Antigen for detecting HEV (Hepatitis E Virus) IgM (Immunoglobulin M) as well as preparation method and application thereof

Abstract

The invention provides an antigen for detecting hepatitis E virus IgM as well as a preparation method and application thereof, and relates to the technical field of biology. (A) a protein composed of an amino acid sequence as shown in SEQ ID NO.1; and (B) a protein formed by connecting the C end of the amino acid sequence as shown in SEQ ID NO.2 and the N end of the amino acid sequence as shown in SEQ ID NO.1. It is found that when the ORF2aa417-660 antigen and the ORF2aa417-660 antigen added with the beta folding structure sequence serve as dot membrane raw materials to detect IgM antibodies in colloidal gold products, the positive detection rate is high, the false positive rate is low, and the sensitivity is obviously superior to that of the ORF2aa459-606 antigen. Especially, the ORF2aa417-660 antigen added with a beta folding structure sequence is helpful for the antigen to form a dimer and a polymer, and the immunogenicity of the antigen is further enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an antigen for detecting hepatitis E virus IgM, a preparation method and application thereof. Background Art

[0002] Hepatitis E is an infectious disease with hepatitis E virus (HEV) as the pathogen. The virus is mainly transmitted through the fecal-oral route, mainly causing liver damage and with severe symptoms. It is more common in young and elderly people clinically.

[0003] The main methods for detecting hepatitis E include nucleic acid testing, liver function testing, and serum testing. Since nucleic acid testing and liver function testing require more instruments and equipment and take a long time, serum testing is convenient and fast, so serum testing has become the preferred method for detecting hepatitis E virus. Serum testing mainly detects IgM antibodies. Hepatitis E virus is a non-enveloped single-stranded positive-strand RNA virus. Its genome is about 7500kB long and consists of three open protein coding frames. ORF2 encodes a single structural capsid protein. This region includes most immune antigen regions, which are commonly used sequences for disease diagnosis. Among them, amino acids 459 to 606 are the main immune epitopes. Most of the antigens designed on the market are prepared as diagnostic antigens based on this fragment, but the IgM positive detection rate is not high and there will be false positives.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide an antigen for detecting hepatitis E virus IgM, so as to at least solve one of the technical problems existing in the prior art.

[0006] The second object of the present invention is to provide a polynucleotide encoding the above-mentioned antigen.

[0007] The third object of the present invention is to provide a carrier.

[0008] A fourth object of the present invention is to provide a host cell.

[0009] A fifth object of the present invention is to provide a method for preparing the above-mentioned antigen.

[0010] The sixth object of the present invention is to provide the use of the above-mentioned antigen, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method in the preparation of a product for detecting or assisting in the detection of hepatitis E virus IgM or in the preparation of a product for detecting or assisting in the detection of whether a sample to be tested is infected with hepatitis E virus.

[0011] A seventh object of the present invention is to provide a hepatitis E virus IgM detection test strip.

[0012] An eighth object of the present invention is to provide a hepatitis E virus IgM detection kit.

[0013] The ninth object of the present invention is to provide a method for detecting hepatitis E virus IgM for the purpose of non-disease diagnosis.

[0014] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0015] In a first aspect, the present invention provides an antigen for detecting hepatitis E virus IgM, comprising the following (A) or (B);

[0016] (A) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0017] (B) A protein composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.2 connected to the N-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0018] In a second aspect, the present invention provides a polynucleotide encoding the above-mentioned antigen.

[0019] Furthermore, the polynucleotide is a gene encoding the above antigen; the nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0020] In a third aspect, the present invention provides a vector comprising the above-mentioned polynucleotide.

[0021] In a fourth aspect, the present invention provides a host cell comprising the above-mentioned vector.

[0022] In a fifth aspect, the present invention provides a method for preparing the above-mentioned antigen, wherein an expression vector containing a nucleotide sequence encoding the antigen is transferred into a host, and then induced to express.

[0023] In a sixth aspect, the present invention provides the use of the above-mentioned antigen, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method in the preparation of a product for detecting or assisting in the detection of hepatitis E virus IgM or in the preparation of a product for detecting or assisting in the detection of whether a sample to be tested is infected with hepatitis E virus.

[0024] In a seventh aspect, the present invention provides a hepatitis E virus IgM detection test strip, comprising the above-mentioned antigen, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method.

[0025] In an eighth aspect, the present invention provides a hepatitis E virus IgM detection kit, comprising the above-mentioned antigen, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method.

[0026] In a ninth aspect, the present invention provides a method for detecting hepatitis E virus IgM for purposes other than disease diagnosis, comprising utilizing the above-mentioned test strip or the above-mentioned kit.

[0027] The invention provides an antigen for detecting hepatitis E virus IgM. By analyzing the published antigen epitopes, an amino acid sequence ORF2 aa417-660 antigen is obtained, an ORF2 aa417-660 fragment is selected, and a lysozyme beta-folded structure sequence SCDVTKLLNCGIPGS is added to the N-terminus of the sequence. The amino acid sequence of the lysozyme dimer is composed of amino acids such as serine (Ser), aspartic acid (Asp), alanine (Ala), lysine (Lys), etc. The addition of the sequence is conducive to the antigen forming dimers and polymers. Through a later antigen verification experiment, it is found that the ORF2 aa417-660 antigen and the ORF2aa417-660 antigen with the beta-folded structure sequence are used as spot film raw materials to detect IgM antibodies in colloidal gold products, and the positive detection rate is high, the false positive rate is low, and the sensitivity is significantly better than that of the ORF2 aa459-606 antigen. In particular, the ORF2 aa417-660 antigen with the addition of a β-folded structure sequence helps to further enhance the immunogenicity of the antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The agarose gel electrophoresis diagram of the amplified band of the expression strain of the HEV-pET32a-1 recombinant antigen provided in Example 1 of the present invention;

[0030] Figure 2 A comparison chart of antigen expression in the supernatant of the HEV-pET32a-1 recombinant antigen expression strain under different induction conditions provided in Example 1 of the present invention;

[0031] Figure 3 This is a comparison chart of the dimer verification results of different antigens provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0033] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0034] The term "polynucleotide" herein refers to a polymer form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the above-mentioned SS-B binding protein, and the encoding is optionally for encoding the sense strand or the antisense strand. The polynucleotide can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0035] The term "vector" as used herein refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a cell by transformation, transfer or transfection so that the genetic material elements it carries are expressed in the cell.

[0036] As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Progeny may not necessarily be completely identical to the original cell due to natural, accidental or deliberate mutation, and may differ from the original cell in morphology and / or in genomic DNA.

[0037] The term "amino acid" herein refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof. Common natural amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V). In this article, the Chinese names, three-letter abbreviations and single-letter abbreviations of amino acids are used interchangeably.

[0038] In one aspect, the present invention provides an antigen for detecting hepatitis E virus IgM, comprising the following (A) or (B);

[0039] (A) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0040] (B) A protein composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.2 connected to the N-terminus of the amino acid sequence shown in SEQ ID NO.1.

[0041] By analyzing the published antigen epitopes, the ORF2aa417-660 antigen with an amino acid sequence as shown in SEQ ID NO.1 was obtained, the ORF2 aa417-660 fragment was selected, and a lysozyme β-folded structure sequence SCDVTKLLNCGIPGS was added to the N-terminus of the sequence. The amino acid sequence of the lysozyme dimer is composed of amino acids such as serine (Ser), aspartic acid (Asp), alanine (Ala), and lysine (Lys). The addition of this sequence helps the antigen to form dimers and polymers. Through subsequent antigen verification experiments, it was found that the ORF2 aa417-660 antigen and the ORF2aa417-660 antigen with a β-folded structure sequence were used as spot film raw materials to detect IgM antibodies in colloidal gold products, with a high positive detection rate and a low false positive rate, and the sensitivity was significantly better than that of the ORF2aa459-606 antigen. In particular, the ORF2 aa417-660 antigen with the addition of a β-folded structure sequence helps the antigen to form dimers and polymers, further enhancing the immunogenicity of the antigen.

[0042] According to another aspect of the present invention, a polynucleotide encoding the above antigen is also provided.

[0043] In some specific embodiments, the polynucleotide is a gene encoding the above antigen; the nucleotide sequence of the gene is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0044] According to another aspect of the present invention, a vector is provided, comprising the above-mentioned polynucleotide.

[0045] The vector is well known to those skilled in the art, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as lambda phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some specific embodiments, the vector of the present invention can be selected from pET32a, pET32b, or pET32c, preferably pET32a as an expression vector.

[0046] According to another aspect of the present invention, a host cell is provided, comprising the above-mentioned vector.

[0047] Wherein, the host includes Escherichia coli, and the Escherichia coli expression host bacteria include BL21 (DE3), BLR (DE3), BL21 (DE3) pLysS, Rosetta (DE3), and Rosetta (origami), preferably Rosetta (origami).

[0048] According to another aspect of the present invention, a method for preparing the above antigen is also provided, wherein an expression vector containing a nucleotide sequence encoding the antigen is transferred into a host, and then induced to express.

[0049] Specifically, the inducing expression includes adding IPTG to induce expression at 20-37°C, preferably 30°C.

[0050] In some specific embodiments, pET32a can be selected as the expression vector, Rosetta (DE3) expression strain, protein expression can be induced at 30°C, and gradient renaturation can be selected to promote the formation of more dimers and polymers of the protein.

[0051] According to another aspect of the present invention, there is also provided the use of the above-mentioned antigen, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method in preparing a product for detecting or assisting in detecting hepatitis E virus IgM or preparing a product for detecting or assisting in detecting whether a sample to be tested is infected with hepatitis E virus.

[0052] According to another aspect of the present invention, a hepatitis E virus IgM detection test strip is also provided, comprising the above-mentioned antigen, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method.

[0053] According to another aspect of the present invention, there is also provided a hepatitis E virus IgM detection kit, comprising the above-mentioned antigen, the above-mentioned vector, the above-mentioned host cell or the antigen prepared by the above-mentioned preparation method.

[0054] According to another aspect of the present invention, a method for detecting hepatitis E virus IgM for non-disease diagnosis purposes is also provided, comprising using the above-mentioned test strip or the above-mentioned kit.

[0055] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] 1. Strains and vectors: Escherichia coli Rosetta (origami) was purchased from Shanghai Weidi Biotechnology, and pET32a vector was purchased from Shanghai Shenggong Biotechnology;

[0057] 2. Enzymes and kits: Recombinant enzymes were purchased from Nanjing Novozymes Biopharmaceutical Technology Co., Ltd., plasmid extraction kits were purchased from Beijing Qingke Biotechnology Co., Ltd., and purification and recovery kits were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0058] 3. LB medium: 0.5% yeast extract, 1% peptone, 0.5% NaCl;

[0059] 4. Cell lysis buffer: 150 mM Tris-HCl, 100 mM NaCl, 1 mM PMSF and 1 mM EDTA, pH 8.5;

[0060] 5. Balance solution: 150 mM NaCl, 10 mM Na2HPO4, 0.17 mM KH2PO4, 0.26 mM KCl and 8 M Urea, pH 8.5;

[0061] 6. The nucleotide sequence of the ORF2 aa459-606 antigen is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO.9. The vector is pET32a, and the restriction site is BamHI / HindIII. The purification method is the same as that of ORF2 aa417-660.

[0062] 7. Colloidal gold products include colloidal gold products that use colloidal gold as an indicator / tracer to qualitatively or quantitatively detect specific substances, such as immunochromatographic test strips or immunochromatographic kits.

[0063] In the following embodiments, the colloidal gold product is an immunochromatographic test strip, which specifically includes a base plate (polyester plate), absorbent paper, nitrocellulose membrane, gold label pad, sample pad and tape, and the overlapping order from top to bottom is: absorbent paper, nitrocellulose membrane, gold label pad and sample pad.

[0064] Example 1 Design and expression of antigens

[0065] The recombinant hepatitis E nucleotide full-length fragment of the present invention is obtained by artificial synthesis, and the required fragment is amplified by PCR using the full-length fragment as a template, and then the fragment is connected to a cloning vector TOP10 and then transferred into an expression vector.

[0066] The technical solution adopted by the present invention is as follows:

[0067] 1.1 Design of HEV recombinant antigens

[0068] According to the HEV sequence published in the NCBI database, the ORF2aa417-660 protein was screened by analyzing the published antigenic epitopes.

[0069] Amino acid sequence of ORF2 aa417-660:

[0070] ENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLP LRGKLSFWEAGTTKAGYPYNYNTTASDQLLIENAAGHRVAISTYTTSLGAGPVAISAVAVLAPHSALALLEDTMDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTREL(SEQ ID NO.1).

[0071] Experiments have shown that lysozyme dimers can enhance the immunogenicity of antigens. The present invention adds a lysozyme β-folded structure sequence SCDVTKLLNCGIPGS (SEQ ID NO.2) at the N-terminus of the expressed sequence to promote the antigen to form more dimers (polymers). According to the codon preference of Escherichia coli, the desired fusion nucleotide sequence is designed and synthesized. The nucleotide sequence of ORF2 aa417-660 is shown in SEQ ID NO.3, and the nucleotide sequence of ORF2 aa417-660 after adding a lysozyme β-folded structure sequence is shown in SEQ ID NO.4.

[0072] The ORF2 aa368-660 protein and a recombinant protein with a SCDVTKLLNCGIPGS (SEQ ID NO. 2) added to the ORF2 aa368-660 protein sequence were expressed at the same time, and were named ORF2 aa368-660-1 and ORF2aa368-660-2, respectively.

[0073] The amino acid sequence of ORF2 aa368-660 is shown in SEQ ID NO.5, the nucleotide sequence of ORF2 aa368-660-1 is shown in SEQ ID NO.6, and the nucleotide sequence of ORF2 aa368-660-2 is shown in SEQ ID NO.7.

[0074] 1.2 Construction of HEV-pET32a-1 recombinant antigen vector

[0075] Gene fragments were obtained by PCR amplification technology, and BamH and Hind restriction sites were added to the nucleotide sequences at both ends. The purified gene fragments were connected to the pET32a(-) vector under the action of recombinase. The vector with the β-folded structure sequence was named HEV-pET32a-1, and the vector without the β-folded structure sequence was named HEV-pET32a-2. The two were subsequently expressed and purified using the same experimental method. The ligation product was transferred into TOP10 competent cells by heat shock method, and the competent cells were spread on LB solid culture medium containing ampicillin, and a single clone was picked for bacterial liquid identification (the results are shown in Figure 1 As shown, 1 is the nucleic acid Maker and 2 is the bacterial solution PCR band), the bacterial solution with correct detection is sent for sequencing, the strain with correct sequencing is extracted with plasmid, and the recombinant plasmid is transformed into the Escherichia coli Rosetta (origami) expression strain.

[0076] 1.3 Small-scale expression of HEV-pET32a-1 recombinant antigen

[0077] The two bacterial solutions were respectively transferred into 4 EP tubes containing 10 ml LB liquid culture medium, and shaken in a 37°C shaker until the OD was between 0.4 and 0.6, and 1 mM IPTG was added to induce expression. One tube of bacterial solution was used as a control and continued to be shaken in a 37°C shaker without adding IPTG. The remaining three tubes of bacterial solution were all added with 1 mM IPTG and shaken in a 37°C, 30°C and 20°C shaker for 6 hours. Collect each tube of bacterial solution, dissolve the bacterial solution with 20 mM PBS, perform ultrasound and centrifugation at 12000 rpm, and keep the supernatant for later use. Dissolve the inclusion bodies after centrifugation with 8 M urea, centrifuge after complete dissolution, and run SDS-PAGE gel verification on the supernatant after ultrasound and the supernatant after inclusion body centrifugation. The results are as follows: Figure 2 As shown, 1 is the supernatant of uninduced bacterial solution at 37°C, 2 is the supernatant of induced bacterial solution at 37°C, 3 is the supernatant of induced bacterial solution at 30°C, 4 is the supernatant of induced bacterial solution at 25°C, 5 is the protein maker, 6 is the inclusion body of uninduced bacterial solution at 37°C, 7 is the inclusion body of induced bacterial solution at 37°C, 8 is the inclusion body of induced bacterial solution at 30°C, and 9 is the inclusion body of induced bacterial solution at 25°C. It can be seen that the protein is expressed when induced at 37°C, 30°C, and 20°C, and the protein expression at 30°C is higher, so 30°C is selected for shaking purification.

[0078] 1.4 Expression and purification of HEV recombinant antigens

[0079] Two kinds of E. coli were inoculated into low sodium LB medium, shaken at 37℃ until OD was 0.4-0.6, 1mM IPTG was added, induced at 30℃ for 6 hours, and the cells were collected. The cells were dissolved with cell lysis buffer and fully dissolved, and the dissolved bacterial solution was broken by ultrasonic method, then centrifuged at 14000rpm for 30 minutes, and the supernatant was poured out. The precipitate after centrifugation, i.e., the inclusion body, was dissolved with 8M urea overnight, and centrifuged at 14000rpm for 30 minutes the next day, and the supernatant was taken for purification. Ni-excel column was used for purification, and the column was balanced with equilibrium buffer, and the supernatant after centrifugation was loaded at a speed of 2ml / mim. After loading, the Ni-excel column was first balanced with equilibrium solution, and 30mM, 60mM and 300mM imidazole were added to the equilibrium solution, and the target protein was collected respectively. After SDS-PAGE verification, the target protein was eluted at 300mM imidazole.

[0080] The collected inclusion body proteins were renatured by gradient renaturation method, renaturation solution 1: 150mM NaCl, 10mM Na2HPO4, 0.17mM KH2PO4, 0.26mM KCl, 5% glycerol, 1% glycine and 4M Urea, pH 8.5; renaturation solution 2: 150mM NaCl, 10mM Na2HPO4, 0.17mM KH2PO4, 0.26mM KCl, 5% glycerol, 1% glycine and 2M Urea, pH8.5; renaturation solution 3: 150mM NaCl, 10mM Na2HPO4, 0.17mM KH2PO4, 0.26mM KCl, 5% glycerol, 1% glycine and 2M Urea, pH 8.5; final storage solution: 50mM NaCl, 10mM Na2HPO4, 0.17mM KH2PO4, 0.26mM KCl, 5% glycerol and 1% glycine, pH 8.5. The expression and purification procedures of HEV-pET32a-2 protein, ORF2 aa368-660-1 protein and ORF2 aa368-660-2 protein were the same as those of HEV-pET32a-1 protein.

[0081] 1.5 Validation of recombinant antigen dimers

[0082] The protein bands were verified by running a non-denaturing SDS-PAGE gel. The results showed that the dimers (polymers) with polypeptide series antigens were more than the original sequence, such as Figure 3 As shown, in A, 1 is HEV-pET32a-1 antigen, 2 is HEV-pET32a-2 antigen, and the right side is protein Maker; in B, 1 is ORF2aa368-660-1 antigen, 2 is ORF2 aa368-660-2 antigen, and the left side is protein Maker.

[0083] Example 2 Verification of recombinant antigen activity

[0084] The indirect Elisa experiment initially verified the activity, and the steps are as follows:

[0085] (1) Antigen coating: HEV-pET32a-1 antigen, HEV-pET32a-2 antigen, ORF2 aa368-660-1 antigen, ORF2 aa368-660-2 antigen, and ORF2 aa459-606 antigen were diluted to 1 ug / ml with coating solution CBS, 100 ul / well, pH 9.6, and coated overnight;

[0086] (2) Blocking: Add 300 μl of blocking solution (PBS containing 1% BSA) to each well and block at 37°C for 2 hours;

[0087] (3) Discard the blocking solution and wash three times with PBST, patting dry on a clean paper towel after each wash;

[0088] (4) Adding primary antibody and serum: dilute the serum 500 times with 0.5% BSA, add 50 μl of diluted serum to each well and place at 37°C for 1 hour;

[0089] (5) Washing: Aspirate the serum with a pipette and wash three times with PBST, patting dry on a clean paper towel after each wash;

[0090] (6) Adding secondary antibody: dilute IgM with 0.5% BSA at a ratio of 1:1000, add 50 μl of diluted IgM to each well, and incubate at 37°C for 30 minutes;

[0091] (7) Washing: Aspirate the IgM with a pipette and wash three times with PBST, patting dry on a clean paper towel after each wash;

[0092] (8) Color development: Add 50ul TMB substrate buffer (0.005mol / L sodium acetate-citrate buffer pH 3.6) and 50ul TMB substrate solution (0.08g of 3,3',5,5'-tetramethylbenzidine (TMB) dissolved in 40mL dimethyl sulfoxide (DMSO), add 60mL methanol, mix well, add 100mL substrate buffer, stir in a dark place for 2 hours until dissolved, and store at 4℃ in dark.) to each well in turn and incubate in dark place for 5-10 minutes.

[0093] (9) Termination: Add 50ul 2mol / L H2SO4 to each well;

[0094] (10) The OD450 value was read using an ELISA reader. The results are shown in Table 1.

[0095] Table 1 Results of different antigen activity tests

[0096]

[0097] The preliminary results of Elisa experiment show that: for positive sample detection, the positive sample detection values ​​of the two antigens HEV-pET32a-1 and HEV-pET32a-2 expressed by the present invention are higher than those of ORF2 aa459-606 antigen, and the positive value of HEV-pET32a-1 antigen is higher than that of HEV-pET32a-2; for negative sample detection results, the negative value of HEV-pET32a-1 antigen is lower than that of HEV-pET32a-2 and ORF2aa459-606 antigen, and the two antigens expressed by the present invention can be verified by colloidal gold products. The positive sample detection values ​​of ORF2aa368-660-1 and ORF2 aa368-660-2 are lower than those of ORF2aa459-606 antigen, and the negative sample detection values ​​are significantly higher than those of ORF2 aa459-606 antigen, resulting in false positives.

[0098] Example 3 Preliminary verification of recombinant HEV antigen in colloidal gold products

[0099] The obtained HEV-pET32a-1 antigen, HEV-pET32a-2 antigen, ORF2 aa368-660-1 antigen, ORF2aa368-660-2 antigen and ORF2 aa459-606 antigen were used as the raw materials for spotting, and IgM antibody was used as the gold-labeled raw material to detect serum samples in HEV colloidal gold products. The principle of dripping all samples was 5 μL sample + three drops of whole blood solution. After 15 minutes, the results were judged and recorded, and the color card readings were used (colloidal gold values ​​were read with standard color cards). The experimental results are shown in Table 2. Among them, there are 10 negative references (N1-N10), 2 positive references (P1, P2), and 3 detection limit references (L1-L3).

[0100] Table 2 Detection results of different antigens in the application of HEV colloidal gold products

[0101]

[0102] In the colloidal gold product, the HEV-pET32a-1 and HEV-pET32a-2 antigens expressed by the present invention can be detected in two positive serum samples, and the detection value of HEV-pET32a-1 is the highest, and no false positives appear in 10 negative serum samples, and the detection limit is two out of three reference samples; ORF2 aa459-606 antigen can be detected in two positive serum samples, and the detection limit is two out of three reference samples, but there are weak false positives in negative serum samples and the detection value of positive samples is lower than that of the antigen expressed by the present invention. The preliminary verification results in the colloidal gold product show that the two antigens expressed by the present invention are superior to the ORF2 aa459-606 antigen, and the HEV-pET32a-1 antigen with a high dimer content has the best result. When ORF2 aa368-660-1 and ORF2aa368-660-2 antigens were tested in negative serum samples, 10 negative sera tested positive, and the positive values ​​were high. The antigens showed obvious false positive phenomenon in the colloidal gold product, and no verification experiments will be carried out subsequently.

[0103] Example 4 Clinical positive detection rate and specificity experiment

[0104] In order to further verify the performance of the antigen expressed by the present invention in the colloidal gold product, the HEV-pET32a-1 antigen, HEV-pET32a-2 antigen and ORF2 aa459-606 antigen were respectively used for clinical specific experiments on 14 positive serum samples and 126 negative serum samples provided by the testing agency, and the serum positive and negative samples were determined according to the standard color card reading. Among them, sera No. 3, No. 25, No. 30, No. 38, No. 44, No. 53, No. 66, No. 72, No. 88, No. 90, No. 93, No. 110, No. 112, and No. 138 in the 140 samples were positive sera, and the rest were negative sera. The detection results of the kits for different antigens are shown in Table 3.

[0105] Table 3 Detection results of colloidal gold kits with different antigens on clinical samples

[0106]

[0107] The results of clinical specificity experiments showed that the positive detection rate of HEV-pET32a-1 antigen in colloidal gold products was 13 / 14, with a specificity of 99.21%; the positive detection rate of HEV-pET32a-2 antigen in colloidal gold products was 8 / 14, with a specificity of 96.83%; the positive detection rate of ORF2 aa459-606 antigen was 7 / 14, with a specificity of 92.06%.

[0108] Wherein, specificity = true negative serum sample / (true negative serum sample+false negative serum sample)*100%.

[0109] Example 5 Repeatability and stability verification

[0110] 1.1 Antigen repeatability verification

[0111] The HEV-pET32a-1 antigen was verified for repeatability, and the results are shown in Table 4. All three batches of positive and negative samples met the test criteria, with no significant differences between batches.

[0112] Table 4 HEV-pET32a-1 antigen repeatability test results

[0113] Group 1 2 3 Buffer 0 0 0 P1 10 10 10 P2 6+ 7 7 L1 8 8 8 L2 5 5 5 L3 0 0 0 N1 0 0 0 N2 0 0 0 N3 0 0 0 N4 0 0 0 N5 0 0 0 N6 0 0 0 N7 0 0 0 N8 0 0 0 N9 0 0 0 N10 0 0 0

[0114] 5.2 Accelerated stability test

[0115] The three batches of HEV-pET32a-1 antigen obtained were used as dot-film raw materials, and IgM antibody was used as gold-labeled raw materials in HEV colloidal gold products. The products were placed in a 60°C incubator for accelerated stability testing. Three months later, the products were taken out for testing serum samples. The results are shown in Table 5, which are basically consistent with the data without accelerated stability.

[0116] Table 5 HEV-pET32a-1 antigen stability test results

[0117] Group 1 2 3 Buffer 0 0 0 P1 10 10 10 P2 6 7 7 L1 8 8 8 L2 5 5 5 L3 0 0 0 N1 0 0 0 N2 0 0 0 N3 0 0 0 N4 0 0 0 N5 0 0 0 N6 0 0 0 N7 0 0 0 N8 0 0 0 N9 0 0 0 N10 0 0 0

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antigen for detecting hepatitis E virus IgM, characterized in that: Includes (A) or (B) below; (A) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (B) A protein composed of the C-terminus of the amino acid sequence shown in SEQ ID NO.2 connected to the N-terminus of the amino acid sequence shown in SEQ ID NO.

1.

2. A polynucleotide encoding the antigen according to claim 1.

3. The polynucleotide according to claim 2, characterized in that The polynucleotide is a gene encoding the antigen according to claim 1; the nucleotide sequence of the gene is shown as SEQ ID NO.3 or SEQ ID NO.

4.

4. A carrier, characterized in that Comprising the polynucleotide according to claim 2 or 3.

5. A host cell, characterized in that Comprising the vector of claim 4.

6. A method for preparing the antigen according to claim 1, characterized in that: The expression vector containing the nucleotide sequence encoding the antigen is transferred into the host and then induced for expression.

7. Use of the antigen according to claim 1, the polynucleotide according to claim 2 or 3, the vector according to claim 4, the host cell according to claim 5 or the antigen prepared by the preparation method according to claim 6 in preparing a product for detecting or assisting in detecting hepatitis E virus IgM or preparing a product for detecting or assisting in detecting whether a sample to be tested is infected with hepatitis E virus.

8. A hepatitis E virus IgM test strip, characterized in that: The antigen comprises the antigen according to claim 1, the vector according to claim 4, the host cell according to claim 5 or the antigen prepared by the preparation method according to claim 6.

9. A hepatitis E virus IgM detection kit, characterized in that: The antigen comprises the antigen according to claim 1, the vector according to claim 4, the host cell according to claim 5 or the antigen prepared by the preparation method according to claim 6.

10. A method for detecting hepatitis E virus IgM for non-disease diagnosis purposes, characterized in that: The method comprises utilizing the test strip according to claim 8 or the test kit according to claim 9.

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