPDL1-aa147-160(即，来源于人PD-L1蛋白的第147-160位氨基酸，其氨基酸序列如SEQ ID NO:21所示)；多肽HBsAg-aa113-135(即，来源于人HBV表面抗原主蛋白(HBsAg)的第113-135位氨基酸，其氨基酸序列如SEQ ID NO:22所示)。
直接合成所述3种目的多肽的正义和反义编码序列(如表3所示)，并进行退火，从而获得具有粘性末端的、编码目的多肽的基因片段。
表3：3种目的多肽的正义和反义编码序列
Figure PCTCN2016096481-appb-000008
将实施例1中获得的6种质粒(RBHBcAg189、RBHBcAg149、TBHBcAg188、TBHBcAg153、HBHBcAg189和HBHBcAg149)分别用BamHI和EcoRI双酶切，获得6种线性化的载体。然后，将如上制备的3种具有粘性末端的、编码目的多肽的基因片段分别连接入各个线性化载体中，获得编码重组蛋白的表达质粒(一共18种：RBHBcAg189-SEQ20，RBHBcAg149-SEQ20，TBHBcAg188-SEQ20，TBHBcAg153-SEQ20，HBHBcAg189-SEQ20，HBHBcAg149-SEQ20，RBHBcAg189-SEQ21，RBHBcAg149-SEQ21，TBHBcAg188-SEQ21，TBHBcAg153-SEQ21，HBHBcAg189-SEQ21，HBHBcAg149-SEQ21，RBHBcAg189-SEQ22，RBHBcAg149-SEQ22，TBHBcAg188-SEQ22，TBHBcAg153-SEQ22， HBHBcAg189-SEQ22，和HBHBcAg149-SEQ22)。
2.2重组蛋白的表达、纯化和组装
使用前一步骤构建的18种表达质粒，通过相同的方法来表达和纯化所述表达质粒编码的重组蛋白。下面以RBHBcAg149-SEQX(SEQX表示SEQ20、SEQ21或SEQ22)为例，阐述这些重组蛋白的表达和纯化过程。
(2.2.1)用于表达重组蛋白的菌株的制备：将2.1中获得的表达质粒RBHBcAg149-SEQX转化入大肠杆菌ER2566菌株，从而获得表达菌株。
(2.2.2)重组蛋白RBHBcAg149-SEQX的表达：将表达菌株接种至500mL三角瓶中，于37℃定温摇床培养至OD＝1.0左右；随后加入异丙基-beta-D-硫代半乳糖苷(IPTG)至终浓度0.5mM，并于25℃继续表达6小时。
(2.2.3)重组蛋白RBHBcAg149-SEQX的纯化：
(2.2.3.1)菌体超声破碎：离心收取2.2.2中的菌体，并进行超声破碎。超声缓冲液为：20mM磷酸盐缓冲液(PH6.0)+300mM NaCl。
(2.2.3.2)重组蛋白的初步纯化：将超声破碎后的混合物于65℃水浴中温育30分钟，随后离心收取上清；按1:1的体积比向上清中加入饱和硫酸铵，离心收取沉淀；加入适当体积的缓冲液(20mM磷酸盐缓冲液(pH＝7.4)+150mM NaCl)将沉淀重悬，从而获得初步纯化的重组蛋白RBHBcAg149-SEQX。
(2.2.3.3)重组蛋白的层析纯化：按照制造商的说明书，利用Sepharose 4FF(GE)分子筛柱层析进一步纯化2.2.3.2中获得的蛋白，从而获得经纯化的重组蛋白。对经纯化的目的蛋白进行SDS-PAGE检测，并用透射电镜观察由重组蛋白形成的VLP的状态。
图2显示了，所构建的18种重组蛋白的SDS-PAGE结果，以及由所述重组蛋白形成的病毒样颗粒的透射电镜观察结果。结果显示，所获得的18种重组蛋白的纯度均大于85％，并且均能够组装成直径约30nm病毒样颗粒。这些结果表明，本发明所构建的多肽载体具有广泛 的通用性，能够用于展示各种目的多肽，并且能够形成VLP。
实施例3.病毒样颗粒的免疫原性的评估
在本实施例中，我们验证了由本发明的重组蛋白形成的病毒样颗粒的免疫原性。此类病毒样颗粒均能够诱导机体产生特异性结合靶抗原的抗体。
3.1小鼠免疫
用实施例2制备的18种病毒样颗粒分别免疫BALB/C小鼠。免疫方案如下：免疫佐剂为氢氧化铝佐剂；免疫剂量为3ug/dose；免疫方式为后肢大腿外侧肌肉注射；免疫程序为，初次免疫+2周后的加强免疫，共免疫2次。
3.2血清中特异性结合靶抗原的抗体的滴度的检测
3.2.1反应板的制备
反应板的包被抗原是，分别与上述3种目的多肽对应的靶抗原，即：HIV-1的gp120蛋白(购自北京义翘神州生物技术有限公司，目录号11233-V08H)，人PD-L1蛋白(购自北京义翘神州生物技术有限公司)，CHO细胞重组表达的人乙型肝炎病毒表面抗原主蛋白(HBsAg，购自北京万泰生物药业股份有限公司)。
将3种重组蛋白分别用pH9.6的50mM CB缓冲液(NaHCO3/Na2CO3缓冲液，终浓度为50mM，pH值为9.6)稀释，终浓度为2μg/mL，以获得包被液。在96孔酶标板的各个孔中加入100μL的包被液，并在2-8℃包被16-24小时，然后在37℃继续包被2小时。然后，用PBST洗涤液(20mM PB7.4，150mM NaCl，0.1％Tween20)洗涤孔1次；随后每孔加入200μL的封闭液(含20％小牛血清及1％酪蛋白的20mM Na2HPO4/NaH2PO4缓冲液溶液，pH值为7.4)，并在37℃封闭2小时。弃去封闭液。然后，将酶标板干燥，并装入铝箔袋，于2-8℃保存备用。
3.2.2血清中Ant i-HBsAg抗体滴度的ELISA检测
血清样品的收集：在0、2、4周采集小鼠眼眶血，分离血清并冻存于-20℃，直至检测。
样品稀释：用含20％新生牛血清的PBS溶液将小鼠血清稀释成1:100、1:500、1:2500、1:12500、1:62500、1:312500、1:1562500共7个稀释梯度。
ELISA检测：向经包被的酶标板的各孔中加入100μL已稀释的血清样品，于37℃孵育30分钟。然后，用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)将酶标板洗涤5遍。洗涤后，向酶标板的各孔中加入100μL GAM-HRP反应液，于37℃孵育30分钟。然后，用PBST洗液(20mM PB7.4，150mM NaCl，0.1％Tween20)将酶标板洗涤5遍。洗涤后，向酶标板的各孔中加入50μL TMB显色剂(由北京万泰生物药业股份有限公司提供)，于37℃孵育15分钟。孵育完成后，向酶标板的各孔中加入50μL终止液(由北京万泰生物药业股份有限公司提供)，并在酶标仪上读取各孔的OD450/630值。
抗体滴度的计算：取读值在0.2-2.0之间的样品孔进行分析，对样品的稀释倍数与读值作回归曲线，计算出读值等于2倍本底值时的样品稀释倍数，并将该样品稀释倍数作为血清中的特异性抗体的滴度。
图3展示了，用18种重组蛋白形成的病毒样颗粒免疫BALB/C小鼠后，小鼠血清中抗靶抗原的抗体滴度随时间的变化情况。图3A：所使用的目的多肽为SEQ ID NO:20，且检测抗GP120抗体的滴度；图3B：所使用的目的多肽为SEQ ID NO:21，且检测抗PD-L1抗体的滴度；图3C：所使用的目的多肽为SEQ ID NO:22，且检测抗HBsAg抗体的滴度。结果显示，18种重组蛋白形成的病毒样颗粒均具有良好的免疫原性，能够诱导小鼠产生高滴度的特异性结合靶抗原的抗体。
实施例4.展示HBsAg表位(SEQ ID NO:22)的病毒样颗粒的抗HBV治疗效果的评估
在本实施例中，我们评估了基于不同多肽载体构建的、展示相同表位肽(SEQ ID NO:22)的病毒样颗粒的抗HBV治疗效果。
4.1小鼠免疫
按照实施例1-2描述的方案，制备了基于人HBV的HBcAg构建的、 展示HBsAg表位(SEQ ID NO:22)的2种重组蛋白(即，HBcAg183-SEQ22，其氨基酸序列如SEQ ID NO:41所示；和HBcAg149-SEQ22，其氨基酸序列如SEQ ID NO:42所示)，并制备了由这2种重组蛋白形成的病毒样颗粒。
随后，利用HBV转基因小鼠模型来评价实施例2中制备的5种展示HBsAg表位(SEQ ID NO:22)的病毒样颗粒以及本实施例制备的2种病毒样颗粒的抗HBV治疗效果。
免疫方案如下：免疫佐剂为氢氧化铝佐剂；免疫剂量为12μg/dose；免疫方式为后肢大腿外侧肌肉注射；免疫程序为，0、2、3、4、5、6周各免疫一次，共免疫6次。
4.2血清抗体滴度与病毒学指标的检测
按实施例3.2中描述的方法进行血清中Ant i-HBsAg抗体滴度的检测，并检测小鼠血清中的病毒学指标，即，HBV DNA和HBsAg的水平。
4.3重组蛋白的治疗效果分析
检测结果示于图4-6中。图4展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性(图4A)和雌性(图4B)小鼠后，小鼠血清中HBsAg的水平随时间的变化情况。图5展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性小鼠后，小鼠血清中HBV DNA的水平随时间的变化情况。图6展示了，用展示相同表位肽(SEQ ID NO:22)的不同病毒样颗粒治疗HBV转基因雄性(图6A)和雌性(图6B)小鼠后，小鼠血清中抗HBsAg抗体的滴度随时间的变化情况。
结果显示，在用VLP进行免疫治疗的组中，在免疫后，小鼠血清中均可检出Ant i-HBsAg抗体，并且小鼠血清中的HBV DNA和HBsAg的水平均出现了不同程度的下降。相比之下，对照组小鼠(未用VLP进行免疫)的血清中均没有产生Anti-HBsAg抗体，并且血清中的HBV DNA和HBsAg的水平均未出现下降。
这些结果显示，基于蝙蝠乙型肝炎病毒核心蛋白所构建的6种多肽载体均可以用于高效展示来自人HBV的HBsAg的表位肽(例如， HBsAg-aa113-135)，其能够形成VLP，并在宿主体内诱导机体产生高滴度的ant i-HBsAg抗体，并由此抑制小鼠体内的HBV DNA和HBsAg的水平(即，HBV DNA和HBsAg显著下降)。此外，图4-6的实验数据还显示，基于本发明的多肽载体(例如RBHBcAg149和TBHBcAg153)的病毒样颗粒具有特别显著的抗HBV治疗效果，其优于基于人HBV的HBcAg构建的病毒样颗粒。
因此，本实施例的实验结果表明：(1)本发明的多肽载体能够形成VLP，适合于展示各种目的多肽，并能够诱导机体产生高滴度的针对目的多肽的抗体；(2)本发明的多肽载体特别适合于展示人HBV的表位(例如，人HBV的HBsAg的表位)，其能够诱导机体产生高滴度的针对HBsAg的抗体，清除或抑制体内的HBV DNA和HBsAg的水平，并且其效果优于基于人HBV的HBcAg构建的多肽载体。因此，本发明的展示人HBV的表位的重组蛋白具有治疗HBV感染的潜力，特别适合用于诱发有效的、特异的、治疗性的抗HBV免疫。
实施例5.展示来自不同基因型的HBV的HBsAg表位的病毒样颗粒的制备和评估
实施例2-4中所使用的HBsAg表位(SEQ ID NO:22)来自HBV基因型B。为了证实本发明的多肽载体对各种基因型的HBV的广泛适应性，我们还以RBHBcAg149和TBHBcAg153为示例性多肽载体，构建了展示来自不同基因型(基因型A、C和D)的HBV的HBsAg表位的重组蛋白，并评估了所构建的重组蛋白组装成病毒样颗粒的能力，以及所产生的病毒样颗粒的免疫原性和抗HBV感染的治疗效果。
5.1编码含有目的多肽和多肽载体的重组蛋白的表达质粒的构建
在本实施例中，除了实施例2-4中所使用的HBsAg表位(来自HBV基因型B，SEQ ID NO:22)之外，目的多肽还包括来自HBV基因型A、C和D的HBsAg蛋白表位(第113-135位氨基酸)，其分别被命名为：HBsAg-aa113-135-A，HBsAg-aa113-135-C和HBsAg-aa113-135-D，并且其序列(SEQ ID NO:60-62)示于表4中。
表4：来自HBV基因型A、C和D的HBsAg蛋白的第113-135位氨基酸的序列
SEQ ID NO 名称 序列信息
60 HBsAg-aa113-135-A STTTSTGPCKTCTTPAQGNSMFP
61 HBsAg-aa113-135-C TSTTSTGPCKTCTIPAQGTSMFP
62 HBsAg-aa113-135-D SSTTSTGPCRTCTTPAQGTSMYP
直接合成所述3种目的多肽的正义和反义编码序列(如表5所示)，并进行退火，从而获得具有粘性末端的、编码目的多肽的基因片段。
表5：3种目的多肽的正义和反义编码序列
Figure PCTCN2016096481-appb-000009
如实施例2中所述，将如上制备的3种具有粘性末端的、编码目的多肽的基因片段分别连接入线性化载体RBHBcAg149和TBHBcAg153中，从而获得编码重组蛋白的表达质粒(共6种：RBHBcAg149-SEQ60，RBHBcAg149-SEQ61，RBHBcAg149-SEQ62，TBHBcAg153-SEQ60，TBHBcAg153-SEQ61，TBHBcAg153-SEQ62)。这些表达质粒所编码的重组蛋白的氨基酸序列如表6所示。
表6：6种重组蛋白的氨基酸序列
Figure PCTCN2016096481-appb-000010
Figure PCTCN2016096481-appb-000011
5.2重组蛋白的表达、纯化和组装
如实施例2中所述，使用前一步骤构建的6种表达质粒，表达和纯化了所述表达质粒编码的重组蛋白。随后，使用透射电镜观察由这些重组蛋白形成的VLP的状态。
图7显示了，由所构建的6种重组蛋白形成的病毒样颗粒的透射电镜观察结果。结果显示，所获得的6种重组蛋白均能够组装成直径约30nm病毒样颗粒。这些结果表明，本发明所构建的多肽载体具有广泛的通用性，能够用于展示来自各种基因型的HBV的表位肽(例如HBsAg蛋白的aa113-135)，并且能够形成良好的VLP。
5.3病毒样颗粒的免疫原性的评估
使用实施例3中描述的方法，评估了如上构建的6个重组蛋白以及实施例2中的重组蛋白RBHBcAg149-SEQ22和TBHBcAg153-SEQ22形成的病毒样颗粒的免疫原性。实验结果示于图8中。
图8展示了，用8种重组蛋白形成的病毒样颗粒免疫BALB/C小鼠 3周后，小鼠血清中抗相应靶多肽(SEQ ID NO：60，22，61，62)的抗体滴度；其中，用来自HBV基因型A的HBsAg蛋白的表位肽(SEQ ID NO：60)来检测用RBHBcAg149-SEQ60和TBHBcAg153-SEQ60免疫的小鼠血清的抗体滴度；用来自HBV基因型B的HBsAg蛋白的表位肽(SEQ ID NO:SEQ22)来检测用RBHBcAg149-SEQ22和TBHBcAg153-SEQ22免疫的小鼠血清的抗体滴度；用来自HBV基因型C的HBsAg蛋白的表位肽(SEQ ID NO:61)来检测用RBHBcAg149-SEQ61和TBHBcAg153-SEQ61免疫的小鼠血清的抗体滴度；并且，使用来自HBV基因型D的HBsAg蛋白的表位肽(SEQ ID NO:62)来检测用RBHBcAg149-SEQ62和TBHBcAg153-SEQ62免疫的小鼠血清的抗体滴度。结果显示，8种重组蛋白形成的病毒样颗粒均具有良好的免疫原性，能够诱导小鼠产生高滴度的特异性结合靶表位的抗体。
5.4病毒样颗粒的抗HBV治疗效果的评估
使用实施例4中描述的方法，评估了4种重组蛋白(SEQ ID NO：36，69，70，71)形成的病毒样颗粒的抗HBV治疗效果。实验结果示于图9中。
图9展示了，用如上构建的4种重组蛋白(RBHBcAg149-SEQ22，RBHBcAg149-SEQ60，RBHBcAg149-SEQ61，RBHBcAg149-SEQ62；其序列分别SEQ ID NO：36，69，70，71)形成的病毒样颗粒治疗HBV转基因雄性(图9A)和雌性(图9B)小鼠后，小鼠血清中HBsAg的水平随时间的变化情况，其中，纵轴：HBsAg的水平(IU/ml)；横轴：时间(周)。结果显示，在用VLP进行免疫治疗的小鼠中，在免疫后，小鼠血清中的HBsAg的水平均出现了显著的下降。
这些实验结果显示，本发明的多肽载体(例如RBHBcAg149和TBHBcAg153)可用于高效展示来自不同基因型(例如基因型A、B、C和D)的人HBV的HBsAg的表位肽(例如，HBsAg-aa113-135)。基于本发明的多肽载体和HBsAg的表位肽所构建的重组蛋白能够形成VLP，并且能够在宿主体内诱导机体产生高滴度的anti-HBsAg抗体，并由此 抑制小鼠体内的HBsAg的水平(即，HBsAg的水平显著下降)。这表明，含有本发明的多肽载体和HBsAg的表位肽的重组蛋白能够用于预防和治疗各种基因型的HBV的感染，从而可用于开发新的抗HBV疫苗和药物。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (16)

  1. 一种核酸分子，其包含编码多肽载体的核苷酸序列或者其变体，所述变体与所述核苷酸序列具有至少90％(例如，至少91％、92％、93％、94％、95％、96％、97％、98％、或99％)的同一性，或者在严紧条件或者高严紧条件下，能够与所述核苷酸序列杂交，其中，所述多肽载体选自：
    (1)RBHBcAg载体，其与蹄蝠乙型肝炎病毒的核心抗原蛋白(RBHBcAg；例如，其氨基酸序列如SEQ ID NO:1所示)的区别在于：(a)RBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个，5个或6个氨基酸残基；例如，第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、或第78-80位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，RBHBcAg蛋白的C端缺失1-40个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨基酸残基)；
    (2)TBHBcAg载体，其与筑帐蝠乙型肝炎病毒的核心抗原蛋白(TBHBcAg；例如，其氨基酸序列如SEQ ID NO:2所示)的区别在于：(a)TBHBcAg蛋白的N端第80-84位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个或5个氨基酸残基；例如，第80-84位氨基酸残基、第80-83位氨基酸残基、或第80-82位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，TBHBcAg蛋白的C端缺失1-35个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，或30-35个氨基酸残基)；或
    (3)HBHBcAg载体，其与菊头蝠乙型肝炎病毒的核心抗原蛋白(HBHBcAg；例如，其氨基酸序列如SEQ ID NO:3所示)的区别在于：(a)HBHBcAg蛋白的N端第78-83位氨基酸残基中的一个或多个氨基酸残基(例如，1个，2个，3个，4个，5个或6个氨基酸残基；例如，第78-83位氨基酸残基、第78-82位氨基酸残基、第78-81位氨基酸残基、或第 78-80位氨基酸残基)被缺失或被置换为连接体(例如，柔性连接体；例如，如SEQ ID NO:43所示的连接体)；以及(b)任选地，HBHBcAg蛋白的C端缺失1-40个氨基酸残基(例如，1-5个，5-10个，10-15个，15-20个，20-25个，25-30个，30-35个，或35-40个氨基酸残基)；
    优选地，在编码所述被缺失的一个或多个氨基酸残基的位置处引入限制性酶切位点；
    优选地，在编码所述连接体的核苷酸序列中和/或其一端或两端引入限制性酶切位点；
    优选地，所述多肽载体的氨基酸序列选自SEQ ID NO:4-9；
    优选地，所述核酸分子包含选自SEQ ID NO:12-17的核苷酸序列；
    优选地，所述核酸分子用于插入编码目的多肽的核苷酸序列，例如所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端(例如，通过限制性酶切位点)；
    优选地，所述核酸分子还包含，编码目的多肽的核苷酸序列，其中，所述目的多肽相对于所述多肽载体而言是异源的，并且所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端(例如，通过限制性酶切位点)；优选地，所述目的多肽是表位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白的第113-135位氨基酸)，HIV的GP120蛋白的表位(例如线性表位，例如GP120蛋白的第361-375位氨基酸)，或人PD-L1的表位(例如线性表位，例如人PD-L1蛋白的第147-160位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62；优选地，所述核酸分子包含或者由编码选自SEQ ID NO:23-40和69-74的氨基酸序列的核苷酸序列组成。
  2. 一种载体(例如克隆载体或表达载体)，其包含权利要求1的核 酸分子。
  3. 一种宿主细胞，其包含权利要求1的核酸分子或权利要求2的载体。
  4. 一种展示目的多肽的方法，其包括:
    (1)将编码所述目的多肽的核苷酸序列插入至权利要求1的核酸分子中，从而获得编码重组蛋白的核酸分子；优选地，所述编码目的多肽的核苷酸序列被插入至编码所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至编码所述连接体的核苷酸序列中或其一端或两端(例如，通过限制性酶切位点)；和
    (2)将步骤(1)中的编码重组蛋白的核酸分子表达为重组蛋白；
    优选地，所述目的多肽是表位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白的第113-135位氨基酸)，HIV的GP120蛋白的表位(例如线性表位，例如GP120蛋白的第361-375位氨基酸)，或人PD-L1的表位(例如线性表位，例如人PD-L1蛋白的第147-160位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62；优选地，所述编码重组蛋白的核酸分子包含或者由编码选自SEQ ID NO:23-40和69-74的氨基酸序列的核苷酸序列组成。
  5. 一种重组蛋白，其包含多肽载体和目的多肽，其中，所述多肽载体如权利要求1所定义，并且，所述目的多肽被插入至所述被缺失的一个或多个氨基酸残基的位置处，或者被插入至所述连接体中或其一端或两端；
    优选地，所述多肽载体的氨基酸序列选自SEQ ID NO:4-9；
    优选地，所述目的多肽是表位肽，例如包含来自HIV、PDL1或HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例 如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白的第113-135位氨基酸)，HIV的GP120蛋白的表位(例如线性表位，例如GP120蛋白的第361-375位氨基酸)，或人PD-L1的表位(例如线性表位，例如人PD-L1蛋白的第147-160位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:20-22和60-62；
    优选地，所述重组蛋白包含或者由选自SEQ ID NO:23-40和69-74的氨基酸序列组成。
  6. 一种病毒样颗粒，其包含或者由权利要求5的重组蛋白组成。
  7. 一种药物组合物(例如疫苗)，其包含权利要求5的重组蛋白或权利要求6的病毒样颗粒，和任选地，一种或多种药学上可接受的载体或赋形剂(例如佐剂)。
  8. 一种预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)的方法，其包括，给有此需要的受试者施用权利要求5的重组蛋白或权利要求6的病毒样颗粒或权利要求7的药物组合物，其中所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白的第113-135位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62；优选地，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
  9. 权利要求5的重组蛋白或权利要求6的病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)，其中所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白 的第113-135位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62；优选地，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
  10. 权利要求5的重组蛋白或权利要求6的病毒样颗粒或权利要求7的药物组合物，其用于预防或治疗HBV感染或与HBV感染相关的疾病(例如，乙型肝炎)，其中所述目的多肽是包含来自HBV(特别是人HBV)的抗原表位的表位肽；优选地，所述表位肽包含：人HBV(例如HBV基因型A、B、C或D)的HBsAg的表位(例如线性表位，例如HBsAg蛋白的第113-135位氨基酸)；优选地，所述目的多肽的氨基酸序列选自SEQ ID NO:22和60-62；优选地，所述重组蛋白包含或者由选自SEQ ID NO:35-40和69-74的氨基酸序列组成。
  11. 一种多核苷酸，其编码权利要求5的重组蛋白。
  12. 一种载体(例如克隆载体或表达载体)，其包含权利要求11的多核苷酸。
  13. 一种宿主细胞，其包含权利要求11的多核苷酸或权利要求12的载体。
  14. 制备权利要求5的重组蛋白的方法，其包括，在适合表达所述重组蛋白的条件下培养权利要求13的宿主细胞，和，回收所述重组蛋白。
  15. 权利要求5的重组蛋白或权利要求6的病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗HIV感染或与HIV感染相关的疾病(例如，AIDS)，其中所述目的多肽包含来自HIV的抗原表位；优选地，所述目的多肽是包含来自HIV的抗原表位的表位肽；优选地，所述表位 肽包含：HIV的GP120蛋白的表位(例如线性表位，例如GP120蛋白的第361-375位氨基酸)；优选地，所述目的多肽的氨基酸序列如SEQ ID NO:20所示；优选地，所述重组蛋白包含或者由选自SEQ ID NO:23-28的氨基酸序列组成。
  16. 权利要求5的重组蛋白或权利要求6的病毒样颗粒用于制备药物的用途，所述药物用于预防或治疗癌症(例如，非小细胞肺癌)，其中所述目的多肽包含人PD-L1蛋白的抗原表位；优选地，所述目的多肽是包含人PD-L1蛋白的抗原表位的表位肽；优选地，所述表位肽包含：人PD-L1蛋白第147-160位氨基酸；优选地，所述目的多肽的氨基酸序列如SEQ ID NO:21所示；优选地，所述重组蛋白包含或者由选自SEQ ID NO:29-34的氨基酸序列组成。
PCT/CN2016/096481 2015-08-25 2016-08-24 一种用于展示目的多肽的多肽载体及其用途 WO2017032303A1 (zh)

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