WO2014127741A1 - Antigène permettant de détecter des anticorps anti-virus du papillome humain, kit d'essai et leurs applications - Google Patents

Antigène permettant de détecter des anticorps anti-virus du papillome humain, kit d'essai et leurs applications Download PDF

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WO2014127741A1
WO2014127741A1 PCT/CN2014/072407 CN2014072407W WO2014127741A1 WO 2014127741 A1 WO2014127741 A1 WO 2014127741A1 CN 2014072407 W CN2014072407 W CN 2014072407W WO 2014127741 A1 WO2014127741 A1 WO 2014127741A1
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peptide
hpv
seq
human papillomavirus
antigen
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PCT/CN2014/072407
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Chinese (zh)
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常小迦
张杰成
许虹
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艾托金生物医药（苏州）有限公司
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Publication of WO2014127741A1 publication Critical patent/WO2014127741A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • Antigen, kit and application thereof for detecting anti-human papillomavirus antibody Antigen, kit and application thereof for detecting anti-human papillomavirus antibody
  • the invention relates to the technical field of antibody detection, in particular to the technical field of detection of anti-human papillomavirus (HPV) antibody, in particular to an antigen for detecting anti-human papillomavirus antibody, related immunoassay kit and the same Applications, especially for the high-risk human papillomavirus (HPV) subtypes HPV16 and HPV18.
  • HPV high-risk human papillomavirus
  • Cervical cancer is the second most common female malignancy, and human papillomavirus (HPV) is a necessary condition for its occurrence.
  • HPV infection is common in the population, such as: Women with high incidence of cervical cancer still have an infection rate of 20.8% after the age of 35.
  • HPV DNA detection defects are incapable of distinguishing between transient infections and infections that can cause cervical lesions, resulting in higher false positive rates.
  • HPV oncogene expression products oncoprotein oncoprotein
  • the expression levels of high-risk HPV E2, E6, and E7 oncoproteins are directly related to different stages of cervical precancerous lesions. Using this feature, a new molecular screening index for cervical cancer can be constructed.
  • HPV E6 and E7 proteins are generally overexpressed in cervical epithelial cells of cervical lesions and cancer patients, and persistently expressed and overexpressed E6, E7 oncoproteins interact with host cell regulatory proteins (eg, P53 and Rb proteins) Inactivates tumor suppressor, leading to immortalization and malignant transformation of cells.
  • host cell regulatory proteins eg, P53 and Rb proteins
  • HPV antibodies in blood have also been shown to be highly relevant for HPV infection and the history of cervical cancer.
  • HPV L1 blood antibodies can be detected by standard ELISA methods to provide HPV infection information.
  • HPV infection time prolongs HPV induces anti-HPV antibodies, so anti-HPV antibodies in serum can be detected.
  • Wikstrom et al tested serum IgG antibodies to HPV6 and HPV11 capsid proteins in 47 male patients with condyloma acuminata and 32 men with a history of condyloma acuminata, and compared with 205 men with no history of genital condyloma acuminata.
  • the anti-HPV6 type IgG antibody in the serum was 35% in men who had a history of condyloma acuminata, 27% in the current condyloma acuminata, and 10% in the control group. This indicates that the occurrence of anti-HPV6 IgG antibodies occurred later in the condyloma acuminata, and IgG positive also reflected that the virus had been infected.
  • an object of the present invention is to provide an antigen peptide for detecting an anti-human papillomavirus antibody and a related immunoassay kit, which can detect antigen peptides against human papillomavirus antibodies with high sensitivity
  • Highly specific and highly stable detection of HPV antibodies in blood, saliva or other samples which can provide a basis for doctors to accurately diagnose cervical intraepithelial neoplasia and cervical cancer, and then timely take treatment to prevent cancer , or the spread of cancer, timely relief of patient suffering
  • the immunoassay kit made of it can quickly and accurately detect HPV antibodies in blood, saliva or other samples, suitable for large-scale application.
  • a human papillomavirus antigen peptide is provided, the antigen peptide is derived from an E7 protein of human papillomavirus, and the antigen peptide is 5-50 amino acids in length, and The antigenic peptide can bind to an antibody against human papillomavirus.
  • the antigenic peptide may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 amino acids in length.
  • the antibody comprises a monoclonal antibody, a polyclonal antibody or an antiserum.
  • the m+2th position, the m+3th position, the m+5th position, and the m+6th position of the antigenic peptide are Asp, Ser, Glu, Glu, and/or m+8.
  • the -m+12 amino acids are in turn Asp, Glu, Ile, Asp and Gly, where m ⁇ 0 and m is an integer.
  • the structure of the antigenic peptide is as shown in Formula I
  • XaaO is a peptide consisting of none or 1-5 amino acids
  • Xaal is DS
  • Xaa2 is selected from S or E;
  • Xaa3 is EE
  • Xaa4 is selected from E or N;
  • Xaa5 is DEIDG
  • Xaa6 is a peptide consisting of no or a length of 1 to 30 amino acids.
  • XaaO and/or Xaa6 are not none.
  • the peptide sequence of XaaO and/or Xaa6 is an E7 protein sequence derived from HPV.
  • the "HPV-derived E7 protein sequence” means that the peptide sequence of XaaO is derived from the sequence of the E7 protein of HPV and directly upstream of Xaal and The peptide sequence of Xaa6 is derived from the sequence of the E7 protein of HPV and directly downstream of Xaa5.
  • the E7 protein comprises a wild type and a mutant E7 sequence.
  • the HPV virus comprises HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68.
  • XaaO is a peptide of 1, 2 or 3 amino acids, such as &&0 being 8, LN or IDG.
  • Xaa6 is a peptide consisting of more than 10 (e.g., 10-20, preferably 11-18) amino acids in length. In another preferred embodiment, said Xaa6 is at positions 14-24 of SEQ ID NO.: 3.
  • the Xaa6 is at positions 13-28 of SEQ ID NO.: 5.
  • the antigenic peptides include both salt and non-salt forms.
  • the salt is pharmaceutically acceptable
  • the structure of the antigenic peptide is as shown in Formula II,
  • YaaO is a peptide consisting of no, or 1-30 amino acids
  • Yaal is DS
  • Yaa2 is selected from S or E;
  • Yaa4 is selected from E or N;
  • Yaa5 is a peptide consisting of no, or 1-30 amino acids in length.
  • YaaO and/or Yaa5 are not none.
  • the YaaO is a peptide consisting of 5-25 amino acids and Yaa5 is a peptide consisting of 1-10 amino acids.
  • YaaO is a peptide consisting of 10-25 amino acids and Yaa5 is a peptide consisting of 1-10 amino acids.
  • the YaaO is a peptide consisting of 1-5 amino acids and Yaa5 is a peptide consisting of 10-25 amino acids.
  • YaaO is a peptide consisting of 1-5 amino acids and Yaa5 is a peptide consisting of 15-25 amino acids.
  • the peptide sequence of YaaO and/or Yaa5 is an E7 protein sequence derived from HPV.
  • the "E7 protein sequence derived from HPV” refers to the sequence of the YaaO peptide sequence derived from the E7 protein of HPV and directly upstream of Yaa 1 for YaaO and/or Yaa5.
  • the peptide sequence of and/or Yaa5 is derived from the sequence of the E7 protein of HPV and located directly downstream of Yaa4.
  • the E7 protein comprises a wild type and a mutant E7 sequence.
  • the HPV virus comprises HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 59, HPV 68.
  • the antigenic peptides include both salt and non-salt forms.
  • the salt is a pharmaceutically acceptable salt.
  • the antigenic peptide is selected from the group consisting of SEQ ID NO.: 3, SEQ ID NO.: 5, or a combination thereof. In another preferred embodiment, the antigenic peptide is selected from the group consisting of:
  • the antibody against human papillomavirus is an antibody against the HPV virus E7 protein.
  • the antigenic peptide is obtained by chemical synthesis or bioengineering methods, including recombinant proteins, fusion proteins.
  • a fusion protein comprising the antigenic peptide of the first aspect of the invention and an optional tag sequence.
  • an isolated nucleic acid molecule encoding an antigenic peptide according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.
  • a composition characterized in that it comprises:
  • the composition includes a pharmaceutical composition and a vaccine composition.
  • a pharmaceutical composition in another preferred embodiment, includes a pharmaceutical composition and a vaccine composition.
  • a vaccine composition in another preferred embodiment, includes a pharmaceutical composition and a vaccine composition.
  • the reagent comprises a test strip, a test plate, a protein chip, a liquid-based chip, and a magnetic bead.
  • the drug is selected from a vaccine or an antibody.
  • the anti-human papillomavirus antibody is an anti-human papillomavirus antibody in a human body fluid sample.
  • the body fluid is blood or saliva.
  • an immunoassay kit comprising the antigen peptide for detecting an anti-human papillomavirus antibody according to the first aspect of the invention .
  • the kit may include one or more antigenic peptides for detecting anti-human papillomavirus antibodies according to the first aspect of the invention.
  • the kit further comprises a vector, and the antigenic peptide for detecting an anti-human papillomavirus antibody is coated on the carrier.
  • the immunodetection kit further comprises a labeled secondary antibody against human papillomavirus antibody.
  • the secondary antibody is used to detect antibodies in human serum IgG, IgM, or IgA.
  • the label used may be horseradish peroxidase, alkaline phosphatase, fluorescent molecule FITC (or other fluorescent label), chemiluminescence detection.
  • the detection method may be qualitative, quantitative analysis by chromogenic method, fluorescence method, chemiluminescence, and electrochemiluminescence.
  • the secondary antibody to the labeled anti-human papillomavirus antibody is a secondary antibody to a horseradish peroxidase-labeled anti-human papillomavirus antibody.
  • the immunoassay kit can be quickly and accurately detected in blood or saliva.
  • HPV resistance Body suitable for large-scale promotion and application. detailed description
  • the inventors have extensively and intensively studied, designed and synthesized dozens of human papillomavirus antigen peptides having different sequences, and selected antigen peptides having excellent sensitivity and specificity. Specifically, the inventors applied bioinformatics methods, based on homology analysis and biological characteristics (including immunogenicity), and designed dozens of candidate sequences, which were synthesized and separated by solid phase method. Purification yields high purity polypeptide fragments. For these candidate sequences, peptides were used as ELISA plate antigens, and patient serum (up to 546 patients with cervical cancer and cervical lesions) and healthy subjects (600 cases) were used for detection; and HPV molecular test results and TCT cell morphology were compared.
  • HPV oncoprotein antigen As used herein, the terms “HPV oncoprotein antigen”, “HPV oncoprotein antigen peptide”, “human papillomavirus oncoprotein antigen peptide”, “human papillomavirus oncoprotein antigen”, “antigen of human papillomavirus antibody” "Peptide antigen” is used interchangeably.
  • the polypeptide sequence is derived from a polypeptide of the human papillomavirus (e.g., HPV16, HPV18) E7 protein.
  • HPV antigen of the present invention HPV antigen peptide of the present invention
  • HPV antigen peptide of the present invention human papillomavirus antigen peptide of the present invention
  • human papillomavirus antigen peptide of the present invention an antigen peptide that binds to an anti-human papillomavirus antibody (such as the polypeptides shown in SEQ ID ⁇ : 1 to 5).
  • antigenic peptides can be used to detect oncoprotein antibodies induced by HPV oncogene products.
  • HPV oncoprotein is a cancer cell protein marker that is expressed when HPV virus invades cells.
  • the term also includes variant forms of the sequence of SEQ ID NO: 1-5 having the function of an antibody that binds to anti-human papillomavirus.
  • variants include, but are not limited to, 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions at the C-terminus and/or N-terminus or One or several (usually 3 or less, preferably 2 or less, more preferably 1 or less) amino acids are deleted.
  • the function of the protein is usually not altered.
  • the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the structure and function of the protein.
  • the term also encompasses polypeptides of the invention in both monomeric and multimeric forms. The term also includes both linear as well as non-linear polypeptides (e.g., cyclic peptides).
  • the invention also encompasses active fragments, derivatives and analogs of human papillomavirus antigenic peptides.
  • fragment refers to a polypeptide that substantially retains the function or activity of binding to an antibody against human papillomavirus.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one of the amino acid residues, or (iii) a polypeptide formed by fusion of the antigen peptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence
  • a polypeptide formed by fusion of the polypeptide sequence a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • a preferred class of reactive derivatives means that up to 3, preferably up to 2, and more preferably up to 1 amino acid are replaced by amino acids of similar or similar nature to the amino acid sequence of Formula I or Formula II. Peptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1. Table 1
  • the invention also provides analogs of human papillomavirus antigen peptides.
  • the difference between these analogs and the polypeptides shown in SEQ ID NOS: 1 to 5 may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, hydrazine sulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, amino phthalates or other conventional "prodrugs". Coding sequence
  • the invention also relates to polynucleotides encoding human papillomavirus antigen peptides.
  • the polynucleotide of the present invention may be in the form of DNA or the form of R A .
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the sequence encoding the polypeptides shown in SEQ ID NOS: 1 to 5 or may be a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence which encodes a polypeptide having the sequence of SEQ ID ⁇ : 1 to 5, but differs in the corresponding coding region sequence.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • a DNA sequence encoding the polypeptide of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells produced by genetic engineering using the vectors or polypeptide coding sequences of the invention.
  • the polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide.
  • the polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.
  • a preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods.
  • Solid phase synthesis allows rapid sample acquisition, and the appropriate resin carrier and synthesis system can be selected based on the sequence characteristics of the peptide of interest.
  • a preferred solid phase support in the Fmoc system is a Wang resin with a C-terminal amino acid attached to the peptide, a Wang resin structure is polystyrene, and an arm between the amino acids is 4-alkoxybenzyl alcohol; using 25% hexahydropyridine /Dimercaptoamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus according to the given amino acid sequence. After the completion of the synthesis, the synthesized proinsulin-related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-nonylphenol, and the protecting group was removed.
  • the resin was removed by filtration and the diethyl ether was precipitated to obtain a crude peptide. After the solution of the obtained product was lyophilized, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography.
  • the resin be a PAM resin to which a C-terminal amino acid is linked, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxydecyl phenylacetamide;
  • the protecting group Boc was removed with TFA/dichlorodecane (DCM) and neutralized with diisopropylethylamine (DIEA/dichloromethane).
  • the peptide chain is cleaved from the resin by treatment with hydrogen fluoride (HF) containing p-nonylphenol (5-10%) at 0 ° C for 1 hour while removing the protecting group. 50-80%
  • HF hydrogen fluoride
  • the peptide is extracted with acetic acid (containing a small amount of thiol ethanol), and the solution is further lyophilized and further purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. It can be used in the field of peptide chemistry.
  • Various coupling agents and coupling methods are coupled to each amino acid residue, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1,1,3,3-tetraurea can be used.
  • DCC dicyclohexylcarbodiimide
  • HOBt hydroxybenzotriazole
  • 1,1,3,3-tetraurea can be used.
  • the human papillomavirus antigen peptide of the present invention is prepared by solid phase synthesis according to its sequence, and purified by high performance liquid chromatography to obtain a high-purity peptide freeze-dried powder, which is stored at -20 °C.
  • Another method is to produce a polypeptide of the invention using recombinant techniques.
  • the polynucleotide of the present invention can be utilized to express or produce the human papillomavirus antigen peptide of the present invention by conventional recombinant DNA techniques. Generally there are the following steps:
  • the recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography
  • polypeptide of the present invention is short, it is conceivable to connect a plurality of polypeptides in series, to obtain an expression product in a multimeric form after recombinant expression, and then to form a desired small peptide by enzymatic cleavage or the like.
  • the antigenic peptide or fusion protein of the present invention can induce an antibody against HPV or HPV-positive cells (cancer cells) and lymphocyte T cell activity as an immunogen in vivo, thereby achieving an anticancer therapeutic effect. Further, the antigen peptide of the present invention has excellent specificity and immunological activity, and thus can be used for preparing an anti-cancer vaccine for immunotherapy or prevention.
  • the present invention provides a pharmaceutical composition (including a vaccine composition) comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof (or a coding sequence thereof); b) a pharmaceutically acceptable carrier or excipient.
  • the amount of the polypeptide of the present invention is usually from 10 ⁇ g to 100 mg / dose, preferably from 100 to 1000 ⁇ g / dose.
  • an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg of body weight of the polypeptide of the invention.
  • the polypeptide of the present invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent.
  • pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • the pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the therapeutic composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be prepared as a solid form of liquid carrier suitable for administration in solution or suspension prior to injection.
  • composition of the invention can be administered by conventional routes including, but not limited to, intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the subject to be prevented or treated may be an animal; especially a human.
  • a pharmaceutical composition of various dosage forms can be used depending on the use. It is preferably an injection.
  • compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in the usual manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity Isotonicities, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers and solubilizers, and the formulation process can be carried out in the usual manner according to the dosage form.
  • compositions of the invention may also be administered in the form of sustained release agents.
  • sustained release agents a human papillomavirus antigen peptide or a salt thereof can be incorporated
  • the pellet or microcapsule is loaded with a sustained-release polymer, and the pellet or microcapsule is then surgically implanted into a human tissue.
  • sustained-release polymer examples include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, mercaptocellulose, lactic acid polymer, A lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.
  • the dose of the human papillomavirus antigen peptide or a pharmaceutically acceptable salt thereof as an active ingredient may be according to each subject (patient) to be prevented or treated.
  • the weight, age, sex, and degree of symptoms are reasonably determined.
  • the main advantages of the invention include:
  • the human papillomavirus antigen peptide of the present invention has the ability to specifically bind to an anti-human papillomavirus antibody, and has high sensitivity and specificity;
  • the human papillomavirus antigen peptide of the present invention is moderate in length and easy to prepare;
  • MULTISKAN MK3 microplate reader Thermo
  • ELISA plate COSTAR
  • Coating buffer Potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 12g 2.9g, sodium chloride 8.0g, potassium chloride 0.2g, distilled water to 1L, 4 degrees storage.
  • Wash buffer PBS-Tween 20, 0.1%): Tween-20 lmL, add to 1L of the dilution buffer (PBS), mix and mix, adjust the pH to 7.0 ⁇ 7.2.
  • Blocking solution 5%milk-PBS-Tween
  • TMB display liquid Amresc, CAT NO.64285730, before use TMB background color development A liquid and B liquid volume mixing.
  • Substrate coloring Liquid A Sodium acetate 13.6g, citric acid 1.6g, hydrogen peroxide (30%) 0.3ml, distilled water added to 500ml.
  • Substrate coloring liquid B disodium edetate 0.2g citric acid 0.95g glycerol 50ml tetraterpene Aniline 0.2 g of distilled water was added to 500 ml.
  • TMB color development TMB A liquid and B liquid are mixed in equal volume, lOOul/well, 37 degrees 20 min.
  • This kit consists of the following parts:
  • TMB coloring solution A bottle (6ml), one bottle of sputum (6ml)
  • the polypeptide antigen is as follows
  • sequence alignment of SEQ ID NO.: 2-5 is as follows:
  • SEQ ID NO. : 3 LNDSSEEEDEIDGPAGQAEPDRAH
  • SEQ ID NO. : 4 IDGVNHQHLPARRAEPQR
  • the kit selects 5 polypeptides (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5) as coating selection, serum is detected by ELISA method, and each record is recorded. The OD value of each sample (450 nm wavelength).
  • the healthy human serum was detected by ELISA using an enzyme-labeled plate coated with an antigen, and the raw data of the test were as follows.
  • the patient's serum was detected by ELISA method as the coated and encapsulated ELISA plate.
  • the pathological diagnosis results of the patient are shown in Table 3. Some of the patients were examined by HC2 pathology. The results are as follows:
  • Serum number pathological data NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 Patient No. 1 CIN III 0.917 0.533 0.723 0.852 0.643 Patient No. 2 CIN II 1.290 0.640 0.714 1.225 1.085 Patient No. 3 CIN II 0.782 0.516 0.295 0.822 0.684 Patient No. 4 CIN III 1.100 0.708 0.676 1.413 0.886 Patient No. 5 Cervical Cancer 0.489 0.344 0.447 0.708 0.536 Patient No. 6 Ovarian Cancer 0.472 0.419 0.447 1.035 0.535 Patient No. 7 Uterine Fibroids 0.909 0.326 0.263 0.844 0.395 Patient No.
  • Table 5 HPP E7 5 peptide serum ELISA test data sheet SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 Average 0.292 0.279 0.382 0.301 0.383 Standard deviation 0.098 0.100 0.111 0.076 0.111 cutoff 0.487 0.480 0.604 0.452 0.605
  • Table 7 According to the results of Table 3, the comparison of serum ELISA results and HC2 diagnosis results for patients
  • Table 9 HC2 results and blood ELISA detection accuracy rate and proportion comparison table
  • gynecological diseases are mainly pelvic inflammatory disease, ovarian swelling, uterine fibroids and other gynecological diseases.
  • the HC2 results of the results in Table 8 and the blood ELISA results were statistically analyzed. The results are as follows:
  • Table 10 HC2 results and blood ELISA detection accuracy rate and proportion comparison table Pathological diagnosis results Number of people HC2 detection SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 5 positive positive positive positive positive
  • gynecological diseases are mainly pelvic inflammatory disease, ovarian swelling, uterine fibroids and other gynecological diseases.
  • Table 11 Summary of HPV blood ELISA results for patients without HC2 diagnosis, but with pathological findings
  • Table 12 Number of patients with CIN, cervical cancer and cervical lesions without blood HC2
  • Table 13 Proportion of the number and accuracy of blood ELISA for all patients with CIN, cervical cancer and cervical lesions
  • the above results indicate that the antigen peptides SEQ ID NO: 2, 3, 5 are more sensitive and accurate for CIN II detection; and the antigen peptides SEQ ID NO: 3, 4, 5 are more sensitive and accurate for CIN III and cervical cancer detection. excellent.
  • the above polypeptide of the present invention is selected by the inventors and verified in the patient sample and the normal human sample, and the GST-HPV16-E7 and GST-HPV118E7 full-length fusion proteins (SEQ ID NO: 6 respectively) are directly used.
  • the sequence shown in SEQ ID NO: 7) is not effective, please refer to the following experimental data.
  • the experimental materials and ELISA methods are the same as the first point above, and the GST-HPV16-E7 and GST-HPV118E7 full-length fusion proteins are also used together with the detection of the polypeptide (SEQ ID NO: 3, SEQ ID NO: 5).
  • SEQ ID NO: 3 SEQ ID NO: 5
  • As a detection antigen when the GST-HPV16-E7 and GST-HPV18-E7 full-length fusion proteins were diluted with PBS, the concentration was 2 ug/ml, and the results were as follows:
  • test results and patient pathology report are as follows:
  • the ELISA method is used to detect the full-length HPV 16-E7 protein, or HPV 18-E7 protein.
  • the monoclonal antibody obtained as an immunizing antigen was subjected to specific binding test with each antigen peptide.
  • each monoclonal antibody is a monoclonal antibody which specifically binds to the full-length 16-E7 or 18-E7 antigen.
  • the antigen peptides of SEQ ID NOS: 1-5 can bind to various monoclonal antibodies. This result suggests that the antigenic peptide of the present invention has an epitope capable of specifically binding to a human papillomavirus antibody (see the table below). Also. SEQ ID No.: 1-5 also does not bind to other unrelated antibodies (including antibodies against other viral proteins), suggesting that the antigenic peptide of the present invention is cross-reactive.
  • the antigenic peptide of the present invention can be used not only for detecting (especially early detection) cancer caused by HPV infection and HPV infection, but also for preparing a prophylactic or therapeutic pharmaceutical composition (including a vaccine composition), thereby Effectively induces the body to produce a specific immune response against human papillomavirus HPV oncoprotein, thereby preventing cancer or treating cancer.
  • a prophylactic or therapeutic pharmaceutical composition including a vaccine composition
  • the blood enzyme-labeled immunoassay (ELISA) of the present invention provides an antigen-antibody detection method with high sensitivity, high specificity and high stability based on the principle of immunobiochemistry.
  • the patient develops specific antibody IgM, IgA or IgG molecules in serum and oral saliva after infection by the virus.
  • Antibody-specific anti-HPV antibody molecules in serum or saliva were detected by enzyme-labeled immunoassay ELISA.
  • the invention predicts the antigenicity by bioinformatics analysis through the analysis of HPV virus high-risk HPV16 and HPV18 gene sequences, and uses experimental methods to screen and prove the significance of clinical application.
  • the target antigen HPV16-1 By preparing a plurality of high-purity antigenic polypeptides, and repeatedly screening and verifying the prepared antigens by the serum of the domestic population and the serum of the infected person, the target antigen HPV16-1 with high sensitivity, high specificity and high stability was obtained. HPV 16-2, HPV 16-3, HPV18- HPV18-2. It has also been demonstrated that the binding of these polypeptide antigens to the surface of a solid phase carrier maintains its immunoreactive activity. At the time of measurement, the test specimen is allowed to react with the antigen on the surface of the solid phase carrier.
  • the blood ELISA detection kit of the invention has uniform sampling, non-aggressiveness, quicker operation and simple operation; low cost, easy promotion, and more suitable for the Chinese market; False positives and false negatives greatly improve the accuracy and accuracy of the test results; compared with traditional Pap smear, acetic acid stained VIA, colposcopy, more accurate, easy and fast.
  • ELISA detection method through various parameters and experimental procedures The optimization can be used as a tool for large-scale screening for early screening of cervical cancer.
  • the antigen for detecting an anti-human papillomavirus antibody of the present invention can detect HPV antibodies in blood or saliva with high sensitivity, high specificity and high stability, thereby accurately diagnosing cervical intraepithelial neoplasia for a doctor. And cervical cancer to provide the basis, and then timely treatment to prevent cancer, or cancer spread, timely relief of patient suffering, the immunoassay kit made of it can quickly and accurately detect HPV antibodies in blood or saliva, Suitable for large-scale promotion of applications.

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Abstract

L'invention concerne un peptide antigénique utilisé pour détecter des anticorps anti-virus du papillome humain (HPV), ledit peptide antigénique étant dérivé de la protéine E7 de HPV. Ces protéines peuvent être utilisées pour détecter des anticorps anti-HPV dans le sang ou dans la salive. L'invention concerne également une composition et un kit d'essai comprenant lesdits peptides antigéniques.
PCT/CN2014/072407 2013-02-22 2014-02-21 Antigène permettant de détecter des anticorps anti-virus du papillome humain, kit d'essai et leurs applications WO2014127741A1 (fr)

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US7026443B1 (en) * 1999-12-10 2006-04-11 Epimmune Inc. Inducing cellular immune responses to human Papillomavirus using peptide and nucleic acid compositions
WO2001077142A1 (fr) * 2000-04-05 2001-10-18 Impact Diagnostics, Inc. Methodologie immunologique permettant de discerner le papillomavirus humain
US7314630B2 (en) * 2005-01-07 2008-01-01 Yao-Xiong Hu Compounds and methods of early diagnosis of cervical cancer and genital condyloma with HPV, CHSP60 tumor suppressor H-Ras, K-Ras and PTEN derived peptides modified
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