CN106939034B - Methods and kits for identifying HEV genotypes infected by a subject - Google Patents

Abstract

The present invention is in the fields of molecular biology, immunology and disease diagnosis. Specifically, the invention relates to an HEV epitope polypeptide, which can be used for identifying different genotypes of HEV infection. The invention also relates to a kit for identifying different genotypes of HEV infection. The invention also relates to a method for identifying different genotypes of HEV infection based on immunological detection, and provides a powerful tool for virus diagnosis, epidemiological investigation and the like.

Description

Methods and kits for identifying HEV genotypes infected by a subject

Technical Field

The present invention is in the fields of molecular biology, immunology and disease diagnosis. In particular, the invention relates to an HEV epitope polypeptide which can be used for identifying the genotype of the HEV infected by a subject. The invention also relates to a kit for identifying the genotype of HEV infected by a subject. The invention also relates to a novel method for identifying the genotype of HEV infected by a subject.

Background

Hepatitis E Virus (HEV) is the causative agent of Hepatitis E. Hepatitis E Virus (HEV), caused by hepatitis e virus (HE), has become a serious public health problem that endangers human health. The WHO reports about 2000 million infections, 300 more than ten thousand cases of acute hepatitis and 7 more than ten thousand cases of death worldwide each year. The mortality rate of pregnant women infected with HEV is higher and can reach 20%. At present, more and more researches show that after organ transplantation and HIV infection and other patients with low immune function infect HEV, chronic hepatitis can be delayed. Since 2004, the incidence of hepatitis E in our population has increased year by year, and many cases of chronic hepatitis have also been found. Currently, there are internationally recognized HEV genotypes that can infect humans, type 1, type 2, type 3, and type 4. HEV genotypes 1 and 2 only infect humans, and genotypes 3 and 4 can infect humans, pigs, and other animals. Among them, genotype 2 is popular mainly in africa and central america, and is not found in China at present, and other genotypes are found in China.

Because different genotypes of HEV have obvious epidemiological difference, the definition of the HEV genotypes has important significance for the diagnosis, prevention and epidemiology of hepatitis E. The conventional genotyping method mainly comprises direct sequencing analysis after specific primer amplification, restriction fragment length restriction polymorphism (RFLP) analysis, a specific probe PCR method, a gene chip method and the like, and although the methods have high specificity and sensitivity, the genotyping methods all need RNA extraction and other processes, are complex to operate and have high technical requirements, and are difficult to develop in a common laboratory; and when the RNA in the sample is negative or damaged due to improper storage or handling, genotyping cannot be performed.

Accordingly, there is a need in the art to develop HEV genotyping methods that do not rely on genetic sequencing to achieve more economical, efficient, rapid, and accurate identification of HEV genotypes infected by a subject.

Disclosure of Invention

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of cell culture, biochemistry, cell biology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "HEV ORF3 protein" refers to the protein ORF3 naturally occurring in the hepatitis e virus, encoded by open reading frame 3(ORF3) of the HEV genome. The sequences of the ORF3 proteins of different genotypes of HEV virus are well known in the art, for example, the sequence of the ORF3 protein of type 1HEV virus can be found in various public databases (e.g., GenBank database accession numbers D11092, JQ655734, M73218, or X98292), the sequence of the ORF3 protein of type 3 HEV virus can be found in various public databases (e.g., GenBank database accession numbers AF060668, AP003430, AB073912, or AY115488), and the sequence of the ORF3 protein of type4 HEV virus can be found in various public databases (e.g., GenBank database accession numbers AB197673, AB097811, AY723745, or JQ 655736).

In the present invention, when referring to the amino acid sequence of ORF3 protein of type 1HEV virus, reference is made to SEQ ID NO:1, is described. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of type 1HEV virus" means that the amino acid sequence of SEQ ID NO:1, amino acid residues 66-87 of the polypeptide shown in figure 1. However, it is understood by those skilled in the art that type 1HEV virus can include multiple isolates, and that there may be differences between the amino acid sequences of the ORF3 proteins of the various isolates. Further, it is understood by those skilled in the art that although sequence differences may exist, the ORF3 proteins of different isolates of type 1HEV virus have very high identity in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the expression "ORF 3 protein of type 1HEV virus" includes not only the protein shown in SEQ ID NO:1 but also ORF3 proteins of various type 1HEV virus isolates (for example, ORF3 protein shown in GenBank database accession No. JQ 655734). And, when describing the sequence fragment of ORF3 protein of type 1HEV virus, it includes not only SEQ ID NO:1, and also includes the corresponding sequence fragment in ORF3 protein of various type 1HEV virus isolates. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of type 1HEV virus" includes, SEQ ID NO:1, and the corresponding fragment of the ORF3 protein of each type 1HEV virus isolate.

In the present invention, when referring to the amino acid sequence of ORF3 protein of type 3 HEV virus, reference is made to SEQ ID NO:2, to the sequence shown in figure 2. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of HEV type 3 virus" means that the amino acid sequence of SEQ ID NO:2, amino acid residues 66-87 of the polypeptide shown in figure 2. However, it is understood by those skilled in the art that type 3 HEV virus can include multiple isolates, and that there may be differences between the amino acid sequences of the ORF3 proteins of the various isolates. Further, it is understood by those skilled in the art that although sequence differences may exist, the ORF3 proteins of different isolates of type 3 HEV virus have very high identity in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the expression "ORF 3 protein of type 3 HEV virus" includes not only the protein shown in SEQ ID NO:2 but also ORF3 proteins of various type 3 HEV virus isolates (e.g., ORF3 protein shown in GenBank database accession No. AF 060668). And, when describing the sequence fragment of ORF3 protein of type 3 HEV virus, it includes not only SEQ ID NO:2, and also includes the corresponding sequence fragment in ORF3 protein of various type 3 HEV virus isolates. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of HEV type 3 virus" includes, SEQ ID NO:2, and the corresponding fragment of ORF3 protein of each type 3 HEV virus isolate.

In the present invention, when referring to the amino acid sequence of ORF3 protein of type4 HEV virus, reference is made to SEQ ID NO:3, is described. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of HEV type4 virus" means that the amino acid sequence of SEQ ID NO:3 at amino acid residues 66-87 of the polypeptide. However, it is understood by those skilled in the art that type4 HEV virus can include multiple isolates, and that there may be differences between the amino acid sequences of the ORF3 proteins of the various isolates. Further, it is understood by those skilled in the art that although sequence differences may exist, the ORF3 proteins of different isolates of type4 HEV virus have very high identity in amino acid sequence and have essentially the same biological function. Therefore, in the present invention, the expression "ORF 3 protein of type4 HEV virus" includes not only the protein shown in SEQ ID NO:3 but also ORF3 proteins of various type4 HEV virus isolates (for example, ORF3 protein shown in GenBank database accession No. JQ 655733). And, when describing the sequence fragment of ORF3 protein of type4 HEV virus, it includes not only SEQ ID NO:3, and also includes the corresponding sequence fragment in ORF3 protein of various type4 HEV virus isolates. For example, the expression "amino acid residues 66 to 87 of ORF3 protein of HEV type4 virus" includes, SEQ ID NO:3, and the corresponding fragment of the ORF3 protein of each type4 HEV virus isolate.

In the present invention, the expression "corresponding fragments" refers to the fragments at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e., when the sequences are aligned for the highest percent identity.

As used herein, the term "epitope polypeptide" refers to a polypeptide fragment of an antigenic protein that is recognized by a specific antibody.

As used herein, the expression "epitope polypeptides having amino acid sequences shown as SEQ ID NOS.4 to 6, respectively" refers to a combination of an epitope polypeptide having an amino acid sequence shown as SEQ ID NO.4, an epitope polypeptide having an amino acid sequence shown as SEQ ID NO.5, and an epitope polypeptide having an amino acid sequence shown as SEQ ID NO.6.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide may be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; bacteriophage; cosmids, and the like.

As used herein, the term "detectable label" refers to any composition detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. In the present invention, it is particularly preferred that such labels can be suitably used for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluoroimmunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., Fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), Phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g.,

) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and a biological agent for binding to the label-modified avidin (e.g., streptavidin)And (4) element. Patents that teach the use of such labels include, but are not limited to, U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers encompassed by the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or scintillation calculators, and fluorescent labels can be detected using photodetectors to detect the emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

As used herein, the term "carrier protein" refers to a protein that, upon attachment to an antigen or hapten, induces an immune response in the body against the antigen or hapten, wherein the immune response includes a reaction that produces an antibody that specifically binds to the antigen or hapten. Such proteins are well known in the art and include, but are not limited to, Bovine Serum Albumin (BSA), Keyhole Limpet Hemocyanin (KLH), Ovalbumin (OVA), Bovine Thyroglobulin (BTG), and the like.

As used herein, the expression "detection reagent capable of recognizing and binding to anti-HEV antibodies" refers to a substance capable of specifically binding to anti-HEV antibodies. Such agents are known in the art or may be prepared using methods known in the art, such as antibodies (i.e., secondary antibodies), targeting polypeptides, or aptamers. In general, it is particularly preferred that such reagents are capable of determining the amount of anti-HEV antibodies in a sample by immunological detection. The use of immunological detection is particularly advantageous because it exploits the specific interaction/binding affinity between antigen-antibody. Thus, the reagent can be used to determine the amount of anti-HEV antibody in a sample by immunological detection, as long as the reagent retains reactivity with specific binding to the anti-HEV antibody (i.e., the reagent can be used as a detection reagent capable of recognizing and binding to the anti-HEV antibody). Various agents that retain reactivity for specific binding to anti-HEV antibodies are readily envisioned and readily available to those skilled in the art, including, but not limited to, antibodies or antigen-binding fragments thereof against HEV antibodies, e.g., polyclonal or monoclonal antibodies against HEV antibodies, e.g., antibodies derived from a subject species.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as a reaction between an antibody and an antigen against which it is directed. Binding affinity between two molecules may be represented by K_DThe value describes. K_DThe value refers to the dissociation constant derived from the ratio of kd (the dissociation rate of a particular binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a particular binding molecule-target molecule interaction; also known as kon), or kd/ka expressed as molarity (M). K_DThe smaller the value, the more tightly bound the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen. K_DValues can be determined by methods well known in the art, for example, in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the term "immunological detection" refers to an assay that utilizes specific antigen-antibody interactions/binding affinities, which are generally useful for detecting the presence or level of a particular antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art and include, but are not limited to, Enzyme Immunoassay (EIA), chemiluminescence immunoassay (CLIA), Radioimmunoassay (RIA), Fluorescence Immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, and the like. In certain embodiments, the immunological assay is an Enzyme Immunoassay (EIA), such as an ELISA assay, an Elispot assay, or a CLEIA assay. For a detailed description of immunological assays, see, e.g., Fundamental Immunology, ch.7paul, w., ed., 2 nd edition, Raven Press, n.y. (1989).

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, an "antigen-binding fragment" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen to which the antibody binds (e.g., an anti-HEV antibody), competing with an intact antibody for specific binding to the antigen. See, generally, Fundamental Immunology, ch.7paul, w., ed., 2 nd edition, Raven Press, n.y. (1989), which is incorporated by reference herein in its entirety for all purposes. Antigen-binding fragments can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab ', F (ab')2, Fd, Fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabodies), and polypeptides comprising at least a portion of an antibody sufficient to confer upon the polypeptide the ability to specifically bind an antigen.

As used herein, the term "Aptamer (Aptamer)" refers to a single-stranded oligonucleotide capable of binding a target protein of interest (e.g., an anti-HEV antibody) or other biological target molecule with high affinity and high specificity, which can be folded to form a thermodynamically stable three-dimensional spatial structure such as a Stem-Loop (Stem-Loop), a Hairpin (Hairpin), a Pseudoknot (pseudokinot), or a G-tetramer (G-tetramer), and specifically bind to the target protein of interest or other biological target molecule by, for example, structural complementarity, base stacking forces, van der waals forces, hydrogen bonding, or electrostatic interactions. The aptamer may be DNA or RNA, and may also comprise a nucleic acid analog (e.g., Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), diol nucleic acid (GNA), or Threose Nucleic Acid (TNA)). Methods for obtaining aptamers that bind to a particular target protein are well known in the art, such as the SELEX (systematic evolution of ligands by exogenous genetic engineering) screening technology.

As used herein, the term "targeting polypeptide" refers to a polypeptide molecule that can specifically bind a target protein of interest (e.g., an anti-HEV antibody). In the present invention, the targeting polypeptide may comprise natural amino acids, synthetic amino acids, or amino acid mimetics (mimetics) that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those amino acids which are subsequently modified, for example hydroxyproline, γ -hydroxyglutamate, O-phosphoserine, phosphothreonine or phosphotyrosine. In the present invention, the "specificity" between a targeting polypeptide and its target protein of interest can be determined based on the affinity with which the targeting polypeptide can be usedDissociation equilibrium constant (i.e., K) of the target protein of interest to which it binds_DValue) is described. K_DThe lower the value, the stronger the binding strength between the targeting polypeptide and the target protein of interest to which it binds. Greater than about 10, as is generally known in the art^-3K of M_DValues are generally considered to represent non-binding or non-specific binding. Depending on the particular target protein of interest, the targeting polypeptide that specifically binds to the target protein can be obtained by methods known to those skilled in the art, e.g., screening by phage display technology or protein microarray technology.

As used herein, the term "Secondary antibody" refers to an antibody that can specifically bind to an antibody. Generally, in immunological assays, the Fab domain of a primary antibody (e.g., an anti-HEV antibody) binds to an antigen, resulting in exposure of its Fc domain, and thus secondary antibodies typically bind to the Fc domain of the primary antibody. Since the Fc domain is constant in the same animal class, one type of secondary antibody can bind many types of primary antibodies from the same animal class. The skilled person is well aware of how to select the corresponding secondary antibody depending on the species source of the primary antibody.

As used herein, the term "capture reagent" refers to a substance used for immobilization on a solid support in an immunological assay that specifically binds to an antibody or antigen to be detected. In the invention, the capture reagent is the epitope polypeptide or the complex of the invention, which can specifically bind to the anti-specific genotype HEV antibody in the sample to be detected, and then the amount of the captured anti-HEV antibody is determined to complete the whole immunological detection.

As used herein, the term "positive control sample" refers to a sample containing a known amount of antibodies against a specific genotype HEV, such as a sample containing a known amount of anti-type 1HEV antibodies, a sample containing a known amount of anti-type 3 HEV antibodies, or a sample containing a known amount of anti-type 4 HEV antibodies. In certain embodiments, the amount of test antibody in the test sample can be calculated by comparing the test results of the test sample with the test results of the positive control sample, such methods being well known in the art, for example, by constructing a standard curve by testing different concentrations of the positive control sample. In the present invention, the term "negative control sample" refers to a sample that does not contain anti-HEV antibodies or antibodies against a specific genotype HEV, such as a sample that does not contain anti-type 1HEV antibodies, a sample that does not contain anti-type 3 HEV antibodies, or a sample that does not contain anti-type 4 HEV antibodies.

As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., rhesus monkeys or cynomolgous monkeys), or humans.

The present invention is based, at least in part, on the following unexpected findings of the inventors: the amino acid residues at positions 66-87 of HEV ORF3 protein have obvious genotype specificity, and the epitope polypeptide derived from different genotypes HEV (such as genotype 1, genotype 3 or genotype 4) and consisting of the amino acid residues has obviously higher binding reaction strength to the same type antiserum generated by immunizing mammals with the full-length protein of the same genotype HEV ORF3 or the same type antiserum generated by animals naturally infected with the same genotype HEV than the non-same type antiserum. Based on this finding, the present inventors have developed a novel method for identifying the genotype of HEV infected by a subject.

Accordingly, in one aspect, the present invention provides an isolated epitope polypeptide comprising amino acid residues 66-87 of HEV ORF3 protein.

In certain preferred embodiments, the epitope polypeptide consists of amino acid residues 66-87 of HEV ORF3 protein.

In certain preferred embodiments, the HEV ORF3 protein is an ORF3 protein of a type 1HEV virus, an ORF3 protein of a type 3 HEV virus, or an ORF3 protein of a type4 HEV virus.

In certain preferred embodiments, the ORF3 protein of type 1HEV virus has the amino acid sequence shown as SEQ ID NO. 1.

In certain preferred embodiments, the ORF3 protein of type 3 HEV virus has the amino acid sequence shown in SEQ ID NO. 2.

In certain preferred embodiments, the ORF3 protein of type4 HEV virus has the amino acid sequence shown as SEQ ID NO. 3.

In certain preferred embodiments, the epitope polypeptide has an amino acid sequence selected from the group consisting of: 4-6 of SEQ ID NO.

In the present invention, the epitope polypeptide of the present invention is not limited by any particular method for synthesizing a polypeptide, and can be produced by a conventional technique known to those skilled in the art, such as a DNA recombination technique or a chemical synthesis technique. In certain embodiments, the epitope polypeptides of the invention are obtained by DNA recombination techniques, e.g., by using cell-free expression systems from polynucleotides encoding these proteins or polypeptides (cell-free expression systems include, e.g., reticulocyte lysate-based expression systems, wheat germ extract-based expression systems, and e.coli extract-based expression systems); or by using in vivo expression systems (e.g., E.coli prokaryotic expression systems, yeast eukaryotic expression systems) from polynucleotides encoding these proteins or polypeptides. Alternatively, the epitope polypeptide of the present invention can be produced by chemical synthesis. Methods for the chemical total synthesis of proteins or polypeptides are well known in the art (see, e.g., Raibaut L, et al, Top Curr chem.2015; 363: 103-54; Thapa P, et al. Molecules.2014; 19(9): 14461-83; Dawson PE, et al., Science, 1994; 266(5186): 776-9; and Wang P, et al., Tetrahedron Lett,1998,39(47): 88711-14; incorporated herein by reference) and include, but are not limited to: solid Phase Peptide Synthesis (SPPS) or liquid Phase stepwise Synthesis (e.g., Native Chemical Ligation (NCL), Azide method (Azide method), and Transfer activated Ester method (TAEC)).

In another aspect, the invention provides an isolated nucleic acid encoding an epitope polypeptide as described above.

In another aspect, the invention provides a vector comprising an isolated nucleic acid as described above. Vectors useful for inserting a polynucleotide of interest are well known in the art and include, but are not limited to, cloning vectors and expression vectors. In certain embodiments, the vector is, for example, a plasmid, cosmid, phage, or the like.

In another aspect, the invention also relates to a host cell comprising an isolated nucleic acid or vector as described above. Such 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 (e.g., mammalian cells, e.g., primate cells, human cells, etc.). The host cell of the invention may also be a cell line, such as 293T cells.

In another aspect, the present invention provides a complex comprising an epitope polypeptide of the present invention and a modifying group, wherein the modifying group is a carrier protein or a detectable label, and the epitope polypeptide is coupled, conjugated or fused to the modifying group to give the complex.

In certain preferred embodiments, the modifying group is selected from biotin, avidin, streptavidin, or deglycosylated avidin (e.g.,

). In a specific embodiment, the modifying group is biotin.

In certain preferred embodiments, the modifying group is linked to the N-terminus or C-terminus of the epitope polypeptide through an amino acid residue of the epitope polypeptide. In certain preferred embodiments, the modifying group is linked to the N-terminus or C-terminus of the epitope polypeptide by a linker group. Such linker groups are well known in the art and include, but are not limited to, glycine (Ahx), β -alanine (β -Ala), 4-aminobutyric acid (GABA), 5-aminopentanoic acid (Ava), or polyethylene glycol (PEG). The modification group may be attached to the epitope polypeptide of the present invention using attachment methods well known in the art, including, but not limited to, enzymatic methods, oxidative substitution methods, or covalent coupling methods, such as a carbodiimide method, an active ester method, a glutaraldehyde method, a succinic anhydride method, or a diazotization method; see, e.g., Greg t. hermanson, bioconjugation technology, 3 rd edition, Academic Press, 2013, and c.m. niemeyer, bioconjugation experimental methods-strategies and methods, humamana Press, 2004, all of which are incorporated herein by reference.

In another aspect, the invention provides a kit comprising an epitope polypeptide of the invention or a complex as described above.

In certain preferred embodiments, the kit comprises one, two or three epitope polypeptides of the invention.

In certain preferred embodiments, the kit comprises:

a first epitope polypeptide comprising amino acid residues 66-87 of type 1HEV ORF3 protein;

a second epitope polypeptide comprising amino acid residues 66-87 of type 3 HEV ORF3 protein; and

a third epitope polypeptide comprising amino acid residues 66-87 of type4 HEV ORF3 protein.

In certain preferred embodiments, the kit comprises one, two or three complexes of the invention.

In certain preferred embodiments, the kit comprises:

a first complex comprising an epitope polypeptide having amino acid residues 66-87 of type 1HEV ORF3 protein;

a second complex comprising an epitope polypeptide having amino acid residues 66-87 of type 3 HEV ORF3 protein; and

a third complex comprising an epitope polypeptide having amino acid residues 66-87 of type4 HEV ORF3 protein.

In the present invention, a kit comprising the first epitope polypeptide, the second epitope polypeptide and the third epitope polypeptide as described above or a kit comprising the first complex, the second complex and the third complex as described above is particularly advantageous, which allows one-time identification of whether a subject is infected with HEV of type 1, type 3 or type 4.

In a specific embodiment, the kit comprises epitope polypeptides having amino acid sequences as shown in SEQ ID NOS 4-6, respectively.

In another specific embodiment, the kit comprises:

a first complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO 4;

a second complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO. 5; and

a third complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO 6. In certain preferred embodiments, the kit further comprises a detection reagent capable of recognizing and binding anti-HEV antibodies. Such detection reagents are well known in the art and include, but are not limited to, antibodies, targeting polypeptides or aptamers capable of specifically binding to anti-HEV antibodies. In certain preferred embodiments, the detection reagent is a secondary antibody.

In certain preferred embodiments, the detection reagent carries a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters) or a fluorescent dye. In certain embodiments, when the detectable label is an enzyme, the kit may further comprise a color developing solution, such as o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), ABTS, or luminol-like compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.

In certain preferred embodiments, the kit further comprises a solid support, optionally further comprising a coating reagent, such as a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCL buffer, or borate buffer), for coating the epitope polypeptide or the complex onto the solid support. In certain preferred embodiments, the solid support comprises a well plate, test tube, bead (e.g., latex particle) or thin film (e.g., nitrocellulose membrane) made of or coated with a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with a functional group (e.g., amino, carboxyl, biotin, or avidin). In certain embodiments, the solid support is a microtiter plate, such as a microtiter plate or an elisa plate. Methods for coating proteins or polypeptides onto solid phase supports are well known in the art, e.g., physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, protein A or protein G pre-coated surfaces.

In certain preferred embodiments, the kit comprises a solid support having the epitope polypeptide or the complex coated thereon.

In certain preferred embodiments, the kit further comprises one or more reagents or devices selected from the group consisting of: a diluent for diluting the sample (for example, a phosphate buffer or a phosphate buffer containing a nonionic surfactant); a positive control sample (e.g., a sample containing a known amount of antibodies against a particular genotype HEV); a negative control sample (e.g., a sample that does not contain anti-HEV antibodies or a sample determined to not contain anti-specific genotype HEV antibodies); a stopping solution (e.g., sulfuric acid, hydrochloric acid, or sodium hydroxide solution) for stopping the color reaction of the enzyme-catalyzed substrate; a washing solution for removing a reagent not participating in the reaction (for example, a phosphate buffer or a phosphate buffer containing a nonionic surfactant); a blocking solution for inhibiting non-specific binding; and, a blood collection device (e.g., a pyrogen-free evacuated blood collection tube).

In certain preferred embodiments, to inhibit non-specific interactions between proteins or non-specific adsorption between proteins and a solid support in a sample, a non-ionic surfactant, such as tween-20, tween-40, tween-60, tween-80, Triton X-20, Triton X-100 or Triton X-300; or inert proteins such as albumin (e.g. BSA), serum (e.g. calf serum), skimmed milk powder or casein. In certain preferred embodiments, the pH of the diluent, wash solution or blocking solution is maintained between 7.0 and 10.0.

In certain preferred embodiments, the positive control sample is selected from a sample containing a known amount of anti-type 1HEV antibody, a sample containing a known amount of anti-type 3 HEV antibody, or a sample containing a known amount of anti-type 4 HEV antibody. In certain preferred embodiments, the negative control sample is selected from a sample that does not contain anti-type 1HEV antibodies, a sample that does not contain anti-type 3 HEV antibodies, or a sample that does not contain anti-type 4 HEV antibodies.

In another aspect, the invention also relates to the use of an epitope polypeptide or complex as described above in the preparation of a kit for identifying the genotype of a HEV infected with a subject.

In certain preferred embodiments, the HEV genotype is selected from the group consisting of type 1, type 3, and type 4.

In certain preferred embodiments, the kit comprises one, two or three epitope polypeptides or complexes of the invention.

In certain preferred embodiments, the kit comprises:

a first epitope polypeptide comprising amino acid residues 66-87 of type 1HEV ORF3 protein;

a second epitope polypeptide comprising amino acid residues 66-87 of type 3 HEV ORF3 protein; and

a third epitope polypeptide comprising amino acid residues 66-87 of type4 HEV ORF3 protein.

In certain preferred embodiments, the kit comprises:

a first complex comprising an epitope polypeptide having amino acid residues 66-87 of type 1HEV ORF3 protein;

a second complex comprising an epitope polypeptide having amino acid residues 66-87 of type 3 HEV ORF3 protein; and

a third complex comprising an epitope polypeptide having amino acid residues 66-87 of type4 HEV ORF3 protein.

In the present invention, a kit comprising the first epitope polypeptide, the second epitope polypeptide and the third epitope polypeptide as described above or a kit comprising the first complex, the second complex and the third complex as described above is particularly advantageous, which allows one-time identification of whether a subject is infected with HEV of type 1, type 3 or type 4.

In a specific embodiment, the kit comprises epitope polypeptides having amino acid sequences as shown in SEQ ID NOS 4-6, respectively.

In another specific embodiment, the kit comprises:

a first complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO 4;

a second complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO. 5; and

a third complex comprising an epitope polypeptide having an amino acid sequence as set forth in SEQ ID NO 6.

In certain preferred embodiments, the kit further comprises a detection reagent capable of recognizing and binding anti-HEV antibodies. Such detection reagents are well known in the art and include, but are not limited to, antibodies, targeting polypeptides or aptamers capable of specifically binding to anti-HEV antibodies. In certain preferred embodiments, the detection reagent is a secondary antibody. In certain embodiments, the detection reagent is a polyclonal or monoclonal antibody raised against the subject species.

In certain preferred embodiments, the detection reagent carries a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters) or a fluorescent dye. In certain embodiments, when the detectable label is an enzyme, the kit may further comprise a color developing solution, such as o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), ABTS, or luminol-like compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase.

In certain preferred embodiments, the kit further comprises a solid support, optionally further comprising a coating reagent, such as a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCL buffer, or borate buffer), for coating the epitope polypeptide or the complex onto the solid support. In certain preferred embodiments, the solid support comprises a well plate, test tube, bead (e.g., latex particle) or thin film (e.g., nitrocellulose membrane) made of or coated with a polymeric material (e.g., polyvinyl chloride, polystyrene, polyacrylamide, or cellulose), or magnetic beads pre-coated with a functional group (e.g., amino, carboxyl, biotin, or avidin). In certain embodiments, the solid support is a microtiter plate, such as a microtiter plate or an elisa plate. Methods for coating proteins or polypeptides onto solid phase supports are well known in the art, e.g., physical adsorption, covalent coupling via aminated or carboxylated surfaces, or mediated binding via avidin-biotin systems, polylysine pre-coated surfaces, protein A or protein G pre-coated surfaces.

In certain preferred embodiments, the epitope polypeptide or the complex is coated on the surface of the solid support.

In certain preferred embodiments, the kit further comprises one or more reagents or devices selected from the group consisting of: a diluent for diluting the sample (for example, a phosphate buffer or a phosphate buffer containing a nonionic surfactant); a positive control sample (e.g., a sample containing a known amount of antibodies against a particular genotype HEV); a negative control sample (e.g., a sample that does not contain anti-HEV antibodies or a sample determined to not contain anti-specific genotype HEV antibodies); a stopping solution (e.g., sulfuric acid, hydrochloric acid, or sodium hydroxide solution) for stopping the color reaction of the enzyme-catalyzed substrate; a washing solution for removing a reagent not participating in the reaction (for example, a phosphate buffer or a phosphate buffer containing a nonionic surfactant); a blocking solution for inhibiting non-specific binding; and, a blood collection device (e.g., a pyrogen-free evacuated blood collection tube).

In certain preferred embodiments, the positive control sample is selected from a sample containing a known amount of anti-type 1HEV antibody, a sample containing a known amount of anti-type 3 HEV antibody, or a sample containing a known amount of anti-type 4 HEV antibody. In certain preferred embodiments, the negative control sample is selected from a sample that does not contain anti-type 1HEV antibodies, a sample that does not contain anti-type 3 HEV antibodies, or a sample that does not contain anti-type 4 HEV antibodies.

In certain preferred embodiments, the kit identifies the HEV genotype of the subject by a method comprising the steps of:

(1) contacting a test sample from said subject with a capture reagent, wherein said capture reagent is selected from the epitope polypeptides or complexes of the invention, and said test sample comprises an anti-HEV antibody;

(2) determining the presence or amount of anti-HEV antibody captured by the capture reagent; and

(3) determining the genotype of HEV infected by said subject based on the presence or amount of said anti-HEV antibody in step (2).

In certain preferred embodiments, the sample to be tested is selected from whole blood, serum, plasma, lymph, interstitial fluid or extra-secretory.

In the present invention, methods for determining the presence or amount of said anti-HEV antibody in step (2) are well known in the art, such as immunological assays including, but not limited to, Enzyme Immunoassay (EIA), chemiluminescence immunoassay (CLIA), Radioimmunoassay (RIA), Fluorescence Immunoassay (FIA), Western blot, immunoturbidimetry, or surface plasmon resonance. In certain embodiments, the method used to determine the presence or amount of said anti-HEV antibody in step (2) is an Enzyme Immunoassay (EIA), such as an ELISA assay, an Elispot assay, or a CLEIA assay. In certain embodiments, in step (2), the presence or amount of the anti-HEV antibody is determined using a secondary antibody with a detectable label.

In another aspect, the present invention provides a method for identifying the genotype of a HEV infected with a subject, comprising the steps of: contacting a test sample from said subject with a capture reagent, wherein said capture reagent is selected from the epitope polypeptides or complexes of the invention, and said test sample comprises an anti-HEV antibody.

In certain preferred embodiments, the HEV genotype is selected from the group consisting of type 1, type 3, and type 4.

In certain preferred embodiments, the method for identifying the genotype of an HEV infected with a subject comprises the steps of:

(1) providing a test sample from the subject, wherein the test sample contains anti-HEV antibodies;

(2) contacting said test sample with a capture reagent, wherein said capture reagent is selected from the group consisting of epitope polypeptides or complexes of the invention;

(3) determining the presence or amount of anti-HEV antibody captured by the capture reagent; and

(4) determining the genotype of HEV infected by said subject based on the presence or amount of said anti-HEV antibody in step (3).

In certain preferred embodiments, the sample to be tested is selected from whole blood, serum, plasma, lymph, interstitial fluid or extra-secretory.

In the present invention, methods for determining the presence or amount of said anti-HEV antibody in step (3) are well known in the art, such as immunological assays including, but not limited to, Enzyme Immunoassay (EIA), chemiluminescence immunoassay (CLIA), Radioimmunoassay (RIA), Fluorescence Immunoassay (FIA), Western blot, immunoturbidimetry, or surface plasmon resonance. In certain embodiments, the method used to determine the presence or amount of said anti-HEV antibody in step (3) is an Enzyme Immunoassay (EIA), such as an ELISA assay, an Elispot assay, or a CLEIA assay. In certain embodiments, in step (3), the presence or amount of the anti-HEV antibody is determined using a secondary antibody with a detectable label.

In certain preferred embodiments, the subject is a mammal, e.g., a rodent (e.g., a mouse or rat), a non-human primate (e.g., a cynomolgus monkey or cynomolgus monkey), or a human.

Advantageous effects of the invention

At present, the HEV genotyping method mainly depends on gene sequencing, and the RNA extraction and other processes are complex in operation and high in technical requirement, and are difficult to develop in a common laboratory, so that the requirements of large-scale clinical detection are difficult to meet.

Compared with the prior art, the technical scheme of the invention has at least the following beneficial effects:

(1) the polypeptide sequence consisting of the 66 th to 87 th amino acid residues of HEV ORF3 protein is found to be used for HEV genotyping for the first time, and at least can be used for distinguishing genotype 1, genotype 3 or genotype 4;

(2) the operation is simple and convenient: for example, the detection of the genotype of HEV infected by a subject can be completed by collecting whole blood, separating serum, adding the whole blood and the separated serum into an ELISA plate coated with the epitope polypeptide or the compound of the invention, washing, adding an HRP-labeled secondary antibody, and then adding an enzyme chromogenic substrate;

(3) the requirements on experimental conditions, technical capability of personnel, equipment and environment are not high;

(4) the accuracy of the detection result of the HEV genotyping is high;

(5) low cost, easy popularization and wide application range.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

Fig. 1 shows the results of typing in example 2, in which antisera of mice immunized with ORF3 proteins of different genotypes were detected using HEV capture reagent type 1, HEV capture reagent type 3, and HEV capture reagent type4, respectively, where the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the animal is judged to be positive. The results show that antiserum of mice immunized by type 1HEV ORF3 protein (figure 1A), type 3 HEV ORF3 protein (figure 1B) and type4 HEV ORF3 protein (figure 1C) has obvious combination with the capture reagent of corresponding genotype, and has no cross reaction with other genotype capture reagents.

Fig. 2 shows the results of typing in example 3, in which antisera of macaques immunized with ORF3 type 1 protein were detected using HEV type 1, HEV type 3, and HEV type4 capture reagents, respectively, wherein the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the macaques were determined to be positive. The results show that the type 1HEV capture reagent has strong binding response to the serial sera of two monkeys infected with HEV type 1 (JE12 and JE68), while the type 3 and type4 HEV capture reagents have no positive response to the serial sera.

Fig. 3 shows the results of typing in example 3, in which antisera of macaques immunized with ORF3 type4 protein were detected using HEV type 1, HEV type 3, and HEV type4 capture reagents, respectively, wherein the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the macaques were determined to be positive. The results showed that the type4 HEV capture reagent had a strong positive response to sera from the series of three monkeys infected with HEV type4 (JE13, JE53 and JE54), whereas the type 1 and type 3 HEV capture reagents did not have a significant positive response to sera from the series.

Fig. 4 shows the results of typing in example 4, in which antisera of a person who naturally infects HEV type 3 were detected using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent, respectively, wherein the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the test result is judged to be positive. The results show that the type 3 HEV capture reagent has obvious positive reaction to the serums of 3 people (007B, 002B and 001B) naturally infected with HEV type 3, and other genotype capture reagents have no positive reaction.

Fig. 5 shows the results of typing in example 4, in which antisera from a human naturally infected with HEV type4 were tested using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent, respectively, wherein the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the test result is positive. The results show that type4 HEV capture reagents reacted significantly positively in the serial sera of 3 naturally infected HEV type4 people (YA701, YA866 and YA827), while none of the other genotype capture reagents reacted positively.

Fig. 6 shows the results of typing in clinical serum samples for HEV infection in example 5, in which the HEV assay was performed using the HEV type 1, HEV type 3, and HEV type4 capture reagents, respectively, wherein the dotted line indicates that the S/CO value is 1, and the sample is determined to be positive when the S/CO value is greater than 1. The result shows that 16 of 19 HEV antibody positive serums measured by the commercial HEV antibody detection kit have positive reaction to the type4 HEV capture reagent, and the type 1HEV capture reagent and the type 3 HEV capture reagent are negative.

Fig. 7 shows the results of typing in example 6, in which antisera of cynomolgus monkeys infected with type 1 (JE68) and type4 (JE54) HEVs were detected using the type 1HEV capture reagent, the type4 HEV capture reagent, and the commercial HEV IgM antibody detection reagent and HEV IgG antibody detection reagent, respectively, wherein the dotted line in the figure indicates that the S/CO value is 1, and when the S/CO value is greater than 1, the cynomolgus monkeys were judged to be positive. The results show that in the serum of macaque (JE68) series infected with HEV type 1, the detection time of HEV type 1 capture reagent to HEV antibody is later than that of commercial HEV IgM antibody reagent and earlier than that of commercial IgG antibody reagent; in the serum of macaque (JE54) series infected with HEV type4, the HEV type4 capture reagent has obvious positive reaction, and the detection time of anti-HEV IgG antibody is earlier than that of commercial IgG antibody reagent.

Fig. 8 shows the results of typing of antisera tested against HEV infected with different genotypes in example 7 using other polypeptide fragments of HEV ORF3 protein as capture reagents, where the dotted line indicates an S/CO value of 1, and positive results are determined when the S/CO value is greater than 1. The result shows that RefHEV-3 has negative reaction to type 3 HEV antiserum, and RefHEV-1 and RefHEV-4 have positive reaction to same type antiserum, but the reaction intensity is obviously lower than that of type 1HEV capture reagent and type4 HEV capture reagent.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Sequence 1(SEQ ID NO: 1):

MGTRPCALGLFCCCSSCFCLCCPRHRPVSRLAAVVGGAAAVPAVVSGVTGLILSPSQSPIFIQPTPSPPMSPLRPGLDLVFANPPDHSAPLGVTRPSAPPLPHVVDLPQLGPRR

sequence 2(SEQ ID NO: 2):

MNNMSFASPMGSPCALGLFCCCSSCFCLCCPRHRPVSRLAVAVGGAAAVPAVVSGVTGLILSPSPSPIFIQPTPSPPMSFHNPGLELALDSRPAPSVPLGVTSPSAPPLPPVVDLPQLGLRR

sequence 3(SEQ ID NO: 3):

MAMPPCALGLFCFCSSCFCLCCPRHRPVSRLAVVVGGAAAVPAVVSGVTGLILSPSPSPIFIQPTPSHLTFQPPPGLELALDSRPAHSAPLGVTNPSAPPLPPVVDLPQPGLRR

sequence 4(SEQ ID NO: 4):

PSPPMSPLRPGLDLVFANPPDH

sequence 5(SEQ ID NO: 5):

PSPPMSFHNPGLELALDSRPAP

sequence 6(SEQ ID NO: 6):

PSHLTFQPPPGLELALDSRPAH

sequence 7(SEQ ID NO: 7):

PSPPMSPLRP

sequence 8(SEQ ID NO: 8):

PSPPISFHNP

sequence 9(SEQ ID NO: 9):

PSHLTFQPQP

primer (5 '-3') (SEQ ID NO: 10-13)

SEEO1:CCCTTATCCTGCTGAGCATTCTC(SEQ ID NO：10)

SEBO1:AAYTATGCWCAGTACCGGGTTG(SEQ ID NO：11)

SEBI1:GTYATGYTYTGCATACATGGCT(SEQ ID NO：12)

SEEI1:AGCCGACGAAATYAATTCTGTC(SEQ ID NO：13)

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 establishment of an Indirect ELISA assay for HEV genotyping

1.1 Synthesis and purification of HEV epitope polypeptide

And downloading the amino acid sequences of various genotypes of the HEV ORF3 protein from a GenBank database, wherein the amino acid residues at the 66 th to 87 th positions of each genotype ORF3 protein are shown in the table 2. The epitope polypeptides of the genes 1, 3 and 4 are respectively synthesized by Beijing Zhongke matt Biotechnology GmbH, coupled with biotin at the N-terminal and purified by HPLC. The antigen epitope polypeptides of the biotin marks of the types 1, 3 and 4 of the genes are respectively named as type 1HEV capture reagent, type 3 HEV capture reagent and type4 HEV capture reagent.

TABLE 2 HEV genotype ORF3 protein amino acid residue sequence from 66 th to 87 th positions

1.2 preparation of avidin-coated plates

And (3) coating a microporous plate with biotin, then incubating with streptavidin with proper concentration, sealing, and storing at 4 ℃ for later use.

The plates were first coated with biotin (purchased from thermo) and 100. mu.l of a biotin dilution of 1:1000 in coating buffer was added to each well overnight at 2-8 ℃. A second coating with avidin (from Roche) was then applied, 100. mu.l of avidin diluted 1:2000 in coating buffer per well, overnight at 2-8 ℃. Finally, 200 μ l of blocking solution containing 1% casein was added to each well and incubated at 37 ℃, then the blocking solution in the wells was spun off, patted dry, and stored at 4 ℃ for further use.

1.3 HEV genotyping indirect ELISA detection method based on HEV epitope polypeptide

Firstly, the coating concentration of each genotype epitope polypeptide is optimized by using a gradient dilution method, namely, each genotype epitope polypeptide is subjected to gradient dilution by using PBS and then is respectively coated on the avidin coated plates obtained in the example 1.2, and after pre-incubation at 37 ℃, the avidin coated plates are kept overnight at 4 ℃. Taking the serum of a normal blood donor for confirming the HEV antibody negativity as a negative control, and setting the light absorption value as N; the serum of monkey with antibody positive transfer after experimental infection of HEV of each genotype was selected as positive control, and the light absorption value was set as P, and the P/N value was calculated. As the polypeptide coating concentration is gradually reduced, the P/N value of the polypeptide is larger and larger, and finally a plateau stage is reached, and the corresponding polypeptide concentration is taken as the optimal coating concentration (the optimal coating concentrations of the type 1 epitope polypeptide, the type 3 epitope polypeptide and the type4 epitope polypeptide are 0.5 mu g/mL, 1 mu g/mL and 1.5 mu g/mL respectively). Then each genotype epitope polypeptide is coated on an avidin coated plate with the respective optimal coating concentration, 100 mul of the coating solution is added into each well, the incubation is carried out for 30min at 37 ℃, and PBST (containing 0.05 percent of tween-20) with pH value of 7.4 is used for washing for 5 times to obtain the microplate coated with the different genotype epitope polypeptides. When detecting a sample to be detected, firstly, using PBST containing 5% bovine serum to detect the ratio of the sample to be detected 1:100, then adding 100 mul/hole into a microporous plate coated with polypeptide, and incubating for 30min at 37 ℃; PBST washing 5 times; adding 100 μ l of HRP-labeled antibody (purchased from Beijing Zhonghua Chihua jin Qiao biotechnology Co., Ltd.) against IgG from the species of the antibody to be detected into each well except for blank wells, and incubating at 37 deg.C for 30 min; remove liquid and wash 5 times with PBST; then adding TMB substrate (purchased from BD company in USA), developing at 37 deg.C for 15 min; then 2M H was added₂SO₄Stopping solution using American MD-SThe absorbance A of each well was read at wavelengths of 450nm and 630nm using a spectramax M5 multifunctional microplate reader. And taking the S/CO value as a final detection value of the sample to be detected, wherein the S/CO value is the light absorption value A of the sample divided by the cut off value, and the cut off value is the mean value of the light absorption values of the negative controls of all the plates plus 3 times of standard deviation. And when the S/CO value of the sample to be detected is more than 1, judging the sample to be positive.

Example 2 evaluation of typing Effect of HEV genotyping Indirect ELISA assay in murine antiserum

9-week-old BalB/C mice (purchased from the Chinese food and drug testing institute) were immunized subcutaneously with type 1, type 3, and type4 HEV ORF3 full-length proteins (type 1 GenBank No. JQ655734; type 3 GenBank No. AF060668; type4 GenBank No. JQ655733; produced by Shenzhen Fenpeng Bio-Ltd.) to prepare 3 groups of antisera of type 1, type 3, and type4, respectively. The primary immunization adopts the antigen mixed and emulsified with complete Freund's adjuvant 1:1, the dosage is 100 mu g/mouse, the boosting immunization is carried out after 2 weeks, and the antigen mixed and emulsified with incomplete Freund's adjuvant 1:1, the dosage is 50 mu g/mouse. Antisera were obtained by taking blood from the canthus at 2 weeks post-eye.

The above immunized mouse antiserum was assayed by the methods of examples 1.2 and 1.3 using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent obtained in example 1.1, respectively. Results as shown in FIG. 1, FIG. 1A shows the binding of 3 capture reagents to antisera from 16 mice immunized with the full-length ORF3 type 1 protein, where 13 mice had antisera positive for the type 1HEV capture reagent and all of them were negative for the other genotype capture reagents; FIG. 1B shows the binding of 3 capture reagents to antisera from 9 mice immunized with the full-length protein of ORF3 type 3, wherein the antisera from 3 mice tested positive for the HEV type 3 capture reagent and all tested negative for the other genotype capture reagents; FIG. 1C shows the binding of 3 capture reagents to antisera from 12 mice immunized with the full-length ORF3 protein type4, wherein the antisera from 5 mice tested positive for the HEV type4 capture reagent and all tested negative for the other genotype capture reagents.

The results show that mouse serum immunized by the type 1 ORF3 full-length protein, the type 3 ORF3 full-length protein and the type4 ORF3 full-length protein respectively has obvious combination with the corresponding type capture reagent, and has no cross with other types of capture reagents, and the good typing effect of the capture reagents (type 1HEV capture reagent, type 3 HEV capture reagent and type4 HEV capture reagent) based on the 66 th to 87 th amino acid residues of the HEV ORF3 protein is preliminarily shown.

Example 3 evaluation of typing Effect of HEV genotyping Indirect ELISA assay in macaque antiserum

2 macaques (JE12 and JE68) were each immunized separately with type 1HEV strain (GenBank No. JQ655734) and challenged by intravenous injection, and sera were collected once a week after inoculation, and all sera and stool samples were stored below-60 deg.C, avoiding repeated freeze-thawing. The monkey antisera were assayed by the methods of examples 1.2 and 1.3 using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent obtained in example 1.1. FIGS. 2A and 2B show the results of the detection of the series of antisera from 2 cynomolgus monkeys (JE12 and JE68) with 3 capture reagents, respectively. Wherein 3 weeks after JE12 infects type 1HEV, its antisera begins to show a strong positive response to the type 1HEV capture reagent; JE68 antisera began to show a strong positive response to type 1HEV capture reagent 14 weeks after infection with type 1 HEV; meanwhile, antisera from JE12 and JE68 at each post-inoculation time point did not respond positively to the detection of both type 3 and type4 capture reagents.

Using type4 HEV virus strain (GenBank No. JQ655733) to attack 3 macaques (JE13, JE53 and JE54) by intravenous injection, collecting serum once a week after inoculation, and storing all serum and feces samples below-60 deg.C to avoid repeated freeze thawing. Then, the monkey antiserum of the series was assayed by the methods of examples 1.2 and 1.3 using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent obtained in example 1.1. FIGS. 3A, 3B and 3C show the results of the detection of 3 capture reagents against the series of antisera from 3 macaques (JE13, JE53 and JE54), respectively. The antiserum infected with JE54 at the 7 th week of type4 HEV and the type 3 capture reagent show weaker reactivity, then a competitive EIA method is adopted to further analyze the antiserum sample, a microplate coated with HEV type 1, 3, 4 and rabbit epitope polypeptide mixtures (each polypeptide is used in an amount of 0.1 mu g) is used, the antibody titer of a sample to be determined is determined by using the microplate coated with HEV mixed epitope polypeptides, and a dilution sample with A450/630 of about 1 is selected for serotype analysis. For each sample, 50 μ l of diluted sample is added to each of 5 wells (labeled as non-inhibitory control, type 1, type 3, type4 and rabbit respectively), 50 μ l of epitope polypeptides of type 1, type 3, type4 and rabbit at a certain concentration (50 μ l of sample diluent is added to the non-inhibitory control well) are added to the type 1, type 3, type4 and rabbit respectively, the mixture is mixed, incubated at 37 ℃ for 30min, PBST is washed for 5 times, 100 μ l of anti-human IgG antibody or anti-rabbit IgG antibody is added, incubated at 37 ℃ for 30min, washed for 5 times, 100 μ l of TMB is added, incubated at 37 ℃ for 15min, and finally the reaction is terminated, and the absorbance A of each well is read at the wavelength of 450nm and 630 nm. The inhibition rates of type 1, type 3, type4 and rabbit polypeptides were calculated, respectively. If the inhibition rate of an epitope polypeptide is more than 50%, the type represented by the epitope polypeptide is the serotype of the sample. Calculating the formula: peptide inhibition ═ (non-peptide added sample wells a 450/630)/non-peptide added sample wells a450/630 × 100%. As a result, it was found that only the type4 capture reagent produced a significant inhibitory effect (inhibition rate of 78.16%) on the antiserum sample.

The results show that the trapping reagent (type 1HEV trapping reagent, type 3 HEV trapping reagent and type4 HEV trapping reagent) based on the 66 th-87 th amino acid residues of the HEV ORF3 protein has good typing effect on macaque serum, and has no obvious cross reaction with antiserum with different genotypes.

Example 4 evaluation of typing Effect of HEV genotyping Indirect ELISA assay in human antiserum

Human antisera were obtained from 3 patients naturally infected with type 3 HEV (007B, 002B and 001B) at various time points post infection, purchased from Biomex, germany, cat #: SCP-HEV-007a, SCP-HEV-002a and SCP-HEV-001a, and the information of patient type, age, and infectious virus genotype can be found in the specification. The human-series antisera were then assayed according to the method of example 1.3 using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent obtained in example 1.1, respectively. FIG. 4A, FIG. 4B and FIG. 4C show the results of a series of antisera tested against 3 patients (007B, 002B and 001B) with 3 capture reagents, respectively. Wherein, the 3-type capture reagent shows strong positive reaction to the serial antiserum of three patients of 007B, 002B and 001B; at the same time, antisera at each post-infection time point, 007B, 002B, and 001B, showed no positive response to detection of both type 1 and type4 capture reagents.

Human series antiserum was obtained from 3 patients naturally infected with type4 HEV (YA701, YA866 and YA827) at different time points post-infection, from beijing youan hospital, and was sequenced to confirm that HEV genotype 4. The human-series antisera were then assayed according to the method of example 1.3 using the type 1HEV capture reagent, the type 3 HEV capture reagent, and the type4 HEV capture reagent obtained in example 1.1, respectively. FIGS. 5A, 5B and 5C show the results of a series of antisera from 3 capture reagents for 3 patients (YA701, YA866 and YA827), respectively. Wherein, the 3-type capture reagent shows strong positive reaction on the serial antiserum of three patients of YA701, YA866 and YA 827; while neither type 1 nor type4 capture reagents have a positive reaction.

The results show that the capture reagent based on the 66 th to 87 th amino acid residues of HEV ORF3 protein also has good typing effect on human serum naturally infected with HEV.

Example 5 evaluation of the typing Effect of HEV genotyping Indirect ELISA assay in clinical serum samples

The indirect ELISA assay for HEV genotyping described in this example uses the HEV capture reagent type 1, HEV capture reagent type 3, and HEV capture reagent type4 obtained in example 1.1, and performs assay on the test sera according to the methods of examples 1.2 and 1.3.

Serum samples of 59 clinical hepatitis patients were first tested using a commercial HEV antibody test kit (Beijing Wantai corporation, national institutes of care 20153401411), of which 19 anti-HEV antibody tests were positive. Then, the results of the 19 cases of antisera were tested using 3 HEV capture reagents, and the results are shown in FIG. 6, in which 16 cases tested positive for the type4 capture reagent, and the 19 cases tested negative for both the type 1 and type 3 capture reagents.

To verify the accuracy of the capture reagent, further genetic sequencing was performed on serum samples from 16 patients identified as infected with type4 HEV by the capture reagent. Extraction of RNA was performed using the QIAamp Viral RNA Mini Kit (cat # 52904) followed by SuperScript^TMIII First-Strand Synthesis System reagent (cat # 18080-. The PCR reaction condition is pre-denaturation at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 45 sec, extension at 72 ℃ for 1 min for 35 cycles; finally, the temperature is extended by 10 minutes at 72 ℃. The PCR product was subjected to sequencing by Biotech, Inc., Otological techniques, Olympic, Kyoto, Beijing. Sequence alignment was performed using MEGA5.2 software: the full-length genotyping standard strain and GeneBank accession numbers thereof are respectively as follows: type 1: sar-55(M80581, subtype 1b), type 2: mexico (M74506, subtype 2a), type 3: prototype swing HEV (AF082843, subtype 3a), type 4: MO (JQ655733, subtype 4a), swGX40(EU676172, subtype 4b), HE-JF5(AB220973, subtype 4b), W2-5(JQ655736, subtype4d), W3(JQ655735, subtype4 h).

TABLE 3 nested RT-PCR primers

*Reference strain:AJ272108 (Y＝T or C,W＝A or T)

The result shows that 11 cases of nested RT-PCR method detect positive, and the gene type4 is confirmed by sequencing.

The above results fully indicate that the capture reagent based on amino acid residues 66-87 of HEV ORF3 protein can be used to distinguish the genotype of HEV infected by clinical serum samples.

Example 6 comparison of HEV genotyping Indirect ELISA assays with commercial HEV antibody assays

The indirect ELISA assay for HEV genotyping described in this example uses the HEV capture reagent type 1, HEV capture reagent type 3, and HEV capture reagent type4 obtained in example 1.1, and performs assay on the test sera according to the methods of examples 1.2 and 1.3.

Each genotype capture reagent, a commercial HEV IgM antibody detection reagent (beijing wantai corporation, national food and drug administration (kokai) No. 213, 3400240) and an HEV IgG antibody detection reagent (beijing wantai corporation, national food and drug administration (kokai) No. 20153401411) were used to detect 1 part of cynomolgus monkey series serum (JE68) experimentally infected with HEV 1 type and 1 part of cynomolgus monkey series serum (JE54) experimentally infected with HEV 4 type obtained in example 3, respectively. FIG. 7A shows the results of type 1 capture reagent and commercial reagent detection of type 1 macaque antisera at different time points after virus inoculation, showing that type 1 capture reagent was detected one week later than commercial HEV IgM antibody reagent, but earlier than commercial IgG antibody reagent, and the detected S/CO value was significantly higher than both commercial reagents. FIG. 7B shows the results of testing type4 cynomolgus antiserum with type4 capture reagent and commercial reagent at different time points after virus inoculation, showing that the detection time of type4 capture reagent against HEV IgG antibody is about one week earlier than that of commercial IgG antibody reagent, and the detected S/CO value is significantly higher than that of the latter. The above results suggest that a capture reagent based on amino acid residues 66-87 of HEV ORF3 protein can be used not only to distinguish the genotype of HEV infected by a subject, but also for early diagnosis of HEV infection.

Example 7 evaluation of the Effect of other polypeptide fragments of HEV ORF3 protein for HEV genotyping

In this example, the genotyping effect of other polypeptide fragments of HEV ORF3 protein was examined.

Firstly, the 66 th to 75 th amino acid residues of HEV ORF3 protein are synthesized into the comparison polypeptide of each genotype, wherein the 66 th to 75 th amino acid residues of each genotype ORF3 protein are shown in Table 3. The above comparative polypeptides of types 1, 3 and 4 were synthesized by Beijing Zhongke Sudoku Biotech Co., Ltd, respectively, and biotin was coupled to the N-terminus for HPLC purification. The biotin-labeled comparison polypeptides of genotypes 1, 3, and 4 are designated HEV type 1, 3, and 4, respectively.

Table 4.

In this example, the type 1HEV capture reagent, the type 3 HEV capture reagent and the type4 HEV capture reagent obtained in example 1.1, and the type 1HEV contrast polypeptide, the type 3 HEV contrast polypeptide and the type4 HEV contrast polypeptide are used, and the serum to be tested is tested according to the methods of examples 1.2 and 1.3, wherein the coating concentrations of the type 1HEV contrast polypeptide, the type 3 HEV contrast polypeptide and the type4 HEV contrast polypeptide are all 10 μ g/mL.

Serum samples from HEV type 1, type 3 and type4 sources were obtained, wherein HEV type 1 and type4 serum samples were obtained from sera from experimental infected macaques in example 3 for different time periods, and type 3 was obtained from Biomex corporation serum trays, 6 for each genotype. And (3) detecting the blood serum samples of the same genotype by using each genotype capture reagent and each genotype comparison polypeptide. FIG. 8 shows the results of the type 1 capture reagent and the comparative polypeptide in detecting 6 parts of type 1HEV infection sample, the type 3 capture reagent and the comparative polypeptide in detecting 6 parts of type 3 HEV infection sample, and the type4 capture reagent and the comparative polypeptide in detecting 6 parts of type4 HEV infection sample, and the results show that the type 1 capture reagent, the type 3 capture reagent and the type4 capture reagent of the present invention all have strong positive reactions to isogenic HEV infection antiserum, and the binding strength (S/CO value) of the type 1 capture reagent is even up to more than 20. In contrast, the type 3 control polypeptide did not respond positively to 6 type 3 HEV infection samples, indicating that it could not be used to identify whether a subject was infected with type 3 HEV; meanwhile, although type 1 and type4 control polypeptides showed positive responses to isogenic HEV infection antisera, their binding strength was significantly lower than that of type 1 and type4 capture reagents (P < 0.001).

The above results fully indicate that the capture reagent based on the amino acid residues 66-87 of the HEV ORF3 protein can accurately and efficiently distinguish and identify the HEV genotype infected by the subject, but the polypeptide fragment based on other amino acid residues of the HEV ORF3 protein (e.g., amino acid residues 66-75) cannot completely distinguish the HEV types 1, 3 and 4, and therefore cannot be used for identifying the HEV genotype infected by the subject.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

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Claims (22)

1. The amino acid sequence of the separated epitope polypeptide is shown in any one of SEQ ID NO 4-6.

2. An isolated nucleic acid encoding the epitope polypeptide of claim 1.

3. A vector comprising the isolated nucleic acid of claim 2.

4. A host cell comprising the isolated nucleic acid of claim 2 and/or the vector of claim 3.

5. A complex comprising the epitope polypeptide of claim 1 and a modifying group; wherein the modifying group is a carrier protein or a detectable label, and the epitope polypeptide is coupled, conjugated or fused with the modifying group to obtain the complex.

6. The complex of claim 5, wherein the modifying group is selected from biotin, avidin, or deglycosylated avidin.

7. A kit comprising the epitope polypeptide of claim 1 or the complex of claim 5 or 6.

8. The kit of claim 7, wherein the kit comprises epitope polypeptides as shown in any one of SEQ ID NOs 4-6.

9. The kit of claim 7, wherein the kit comprises:

a first complex comprising an epitope polypeptide as set forth in SEQ ID NO 4;

a second complex comprising an epitope polypeptide as set forth in SEQ ID NO. 5; and

a third complex comprising an epitope polypeptide as set forth in SEQ ID NO 6.

10. The kit of any one of claims 7-9, further comprising a detection reagent capable of recognizing and binding anti-HEV antibodies.

11. The kit of claim 10, wherein the detection reagent is an antibody, targeting polypeptide, or nucleic acid aptamer capable of specifically binding to an anti-HEV antibody.

12. The kit of claim 10, wherein the detection reagent is a secondary antibody.

13. The kit of claim 10, wherein the detection reagent is detectably labeled.

14. The kit of claim 13, wherein the detectable label is selected from the group consisting of an enzyme, a chemiluminescent reagent, or a fluorescent dye.

15. The kit of claim 10, wherein the kit further comprises a solid support.

16. The kit of claim 10, wherein the kit comprises a solid support having the epitope polypeptide or the complex coated thereon.

17. Use of the epitope polypeptide of claim 1 or the complex of claim 5 in the preparation of a kit for identifying an HEV genotype infected by a subject, the HEV genotype selected from the group consisting of type 1, type 3, and type 4.

18. The use of claim 17, wherein the kit identifies the HEV genotype of the subject by a method comprising the steps of:

(1) contacting a test sample from said subject with a capture reagent, wherein said capture reagent is selected from the epitope polypeptide of claim 1 or the complex of claim 5, said test sample comprising an anti-HEV antibody;

(2) determining the presence or amount of anti-HEV antibody captured by the capture reagent; and

(3) determining the genotype of HEV infected by said subject based on the presence or amount of said anti-HEV antibody in step (2).

19. The use of claim 18, wherein the sample to be tested is selected from the group consisting of whole blood, serum, plasma, lymph, interstitial fluid or extra-secretory.

20. The use of claim 18, wherein the presence or amount of anti-HEV antibody captured by the capture reagent is determined in step (2) by an immunological assay.

21. The use of claim 20, wherein the immunological assay is an ELISA assay, an Elispot assay, or a CLEIA assay.

22. The use of claim 20, wherein, in step (2), the presence or amount of the anti-HEV antibody is determined using a secondary antibody with a detectable label.

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