Antigen for detecting hepatitis e virus igm and preparation method and application thereof

Abstract

The application provides an antigen for detecting hepatitis E virus IgM and a preparation method and application thereof, relates to the technical field of biotechnology, and comprises the following (A) or (B); (A) a protein composed of the amino acid sequence shown in SEQ ID NO. 1; (B) a protein composed of the C terminal of the amino acid sequence shown in SEQ ID NO. 2 and the N terminal of the amino acid sequence shown in SEQ ID NO. 1. It is found that the ORF2aa417-660 antigen and the ORF2aa417-660 antigen added with a beta-fold structure sequence can be used as a point membrane raw material for detecting IgM antibodies in a colloidal gold product, the positive detection rate is high, the false positive rate is low, and the sensitivity is obviously better than that of the ORF2aa459-606 antigen. In particular, the ORF2aa417-660 antigen added with the beta-fold structure sequence is helpful to the formation of a dimer and a multimer of the antigen, and further enhances the immunogenicity of the antigen.

Description

TECHNICAL FIELD

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

[0002] Hepatitis E is an infectious disease caused by hepatitis E virus (HEV), and the virus is mainly transmitted through the fecal-oral route. Hepatitis E mainly damages the liver, and the symptoms are severe. Clinically, it is mainly seen in young adults and the elderly.

[0003] The detection methods of hepatitis E mainly include nucleic acid detection, liver function detection, and serum detection. Since nucleic acid detection and liver function detection require more instruments and equipment and take a long time, serum detection is convenient and fast, and therefore serum detection becomes the preferred method for detecting hepatitis E virus. Serum detection mainly detects IgM antibodies. Hepatitis E virus is a single-stranded positive-sense RNA virus without envelope, and its genome is about 7500 kB, which consists of three open reading frames. ORF2 encodes a single structural capsid protein, which includes most of the immunogenic regions. This region is a commonly used sequence for disease diagnosis. Amino acids 459-606 are the main immunogenic epitopes. The market often uses this fragment to design antigens as diagnostic antigens, but the IgM positive detection rate is not high and false positives may occur.

[0004] Therefore, the present application is proposed. SUMMARY

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

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

[0007] The third purpose of the present application is to provide a vector.

[0008] The fourth purpose of the present application is to provide a host cell.

[0009] The fifth purpose of the present application is to provide a preparation method of the above-mentioned antigen.

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

[0011] The seventh purpose of the present application is to provide a hepatitis E virus IgM detection test strip.

[0012] The eighth object of the present application is to provide a HGV IgM detection kit.

[0013] The ninth object of the present application is to provide a method for detecting HGV IgM for non-disease diagnosis.

[0014] In order to achieve the above object of the present application, the following technical solutions are adopted:

[0015] In the first aspect, the present application provides an antigen for detecting HGV IgM, comprising (A) or (B) as follows:

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

[0017] (B) a protein consisting of the C-terminal of the amino acid sequence shown in SEQ ID NO. 2 and the N-terminal of the amino acid sequence shown in SEQ ID NO. 1.

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

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

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

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

[0022] In the fifth aspect, the present application provides a preparation method of the above-mentioned antigen, which comprises the steps of: transferring an expression vector containing a nucleotide sequence encoding the antigen into a host, and then inducing expression.

[0023] In the sixth aspect, the present application 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 detecting HGV IgM or a product for detecting or assisting in detecting whether a sample to be tested is infected with HGV.

[0024] In the seventh aspect, the present application provides a HGV 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 the eighth aspect, the present application provides a HGV 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 application provides a method for detecting IgM of the hepatitis E virus for non-disease diagnosis, comprising using the test strip or the kit as described above.

[0027] The present application provides an antigen for detecting IgM of the hepatitis E virus, and the amino acid sequence ORF2 aa417-660 antigen is obtained by analyzing published antigen epitopes, the ORF2 aa417-660 fragment is selected, and a beta-fold structure sequence SCDVTKLLNCGIPGS of lysozyme is added at the sequence N terminal. The amino acid sequence of the lysozyme dimer is composed of serine (Ser), aspartic acid (Asp), alanine (Ala), lysine (Lys) and other amino acids. The addition of the sequence is helpful for the antigen to form a dimer and a multimer. Through a late antigen verification experiment, it is found that the ORF2 aa417-660 antigen and the ORF2 aa417-660 antigen with the beta-fold structure sequence as a point membrane raw material in the colloidal gold product for detecting IgM antibody have a high positive detection rate and a low false positive rate, and the sensitivity is obviously better than that of the ORF2 aa459-606 antigen. Especially, the ORF2 aa417-660 antigen with the beta-fold structure sequence is helpful for further enhancing the immunogenicity of the antigen. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0029] Figure 1 The agarose gel electrophoresis chart of the expression strain amplification band of the HEV-pET32a-1 recombinant antigen provided in Embodiment 1 of the present application is shown in the following figure:

[0030] Figure 2 The antigen expression comparison chart of the supernatant of the expression strain of the HEV-pET32a-1 recombinant antigen under different induction conditions provided in Embodiment 1 of the present application is shown in the following figure:

[0031] Figure 3 The dimer verification result comparison chart of different antigens provided in Embodiment 1 of the present application is shown in the following figure: DETAILED DESCRIPTION

[0032] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meanings of terms should be clear from the context within which they are used, however, in any potentially uncertain cases, the definition provided herein prevails over any dictionary or extrinsic definition. In this application, the use of "or" means "and / or" unless specifically stated otherwise, e.g., "comprising A or B" means "comprising A or B or both". Also, the use of "comprising" or "including" or other forms for "comprise" or "include" or "including" are not intended to be construed to imply exclusivity of the foregoing elements or indicate a numerical limitation to the composition or process.

[0033] The methods and techniques of the present application 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 throughout the present specification unless otherwise indicated.

[0034] The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, a DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. A polynucleotide can encode the above-mentioned SS-B binding protein, optionally encoding a sense or an antisense strand. The polynucleotide can be naturally occurring, synthetic, or 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 vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. The vector can be introduced into a cell by transformation, transfection, or transduction and the vector's carried genetic material elements are then expressed in the cell.

[0036] The expressions "cell", "cell line", and "cell culture" are used interchangeably and all such designations include the progeny. The progeny can not necessarily be completely identical to the parent cell, due to natural, accidental, or deliberate mutation, and can exist as a population of genetically identical or similar cells. The progeny can be distinguished from the parent 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 the naturally occurring amino acids. Naturally occurring amino acids include the amino acids encoded by the genetic code and modified amino acids thereof. Commonly occurring 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). The Chinese name, three-letter abbreviation and one-letter abbreviation of the amino acid are used interchangeably herein.

[0038] In one aspect of the present application, an antigen for detecting IgM of hepatitis E virus is provided, comprising (A) or (B) as follows:

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

[0040] (B) a protein consisting of the C-terminal of the amino acid sequence shown in SEQ ID NO. 2 and the N-terminal of the amino acid sequence shown in SEQ ID NO. 1.

[0041] By analyzing the published antigen epitopes, the ORF2 aa417-660 antigen with the amino acid sequence shown in SEQ ID NO. 1 is obtained, the ORF2 aa417-660 fragment is selected, and a β-pleated structure sequence SCDVTKLLNCGIPGS of lysozyme is added at the N-terminal of the sequence. The amino acid sequence of the lysozyme dimer is composed of serine (Ser), aspartic acid (Asp), alanine (Ala), lysine (Lys) and other amino acids. The addition of the sequence helps the antigen to form dimers and polymers. Through the later antigen verification experiment, it is found that the ORF2 aa417-660 antigen and the ORF2 aa417-660 antigen with the β-pleated structure sequence as a point membrane raw material in the colloidal gold product for detecting IgM antibody have high positive detection rate and low false positive rate, and the sensitivity is obviously superior to that of the ORF2 aa459-606 antigen. Especially, the ORF2 aa417-660 antigen with the β-pleated structure sequence helps the antigen to form dimers and polymers, and further enhances the immunogenicity of the antigen.

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

[0043] In some embodiments, the polynucleotide is a gene encoding the antigen described above; 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 application, a vector comprising the polynucleotide described above is also provided.

[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. The animal virus that can be used as a vector includes but is not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papovavirus. In some embodiments, the vector of the present application can be selected from pET32a, pET32b, or pET32c, preferably pET32a as an expression vector.

[0046] According to another aspect of the present application, a host cell comprising the vector described above is also provided.

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

[0048] According to another aspect of the present application, a method for preparing the antigen described above is also provided, which comprises transforming an expression vector containing the nucleotide sequence encoding the antigen into a host, and then inducing expression.

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

[0050] In some embodiments, the expression vector pET32a, the expression strain Rosetta(DE3), the induction of protein expression at 30°C, and the selection of gradient renaturation mode, etc. can be selected to promote the formation of more dimers and multimers of the protein.

[0051] According to another aspect of the present application, there is further provided use of the antigen, the polynucleotide, the vector, the host cell or the antigen prepared by the preparation method in the preparation of a product for detecting or assisting in detecting hepatitis E virus IgM or 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 application, there is further provided a hepatitis E virus IgM detection test strip comprising the antigen, the vector, the host cell or the antigen prepared by the preparation method.

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

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

[0055] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

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

[0057] 2. Enzymes and kits: recombinant enzyme was purchased from Nanjing Nuowezan Biological Medicine Technology Co., Ltd., plasmid extraction kit was purchased from Beijing Qike Biological Technology Co., Ltd., and purification recovery kit was purchased from Beijing Qike Biological Technology Co., Ltd.

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

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

[0060] 5. Equilibrium 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 as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 9, the vector is pET32a, the enzyme digestion site is BamHI / HindIII, and the purification method is the same as that of the ORF2 aa417-660.

[0062] 7. The colloidal gold product includes a colloidal gold product for qualitatively or quantitatively detecting a specific substance by using colloidal gold as an indicator / tracer, such as an immunochromatographic test strip or an immunochromatographic kit.

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

[0064] Example 1. Design and expression of the antigen

[0065] The full-length nucleotide fragment of the recombinant hepatitis E virus in the application is obtained by an artificial synthesis method, 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 scheme adopted in the application is as follows:

[0067] 1.1. Design of the HEV recombinant antigen

[0068] According to the HEV sequence published by the NCBI database, the ORF2 aa417-660 protein is screened by analyzing the published antigen epitopes.

[0069] The amino acid sequence of the ORF2 aa417-660 is as follows:

[0070] ENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQSTYGSSTGPVYVSDSVTLVNVATGAQAVARSLDWTKVTLDGRPLSTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQLLIENAAGHRVAISTYTTSLGAGPVAISAVAVLAPHSALALLEDTMDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTREL (SEQ ID NO. 1).

[0071] Experiments have shown that lysozyme dimer can enhance the immunogenicity of antigen, and the present application adds a lysozyme beta-sheet structure sequence SCDVTKLLNCGIPGS (SEQ ID NO. 2) at the N-terminal of the expressed sequence to promote the antigen to form more dimers (polymers). According to the codon bias of Escherichia coli, the nucleotide sequence of the desired fusion is designed and synthesized, and the nucleotide sequence of ORF2 aa417-660 is shown in SEQ ID NO. 3, and the optimized nucleotide sequence of ORF2 aa417-660 added with a lysozyme beta-sheet structure sequence is shown in SEQ ID NO. 4.

[0072] Meanwhile, the ORF2 aa368-660 protein and the recombinant protein added with a SCDVTKLLNCGIPGS (SEQ ID NO. 2) sequence in the ORF2 aa368-660 protein sequence are expressed, and are named as ORF2 aa368-660-1 and ORF2 aa368-660-2, 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 Vector construction of HEV-pET32a-1 recombinant antigen

[0075] The gene fragment is obtained by PCR amplification technology, and BamH and Hind enzyme digestion sites are added at both ends of the nucleotide sequence. The purified gene fragment is connected to the pET32a(-) vector under the action of the recombination enzyme, and the vector with the beta-sheet structure sequence is named as HEV-pET32a-1, and the vector without the beta-sheet structure sequence is named as HEV-pET32a-2. The same experimental method is used for expression and purification of the two vectors. The connection product is transferred into the TOP10 competent cells by heat shock method, and the competent cells are coated on the LB solid medium containing ampicillin, and single colonies are picked for bacterial liquid identification (the results are shown in FIG. 1, wherein 1 is a nucleic acid maker, and 2 is a bacterial liquid PCR band), and the bacterial liquid detection is correct. The correct strain is sent for sequencing, and the recombinant plasmid is extracted from the correct strain, and the recombinant plasmid is transformed into the Escherichia coli Rosetta (origami) expression strain. Figure 1

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

[0077] ​Two kinds of bacteria liquid were respectively connected to 4 tubes of 10ml LB liquid medium EP tube, and were shaken in 37℃ shaker to OD at 0.4-0.6. 1mM IPTG was added to induce expression, and one tube of bacteria liquid was used as control without adding IPTG and continuing to shake in 37℃ shaker. The rest three tubes of bacteria liquid were all added with 1mM IPTG and were respectively placed in 37℃, 30℃ and 20℃ shaker to shake for 6 hours. Each tube of bacteria liquid was collected, and the bacteria liquid was dissolved with 20mM PBS to perform ultrasonic and 12000rpm centrifugation. The supernatant was reserved for use, and the inclusion body after centrifugation was dissolved with 8M urea. After complete dissolution, centrifugation was performed. The supernatant after ultrasonic and the supernatant after centrifugation of inclusion body were subjected to SDS-PAGE to verify the results, as shown in Figure 2 Figure 1. Wherein 1 is 37℃ supernatant of uninduced bacteria liquid, 2 is 37℃ induced supernatant of bacteria liquid, 3 is 30℃ induced supernatant of bacteria liquid, 4 is 25℃ induced supernatant of bacteria liquid, 5 is protein maker, 6 is 37℃ inclusion body of uninduced bacteria liquid, 7 is 37℃ induced inclusion body of bacteria liquid, 8 is 30℃ induced inclusion body of bacteria liquid, and 9 is 25℃ induced inclusion body of bacteria liquid. It can be seen that expression is induced at 37℃, 30℃ and 20℃, and more protein is expressed at 30℃. Therefore, 30℃ is selected to shake and purify bacteria.

[0078] 1.4 Expression and purification of HEV recombinant antigen

[0079] Two kinds of E. coli were connected to low sodium LB medium, and 1mM IPTG was added when the OD was at 0.4-0.6 at 37℃ to induce for 6 hours at 30℃, and the bacteria were collected. The bacteria were dissolved with cell lysis solution and were fully dissolved. The dissolved bacteria liquid was broken by ultrasonic method, and then was centrifuged at 14000rpm for 30 minutes. The supernatant was discarded, and the inclusion body after centrifugation was dissolved with 8M urea overnight. The next day, 14000rpm centrifugation was performed for 30 minutes, and the supernatant was taken for purification. Ni-excel column was used for purification, the column was balanced with equilibrium liquid buffer, and the supernatant after centrifugation was loaded at a speed of 2ml / min. After loading, the Ni-excel column was first balanced with equilibrium liquid, and then 30mM, 60mM and 300mM imidazole were added to the equilibrium liquid. The target protein was collected, and was verified by SDS-PAGE. The target protein was eluted at 300mM imidazole.

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

[0081] 1.5 Verification of recombinant antigen dimer

[0082] The protein bands were verified by non-denaturing SDS-PAGE, and the results showed that the dimer (multimer) of the polypeptide series antigen was more than the original sequence, as shown in Figure 3 Figure 1, where 1 is HEV-pET32a-1 antigen, 2 is HEV-pET32a-2 antigen, and the right side is protein Maker; and Figure 2, where 1 is ORF2 aa368-660-1 antigen, 2 is ORF2 aa368-660-2 antigen, and the left side is protein Maker.

[0083] Example 2 Verification of activity of recombinant antigen

[0084] The activity was preliminarily verified by indirect Elisa experiment, and the steps were as follows:

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

[0086] (2) Blocking: 300 ul of blocking liquid (containing 1% BSA in PBS) was added to each well, and blocked at 37°C for 2 hours;

[0087] (3) Shake off the blocking solution and wash with PBST three times, and pat dry with a clean paper towel after each washing;

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

[0089] (5) Wash: use a pipette to remove the serum, and wash with PBST three times, and pat dry with a clean paper towel after each washing;

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

[0091] (7) Wash: use a pipette to remove the IgM, and wash with PBST three times, and pat dry with a clean paper towel after each washing;

[0092] (8) Color development: add 50ul of TMB substrate buffer (0.005 mol / L sodium acetate-citric acid buffer PH 3.6) and 50ul of TMB substrate solution (3,3',5,5'-tetramethylbenzidine (TMB) 0.08g dissolved in 40mL dimethyl sulfoxide (DMSO), add 60mL methanol, mix well, add 100mL substrate buffer, stir in the dark for 2 hours to dissolve, store at 4°C in the dark.) to each well, and place at room temperature in the dark for 5-10 minutes.

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

[0094] (10) Read the values with an enzyme marker OD450, and the results are shown in Table 1.

[0095] Table 1 Detection results of different antigen activities

[0096]

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

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

[0099] The obtained HEV-pET32a-1 antigen, HEV-pET32a-2 antigen, ORF2 aa368-660-1 antigen, ORF2 aa368-660-2 antigen and ORF2 aa459-606 antigen are used as point membrane raw materials, and IgM antibody is used as standard gold raw material to detect serum samples in HEV colloidal gold products. The sample drop principle is 5 μL sample + three drops of whole blood solution, and the results are read and recorded after 15 minutes. The experimental results are shown in Table 2. Among them, 10 negative reference samples (N1-N10), 2 positive reference samples (P1, P2), and 3 detection limit reference samples (L1-L3).

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

[0101]

[0102] In the colloidal gold product, the HEV-pET32a-1 and HEV-pET32a-2 antigens expressed by the application can be detected in both positive serum samples, and the HEV-pET32a-1 has the highest detection value, and no false positive occurs in 10 negative serum samples, and two of the three detection limit reference products are detected; the ORF2 aa459-606 antigen can be detected in both positive serum samples, and two of the three detection limit reference products are detected, but there is a weak false positive in the negative serum samples, and the positive sample detection value is lower than the antigen expressed by the application. The preliminary verification results in the colloidal gold product show that the two antigens expressed by the application are better than the ORF2 aa459-606 antigen, and the HEV-pET32a-1 antigen with high dimer content has the best result. When the ORF2 aa368-660-1 and ORF2 aa368-660-2 antigens are detected in negative serum samples, 10 negative serum samples are detected with high positive values, and the antigens have obvious false positive phenomenon in the colloidal gold product, and subsequent verification experiments will not be carried out.

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

[0104] In order to further verify the performance of the antigens expressed by the application in the colloidal gold product, the HEV-pET32a-1 antigen, the HEV-pET32a-2 antigen and the ORF2 aa459-606 antigen are respectively subjected to clinical specificity experiments on 14 positive serum samples and 126 negative serum samples provided by a detection agency, and the serum positive and negative samples are determined according to the standard color card reading value. Among them, the 3rd, 25th, 30th, 38th, 44th, 53rd, 66th, 72nd, 88th, 90th, 93rd, 110th, 112th and 138th serum samples in the 140 samples are positive serum, and the rest are negative serum. The detection results of the colloidal gold kit of different antigens are shown in Table 3.

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

[0106]

[0107] The clinical specificity experiment results show that the positive detection rate of the HEV-pET32a-1 antigen in the colloidal gold product is 13 / 14, and the specificity is 99.21%; the positive detection rate of the HEV-pET32a-2 antigen in the colloidal gold product is 8 / 14, and the specificity is 96.83%; and the positive detection rate of the ORF2 aa459-606 antigen is 7 / 14, and the specificity is 92.06%.

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

[0109] Example 5 Reproducibility and stability verification

[0110] 1.1 Reproducibility verification of antigen

[0111] The HEV-pET32a-1 antigen was subjected to reproducibility verification, and the results are shown in Table 4. The three batches of positive and negative samples all met the test standards, and there was no significant batch-to-batch difference.

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

[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 the point film raw material, and IgM antibody was used as the standard gold raw material to prepare the HEV colloidal gold product. The product was placed in a 60°C incubator for accelerated stability test. After three months, the product was taken out and the serum samples were detected. The results are shown in Table 5, which are basically consistent with the data without acceleration.

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

[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 examples are only used to illustrate the technical solutions of the present application, and are not limited thereto. Although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features thereof. Such modifications or substitutions 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 application.

Claims

1. An antigen for detecting hepatitis E virus IgM, characterized in that, It is either (A) or (B) below; (A) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (B) A protein consisting of the C-terminus of the amino acid sequence shown in SEQ ID NO.2 and the N-terminus of the amino acid sequence shown in SEQ ID NO.

1.

2. A polynucleotide encoding the antigen of claim 1.

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

4.

4. A carrier, characterized in that, It comprises the polynucleotide as described in claim 2 or 3.

5. A host cell, characterized in that, It includes the carrier as described in claim 4.

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

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

8. A hepatitis E virus IgM detection test strip, characterized in that, This includes the antigen described in claim 1 or the antigen prepared by the preparation method described in claim 6.

9. A hepatitis E virus IgM detection kit, characterized in that, This includes the antigen as described in claim 1, the vector as described in claim 4, the host cell as described in claim 5, or the antigen prepared by the preparation method described in claim 6.

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