A pan-genotype ORF3 protein of Hepatitis E virus genus A and its application

Abstract

The present invention discloses a pan-genotype ORF3 protein of the Hepatitis E virus genus A and an application thereof. The HEV-A pan-genotype ORF3 protein can be used to detect the content of IgG and IgM antibodies in the serum of hepatitis E patients. After the serum is diluted 500 times, IgG and IgM in the serum can still be detected, with high sensitivity. Since the ORF2 polypeptide is the main component of the HEV vaccine, the ORF2 antibody detection kit cannot distinguish between vaccine-immunized people and infected people. However, HEV-positive cases detected by the ORF3 protein detection kit can all be judged as people naturally infected with HEV.

Description

Technical Field

[0001] The present invention relates to a pan-genotype ORF3 protein of Hepatitis E virus A and application thereof, belonging to the technical field of Hepatitis E virus detection. Background Art

[0002] Hepatitis E virus (HEV) is the leading cause of viral hepatitis worldwide. Clinical manifestations often include acute hepatitis E, including asymptomatic infection, mild to moderate liver dysfunction, and fulminant hepatitis. Persistent hepatitis E can develop in immunocompromised individuals and, if left untreated, can progress to cirrhosis. HEV is a member of the Hepeviridae family, which includes two genera: Orthohepevirus and Piscihepevirus. Hepatitis E in humans is primarily caused by Orthohepevirus species A (HEV-A), which includes eight HEV genotypes, namely HEV genotypes 1-8 (G1-8), which infect humans, pigs, wild boars, deer, rabbits, and camels.

[0003] HEV is a positive-strand RNA virus containing three open reading frames (ORFs): ORF1, ORF2, and ORF3. ORF1 encodes the ORF1 nonstructural protein, which is involved in viral replication. ORF2 encodes the viral capsid protein ORF2, which is mainly involved in the assembly of viral particles, binding to host cells, and inducing the host to produce neutralizing antibodies. ORF3 encodes the multifunctional phosphoprotein ORF3, which is mainly involved in the release of viral particles. Recent studies have shown that ORF3 is believed to form an ion channel that shares key structural features with class I viral particles required for the release of viral particles from cells during infection. In addition, ORF3 interacts with ORF2 and lipids to participate in the packaging, formation, and release of quasi-enveloped HEV viral particles.

[0004] ORF2 and ORF3 can stimulate the body to produce humoral immune responses. The ORF2 capsid protein is highly conserved and has strong immunogenicity, and is also the main target of cellular immune responses. ORF3 can also induce the body to produce immune responses and produce corresponding antibodies. However, there are no reports on its immunogenicity and whether it can be used as an antigen to detect antibody levels in patients. Therefore, at this stage, ORF2 is mainly used as an antigen to detect antibodies in patient serum.

[0005] Several studies have reported that the sensitivity and specificity of HEV serological detection reagents currently used both domestically and internationally vary significantly in detecting both IgM and IgG produced against HEV-ORF2. The difference in antibody positivity can be more than 10-fold, and the reproducibility of the test results needs to be improved. (Chinese Journal of Experimental and Clinical Virology, 2007, 21(1): 59-61. Chinese Journal of Microbiology and Immunology, 2009, 29(9): 854-857. Cli Infect Dis, 2010, 51(3): e24-27. Intervirology, 2015, 58(5): 283-287. J Med Virol, 2017, 89(6): 1055-1061.)

[0006] One of the main reasons for the above problems is the difference in antigens between different HEV genotypes. Although HEV-A has only one serotype, studies have shown that since HEV-A is divided into types 1-8, their amino acid homology varies, and there are certain differences when detecting antibodies using different genotype antigens (Infect Genet Evol, 2015, 34: 211-220. Hepat Mon, 2016, 16(8): e35312.). Currently, most detection reagents used at home and abroad use one genotype of ORF2 (HEV-A genotype 1, 3 or 4) as an antigen to detect antibodies (IgM and IgG) against ORF2 protein in patient serum (Current Status, Problems and Prospects of Hepatitis E Virus Antibody Detection [J]. Zhou Xiaoying, Meng Jihong. Acta Virologica Sinica. 2018(01)).

[0007] The main component of the HEV vaccine is a polypeptide fragment of ORF2, which effectively stimulates the body to produce an immune response and antibodies against ORF2. Therefore, when using existing HEV serological tests to screen the general population for anti-HEV-ORF2 antibodies, it is impossible to distinguish between vaccine immunity and natural infection, which interferes with the assessment of HEV epidemiological status. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention provides a pan-genotypic HEV-A ORF3 protein designed based on the ORF3 sequences of HEV-A genotypes 1-8. This consensus sequence maximizes the immunogenicity of genotypes 1-8. Furthermore, using pan-genotypic ORF3 as an antigen effectively detects and assesses IgG and IgM antibodies against HEV-ORF3 in HEV patients, effectively distinguishing between viral infection and vaccine immunity, and further improving current hepatitis E virus serological diagnostic methods.

[0009] The present invention is achieved through the following technical solution: a pan-genotype ORF3 protein of the Hepacivirus genus A, characterized in that the amino acid sequence of the pan-genotype ORF3 protein of the Hepacivirus genus A is shown in SEQ ID NO: 1.

[0010] The invention relates to an application of the hepatitis E virus genus A pan-genotype ORF3 protein in the detection of HEV infection.

[0011] The monoclonal antibody or polyclonal antibody produced by the pan-genotype ORF3 protein of the Hepatitis E virus genus A is used in HEV laboratory detection.

[0012] A kit for detecting hepatitis E virus, comprising an ORF3 protein of the pan-genotype A of the orthohepatitis E virus;

[0013] The amino acid sequence of the pan-genotype ORF3 protein of the Hepatovirus genus A is shown in SEQ ID NO: 1.

[0014] A pan-genotype ORF3 protein of the Hepacivirus A is composed of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0015] The amino acid sequence of the pan-genotype ORF3 protein of the Hepacivirus genus A is: MGSPCALGLFCCCSSCFCLCCPRHRPVSRLAAVVGGAAAVPAVVSGVTGLILSPSPSPI FIQPTPSPPTSFHNPGLELALGSRPAHSAPLGVTSPSAPPLPPVVDLPQLGLRR.

[0016] According to the present invention, a first amino acid sequence having at least 90% identity to a second amino acid sequence means that the first sequence has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to the second amino acid sequence. Sequence identity is typically measured as a percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Methods for comparing sequences are well known in the art. Various programs and alignment algorithms are described by Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; Pearson et al., Meth. Mol. Biol., 24:307-31, 1994. Altschul et al., Nat. Genet., 6:119-129, 1994, provides a detailed discussion of sequence alignment methods and homology calculations. For example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) can be used for sequence comparisons (Internet 1996, W.R. Pearson and the University of Virginia, fasta version 2.0, released December 1996). ALIGN compares entire sequences to each other, while LFASTA compares similarities in local regions. For example, these alignment tools and their respective tutorials are available on the Internet at the National Center for Supercomputer Applications (NCSA) website. Alternatively, for comparisons of amino acid sequences greater than about 30 amino acids, the Blast 2 sequence function can be performed using the default BLOSUM62 matrix set to default parameters (gap penalty of 11 and per-residue gap penalty of 1). When aligning short peptides (less than approximately 30 amino acids), the alignment should be performed using the PAM30 matrix set to default parameters using the Blast 2 sequence function (open gaps 9, extension gaps 1 penalty).The BLAST sequence comparison system is available, for example, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature. Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic. Acids. Res., 25:3389-3402, 1997; and Zhang & Madden, Genome. Res., 7:649-656, 1997.

[0017] Beneficial technical effects of the present invention:

[0018] 1) The HEV-A pan-genotype ORF3 protein can be used to detect the levels of IgG and IgM antibodies in the serum of hepatitis E patients. Even after the serum was diluted 500-fold, IgG and IgM in the serum could still be detected, showing high sensitivity.

[0019] 2) Serum samples from 11 HEV-infected patients were tested, with a 100% IgG and 90.9% IgM positive rate;

[0020] 3) The HEV-A pan-genotype ORF3 protein can be used to detect the titers of IgG and IgM in the serum of patients infected with HEV genotypes 1-8;

[0021] 4) Since ORF2 polypeptide is the main component of HEV vaccine, the ORF2 antibody detection kit cannot distinguish between vaccine-immunized and infected people; however, HEV-positive cases detected by the ORF3 protein detection kit can all be judged as naturally infected with HEV.

[0022] 5) Immunizing New Zealand white rabbits with the HEV-A pan-genomic ORF3 protein stimulated the production of corresponding antibodies, exhibiting good immunogenicity and potentially serving as a potential vaccine component.

[0023] 6) Monoclonal and polyclonal antibodies generated using HEV-A pan-genomic ORF3 can be used for Western blot, immunofluorescence, and ELISA to detect ORF3 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .pET-21a(+)-HEV-A-ORF3 plasmid map;

[0025] Figure 2.Double restriction enzyme digestion and PCR identification of pET21a(+)-HEV-A-ORF3;

[0026] Figure 3 The ORF3 purification steps were identified using Coomassie Brilliant Blue-stained SDS-PAGE.

[0027] Figure 4 The purified protein was identified using His tag antibodies and ORF3-specific antibodies;

[0028] Figure 5 HEV-A pan-genotype ORF3 protein was used as an antigen to detect HEV IgG and IgM in the serum of HEV-infected patients;

[0029] Figure 6 Determine the effect of different amounts of coated ORF3 antigen on antibody detection levels;

[0030] Figure 7 .Determination of antibody titers in rabbit serum after different immunization times;

[0031] Figure 8 PCR results of .ORF3;

[0032] Figure 9 Pan-ORF3 polyclonal antibody was diluted at 1:1000, 1:2000, 1:3000, and 1:4000 to detect eukaryotic ORF3 protein;

[0033] Figure 10 . Use flag antibody (1:2000) to detect eukaryotic expressed ORF3 protein;

[0034] Figure 11 .Use pan-genomic ORF3 protein polyclonal antibody to detect ORF3 protein expressed in eukaryotic cells.

[0035] Figure 12 The serum produced after immunization with ORF2 could not interact with ORF3. DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples and accompanying drawings.

[0037] Example 1

[0038] Comparison of ORF3 amino acid sequences of HEV-A genotypes 1-8 and design of pan-genotype ORF3 amino acid sequence

[0039] The amino acid sequences of ORF3 of HEV-A gene types 1-8 were downloaded from NCBI, all sequences were imported into Vector NTI (11.5.3), all sequences were aligned using the Alignment function, and the consensus sequence was designed based on its amino acid sequence ( Figure 1 ), which is the amino acid sequence of HEV-A pan-genotype ORF3 protein.

[0040] The amino acid sequence of the HEV-A pan-genotype ORF3 protein is: MGSPCALGLFCCCSSCFCLCCPRHRPVSRLAAVVGGAAAVPAVVSGVTGLILSPSPSPIFIQPTPSPPTSFHNPGLELALGSRPAHSAPLGVTSPSAPPLPPVVDLPQLGLRR.

[0041] The HEV-A genotype 1-8 ORF3 sequence number is as follows:

[0042] HEV-A genotype 1: HEV1a(AF051830), HEV1a(AF076239), HEV1a(AF185822), HEV1a(D10330), HEV1a(DQ459342), HEV1a(FJ457024), HEV1a(JF44 3719), HEV1a(JF443720), HEV1a(LC061267), HEV1a(M73218), HEV1a(X99441), HEV1b(AF444003), HEV1b(D11092), HEV1b(D11093), HEV1b (JQ655734), HEV1b(L08816), HEV1b(L25595), HEV1b(NC001434), HEV1c(JF443717), HEV1c(X98292), HEV1d(AY230202), HEV1e(AY20487 7), HEV1f(AB720035), HEV1f(JF443721), HEV1f(JF443722), HEV1f(JF443723), HEV1f(JF443724), HEV1f(JF443725), HEV1f(JF443726).

[0043] HEV-A genotype 2: HEV2a (M74506).

[0044] HEV-A genotype 3: HEV3a (AB074918), HEV3a (AB074920), HEV3a (AB089824), HEV3a (AB481228), HEV3a (AB591734), HEV3a (AF060668), HEV3a (AF060669), HEV3a (AF082843), HEV3a (FJ426403), HEV3a (FJ426404), HEV3a (JN564006), HEV3a (JN837481), HEV3a (JQ679013), HEV3a (KF303502), HEV3a (KJ507955), HEV3b (AB073912), HEV3b (AB091394), HEV3b (AB189071), HEV3b (AB222182), HEV3b (AB222183), HEV3b (AB222184), HEV3b (AB236320), HEV3b (AB246676), HEV3b (AB291955), HEV3b (AB291962), HEV3b (AB291963), HEV3b (AB301710), HEV3b (AB369689), HEV3b (AB369691), HEV3b (AB481229), HEV3b (AB630971), HEV3b (AB740232), HEV3b (AP003430), HEV3b (FJ527832), HEV3b (KJ507956), HEV3c (FJ705359), HEV3c (KC618402), HEV3c (KJ701409), HEV3e (AB248521), HEV3e (AB248522), HEV3e (AB291958), HEV3e (AB780453), HEV3e (EU360977), HEV3e (FJ998015), HEV3e (HM055578), HEV3e (JQ013795), HEV3e (JQ026407), HEV3e (JQ953665), HEV3e (KF922359), HEV3e (KP698919), HEV3f (AB291961), HEV3f (AB369687), HEV3f (AB850879), HEV3f (EU375463), HEV3f (EU723512), HEV3f (EU723513), HEV3f (EU723514), HEV3f (EU723516), HEV3f (FJ653660), HEV3f (FJ956757), HEV3f (GU937805),<h2 style=";text-align:left;direction:ltr">HEV3f(JN906976)、HEV3f(JQ953666)、HEV3f(KC166971)、HEV3g(AF455784)、HEV3h(JQ013794)、HEV3i(FJ998008)、HEV3j(AY115488)、HEV3ra(AB740220)、HEV3ra(AB740 221) HEV3ra(AB740222) HEV3ra(FJ906895) HEV3ra(FJ906896) HEV3ra(JQ013791) HEV3ra(JQ013792) HEV3ra(JQ013793) HEV3ra(JX565469) HEV3ra(KJ013415)。、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] HEV-A type 4: HEV4a (AB197673), HEV4a (AB197674), HEV4a (EF077630), HEV4a (EU366959), HEV4a (FJ763142), HEV4a (GU119960), HEV4a (HQ634346), HEV4a (JQ655733), HEV4a (KC492825), HEV4a (KC692453), HEV4b (AB291964), HEV4 b(DQ279091), HEV4b(EU676172), HEV4b(JX855794), HEV4b(LC042232), HEV4c(AB074915), HEV4c(AB080575), HEV 4c(AB161717), HEV4c(AB200239), HEV4c(AB481227), HEV4d(AJ272108), HEV4d(AY594199), HEV4d(FJ610232), HEV 4d(GU206559), HEV4d(GU361892), HEV4d(HM152568), HEV4d(JQ655736), HEV4d(KC163335), HEV4d(KF176351), HE V4e (AY723745), HEV4f (AB220974), HEV4g (AB108537), HEV4g (AB369688), HEV4g (AB698654), HEV4h (GU119961), H EV4h (GU188851), HEV4h (JQ655735), HEV4h (JQ740781), HEV4h (KJ155502), HEV4i (AB369690), HEV4i (AB602440), HEV4i(AB909125), HEV4i(DQ450072), HEV4i(EF570133), HEV4i(HM439284), HEV4i(JF915746), HEV4i(JQ993308).

[0046] HEV-A basis type 5: HEV5a (AB573435).

[0047] HEV-A basis type 6: HEV6 (ab856243), HEV6a (AB602441).

[0048] HEV-A root type 7: HEV7 (KJ496143), HEV7 (KJ496144).

[0049] HEV-A genotype 8: HEV8(KX387865), HEV8(KX387866), HEV8(KX387867).

[0050] Example 2: Prokaryotic expression and purification of HEV-A pan-genotype ORF3 protein

[0051] 1. Construction of the expression plasmid for HEV-A pan-genotype ORF3 protein:

[0052] A 342-bp His-tagged ORF3 gene fragment was artificially synthesized. Six His amino acid sites were added before the ORF3 terminator codon as a purification tag (DNA sequence: CATCACCATCACCATCAC), and Xho I (CTCGAGGAGCTC) and Nhe I (GCTAGC) restriction sites were added at the front and back ends of the gene, respectively. This 378-bp sequence was artificially synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd.). The synthetic sequence is as follows:

[0053]

[0054] (Note: The bold letters ATG and TGA represent the start codon and stop codon, respectively. The underlined letters CTCGAGGAGCTC, CACCATCACCATCAC, and GCTAGC represent the Xho I restriction site, 6×His-tag, and Nhe I restriction site, respectively.)

[0055] The synthesized sequence was cloned into the pET21a(+) vector (completed by Suzhou Jinweizhi Biotechnology Co., Ltd.). The plasmid map is as follows Figure 1 , where the red region is the ORF3 sequence, which was cloned into the pET21a vector via Xho I and Nhe I.

[0056] The constructed plasmid was double-digested with Xho I and Nhe I (see Table 1 for the reaction system) and the target band and the vector were found to be correct ( Figure 2 A), the target fragment was identified by PCR and its size was consistent with that of the fragment ( Figure 2 B).

[0057] Table 1. Xho I and Nhe I double enzyme digestion reaction system:

[0058] COMPONENT 25μL REACTION DNA 0.5 μg 10X rCutSmart Buffer 2.5μL (1X) XOt 0.5μL (20 units) NheI-HF 0.5μL (20 units) Nuclease-free Water to 25μL

[0059] Table 2. ORF3 fragments in plasmids of PCR reaction system:

[0060]

[0061] 2. Expression of HEV-A pan-genotype ORF3 protein:

[0062] The recombinant expression plasmid pET21a-ORF3 was transformed into the Escherichia coli expression host strain BL21, and isopropyl-β-D-thiopyran and galactoside were added to induce expression. Finally, the bacterial solution was collected, ultrasonically lysed and centrifuged to harvest the unpurified protein. The specific steps are as follows:

[0063] 1) Transformation: Thaw 50 μL of competent E. coli BL21 cells quickly on ice. Add 1 ng of plasmid pET21a-HEV-A-ORF3, swirl gently to mix, and incubate on ice for 30 minutes. Immediately transfer the cells to a 42°C water bath for 90 seconds without shaking to reach the desired heat shock temperature. After heat shock, quickly transfer the cells to ice and incubate on ice for 1-2 minutes. Add 650 μL of LB culture medium and mix thoroughly. Incubate the transformed competent cells at 37°C in a 37°C incubator with shaking at 200 rpm for 1 hour. Centrifuge the shaken culture medium at 5000 rpm for 1 minute at room temperature. After centrifugation, discard 600 μL of the supernatant and mix the remaining 100 μL. Plate the culture medium evenly onto an Amp antibiotic plate and spread until no liquid flows. Incubate the plate upright for 10 minutes. Incubate the plate upside down at 37°C for 12-16 hours.

[0064] 2) Colony Picking and Expansion: Observe colony growth, pick individual colonies, and inoculate them into 5 mL of liquid selective LB medium. Incubate with shaking at 220 rpm in a 37°C incubator for 12-16 hours. Add 2 mL of the bacterial solution to a flask containing 200 mL of medium (200 μL of AMP, final concentration 100 μg / mL). After 2.5 hours, measure the OD value. When the OD value reaches 0.6-0.8, add 500 μL of 0.4 M IPTG. Return the culture to the 37°C shaker and continue incubation for 3 hours.

[0065] 3) Bacterial Collection: Collect the bacteria in a 50 mL centrifuge tube and centrifuge at 4000 rpm for 10 min to collect the precipitate. After multiple centrifugations, transfer the bacterial precipitate to a 50 mL centrifuge tube and freeze at -20°C. Remove the frozen bacterial solution and add 50 μL of 100× protease inhibitor (EDTA-free) and 50 μL of lysozyme (final concentration of 1 mg / mL) to each tube with 5 mL of PBS. Mix thoroughly and let stand for 30 min.

[0066] 4) Ultrasound: Transfer the bacterial solution containing protease inhibitors and lysozyme to a 10 mL centrifuge tube and sonicate for 45 minutes using an ultrasonic device.

[0067] 5) Centrifugation: The sonicated product was centrifuged at 11,000 rpm at 4°C for 30 min, and the supernatant was discarded.

[0068] 6) Resuspend: Resuspend the pellet in 5.5 mL of Lysis buffer (formula see Table 3), 55 μL of 100× protease inhibitor (EDTA-free), and 27.5 μL of 1 M DTT; incubate at 4°C for 30 min. Mix by pipetting several times with a 1 mL syringe, and then incubate at 4°C for 15 min.

[0069] 7) Protein harvesting by centrifugation: Centrifuge at 12,000 rpm at 4°C for 30 min and collect the supernatant, which contains unpurified protein.

[0070] 3. Utilize Ni + Purification of HEV-A pan-genotype ORF3 protein using -NTA affinity chromatography column and identification of the purified protein:

[0071] 1) Rinse out Ni with 3-5ml deionized water + - Storage buffer in the NTA column, use at least 5 ml of Lysis Buffer (formula see Table 3) to equilibrate the column.

[0072] 2) Add the HEV-A pan-genotype ORF3 protein sample to the column and allow the protein to adsorb to the column. Take 150 μL of the outflow liquid to detect the binding of the protein to the affinity chromatography column.

[0073] 3) Rinse the column with 10-15 ml of Wash Buffer (see Table 3 for the recipe). Take 150 μL of the Wash Buffer that flows out of the column to check the column cleaning status.

[0074] 4) Elute the target protein with 5 ml of Elution buffer (see Table 3 for the recipe). Collect the eluted protein in EP tubes, collecting 700-800 μL per tube, and collect seven times.

[0075] Table 3. Protein purification reagent formula

[0076] Reagent name formula Lysis buffer <![CDATA[8M urea, 50mM NaH2PO4, 300mM NaCl, 10mM imidazole]]> Wash buffer <![CDATA[5M urea, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole]]> Elution buffer <![CDATA[5M urea, 50mM NaH2PO4, 300mM NaCl, 250mM imidazole]]>

[0077] 5) Using 15% SDS-PAGE protein gel, identify the proteins before and after the column, in the outflow washing solution and eluted proteins ( Figure 3 ), found that 75% of the protein was able to bind to the affinity chromatography column and was eluted using the eluent. During the elution process, the amount of protein eluted from No. 2-3 was the highest, and its concentration was quantified to reach 1mg / ml.

[0078] The above-mentioned SDS-PAGE protein gel and Coomassie brilliant blue staining steps are as follows: (1) Prepare 12.5% ​​separation gel and 4% concentration gel. Prepare the separation gel first, mix it thoroughly and pour it into the gel plate, use anhydrous ethanol as the pressure line liquid, wait for about 40 minutes, pour out the upper layer of anhydrous ethanol and absorb the residual liquid with absorbent paper, then prepare the concentration gel, mix it thoroughly and pour it into the upper layer, insert the molding comb, let it stand at room temperature for about 40 minutes, and put it into a 4°C refrigerator after the gel is completely solidified for storage. (2) Place the gel plate in the electrophoresis rack, add the electrophoresis solution and remove the molding comb. Add 10-20 μL of protein sample to each gel well and add protein marker to one well. Place the electrophoresis rack in the electrophoresis tank, add the electrophoresis solution in the inner tank and outer tank, and start electrophoresis. Press the 70V electrophoresis line for about 30 minutes. When the sample is electrophoresed to the boundary between the concentration gel and the separation gel, change the voltage to 120V and stop the electrophoresis when the bromophenol blue runs to the bottom of the separation gel. (3) Prepare Coomassie Brilliant Blue staining solution (formula: 1 g R-250, 250 mL methanol, 100 mL glacial acetic acid, ddH2O to 1 L). Take out the gel block and soak it in the staining solution. Shake gently on a shaker overnight. Take out the gel block and soak it in clean water. Place it in a microwave oven. Use medium-high heat and heat for 1 hour until the sample bands on the gel block are clear. Change the water every 20 minutes.

[0079] 6) Identification of ORF3 expression by Western blot: Since ORF3 carries a His tag, the purified protein will be identified using His antibody and ORF3-specific antibody.

[0080] The specific steps of Western blot are as follows: (1) Use wet transfer to transfer the SDS-PAGE protein gel into a transfer machine and transfer the protein to the PVDF membrane. The voltage is maintained at 100V and the wet transfer is carried out for 100 minutes. (2) The membrane is soaked in blocking solution (5% skim milk powder) and blocked on a shaker at room temperature for 2 hours. (3) After blocking, the PVDF membrane is briefly washed and the primary antibody diluted with 1% BSA is added (His tag antibody dilution is 1:20000, ORF3 specific antibody dilution is 1:2000). After incubation at 4°C on a shaker overnight, the membrane is rewarmed on a shaker at room temperature for 30 minutes, the primary antibody is absorbed, and the membrane is washed three times with TBST washing buffer for 10 minutes each. (4) The diluted fluorescent secondary antibodies R-680 and R-800 are added and incubated on a shaker at room temperature for 2 hours. The membrane is washed three times with TBST washing buffer for 10 minutes each. (5) The membrane is scanned using the fluorescence channel of an Odessey membrane scanner.

[0081] The protein was identified by ORF3 purification and found to be ORF3 protein with His tag ( Figure 4 ).

[0082] Example 3: Establishment of an ELISA method using the purified HEV-A pan-genotype ORF3 protein to detect IgG and IgM antibodies in the serum of HEV-infected patients

[0083] 1. Coating Antigen: Dilute the purified ORF3 protein to a final concentration of 2 μg / mL with coating solution (0.1 M Na3PO4 pH = 9.0), add 100 μL per well to the ELISA plate, and refrigerate at 4°C overnight. The next day, remove the plate and incubate at 37°C for 1 hour. After discarding the liquid, wash the plate three times with 200 μL per well of PBS for 3 minutes each time.

[0084] 2. Blocking: Use 5% skim milk powder in PBS as blocking solution, add 100 μL to each well, incubate at 37°C for 1 hour, and then shake off the liquid.

[0085] 3. Add patient serum samples: Dilute patient serum 1 (see Table 4 for patient information) and negative serum from a healthy individual with PBS to 500, 1500, 4500, 13500, 40500, 121500, 364500, and 1093500-fold dilutions, respectively. Add 50 μL to each well. Incubate at 37°C for 2 h, shake off the liquid, and wash the plate three times with 0.05% PBST.

[0086] 4. Add anti-human IgG and IgM secondary antibodies: Add 100ul of enzyme-labeled antibody to each well (anti-human IgG antibody diluted 1:2000, anti-human IgG antibody diluted 1:500), incubate at 37°C for 1 hour, shake off the liquid, and wash the plate three times with 0.05% PBST.

[0087] 5. Color development: Add 100 μL of substrate color developer TMB to each well, incubate at room temperature in the dark, and observe the color development. After about 2-5 minutes, add 100 μL of stop solution 1 mol / L H2SO4 to each well, mix well, and immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0088] 6. Test results: Both IgG and IgM in the serum of HEV-infected patients can bind to the pan-genotype ORF3 protein ( Figure 5 , green). The binding curve is dose-dependent: the OD value decreases with increasing dilution. Healthy control serum does not bind to ORF3, demonstrating that serum from HEV-infected patients specifically binds to the pan-genotype HEV-A ORF3 protein. Based on these results, serum from HEV-infected patients diluted 1:500 can successfully detect HEV-infected patients.

[0089] 7. Detection of HEV-infected patient serum for specific binding to ORF3 protein

[0090] Serum samples from 11 HEV-infected patients (patient information is shown in Table 4) were tested. The serum was diluted 1:500 and added to an ORF3-coated ELISA plate. The ELISA experimental procedures were the same as Steps 1-5 of Example 3. Serum IgG was found to interact with ORF3 in 11 samples, with a positive rate of 100%. Serum IgM was found to interact with ORF3 in 10 samples, with a positive rate of 90.9% (Table 5).

[0091] Table 4. Summary of information of 11 patients

[0092] Patient serial number gender age <![CDATA[HEV IgG detection 1 > <![CDATA[HEV IgM detection 1 > 1 male 49 <![CDATA[+ 2 ]]> + 2 male 46 + + 3 male 48 + + 4 female 83 + + 5 male 42 + + 6 male 58 + + 7 male 59 + + 8 female 78 + + 9 male 52 + + 10 male 70 + + 11 male 67 + +

[0093] 1 The test results are provided by the confirming hospital, and the detection method is the hepatitis E virus IgG / IgM antibody detection kit.

[0094] 2 + indicates a positive test result, and - indicates a negative test result.

[0095] Table 5. Positive rates of IgG and IgM in sera of 11 positive patients using pan-genotype ORF3 as antigen

[0096] IgG IgM Number of HEV-infected patients with positive antibodies 11 / 11 10 / 11 Antibody detection rate in HEV infected patients 100％ 90.9％

[0097] Example 4: Immunization of New Zealand white rabbits with purified HEV-A pan-genotype ORF3 protein to detect ORF3 immunogenicity

[0098] 1. Immunize New Zealand white rabbits with purified ORF3 protein

[0099] (1) The imported New Zealand white rabbits were placed in a regular animal room and observed for one week to allow them to adapt to the environment.

[0100] (2) Before the injection, 2 mL of blood was collected from the rabbit's ear vein and the serum was separated as a negative control. The steps were as follows: calm the rabbit, carefully shave the hair on the rabbit's ear and smear the blood vessel with an alcohol cotton ball to expand the blood vessel. Use a disposable clean syringe to draw 2 mL of blood from the ear vein, then carefully withdraw the needle, press the wound appropriately to prevent bleeding, and then disinfect the wound with an alcohol cotton ball.

[0101] (3) Immunization process: 1 mg of purified ORF3 protein was diluted to 1 mL with normal saline and emulsified with an equal volume of 1 mL of Freund's complete adjuvant. After spraying alcohol on multiple sites on the rabbit's back for disinfection, the rabbit was subcutaneously injected. The sites were generally divided into four, and 250 μL was injected at each site. Two weeks later, 10 mL of blood was collected from the ear vein for the second time to detect the titer of the antibodies produced by the first injection. 1 mg of purified ORF3 protein was diluted to 1 mL with normal saline and emulsified with an equal volume of 1 mL of Freund's incomplete adjuvant. The second injection was performed at multiple sites on the back. The immunization process required a total of 5 injections of emulsified ORF3 protein. Before each immunization, blood was collected from the ear vein to detect the titer of the antibodies produced after the previous immunization. One week after the last immunization, blood was collected from the ear vein to detect the titer. Afterwards, the rabbit was anesthetized with 10 mL of 10% chloral hydrate, and all blood was collected from the heart.

[0102] (4) Preparation of serum antibodies against HEV-A pan-genotype ORF3 protein:

[0103] After collecting rabbit blood, without adding anticoagulant, the blood was placed at 37°C and allowed to coagulate for 1 to 2 hours. The blood was then placed in a 4°C refrigerator overnight to allow the blood clot to solidify. After the serum naturally precipitated, it was centrifuged at 3000 rpm at 4°C for 10 minutes to separate the serum and discard the insoluble matter. The serum was transferred to a clean test tube, divided into small portions, and stored at -80°C. The serum was named pan-genomic ORF3 polyclonal antibody serum.

[0104] 2. Effect of different concentrations of antigen coating on antibody detection levels

[0105] (1) Coating antigen: Dilute the purified ORF3 protein to a final concentration of 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL with coating solution (0.1 M Na2CO3 / NaHCO3 pH = 9.0), and add 100 μL per well to the ELISA plate. The amount of coating protein in each well is 400 ng, 200 ng, 100 ng, and 50 ng, respectively. The coating solution is used as a negative control (0 ng). The coated ELISA plate is placed in a 4°C refrigerator overnight. The next day, it is removed and incubated at 37°C for 1 hour. After the liquid is removed, the ELISA plate is washed three times with 200 μL of PBS per well for 3 minutes each time.

[0106] (2) Blocking: Use 5% skim milk powder in PBS as the blocking solution, add 100 μL to each well, incubate at 37°C for 1 hour, and then discard the liquid.

[0107] (3) Incubation of rabbit serum: The New Zealand white rabbit serum collected before and after immunization was diluted 10 2 , 10 3 , 10 4 , 10 5 , 106 After incubation at 37°C for 2 h, the liquid was discarded and the plate was washed three times with PBST containing 0.05% Tween.

[0108] (4) Adding anti-rabbit IgG: Add 100 μL of enzyme-labeled antibody to each well (dilute the anti-rabbit IgG antibody 1:2500 with 1% skim milk powder PBS), incubate at 37°C for 1 hour, shake off the liquid, and wash the plate three times with 0.05% PBST.

[0109] (5) Color development: Add 100 μL of substrate color developer TMB to each well, incubate at room temperature in the dark, and observe the color development. After about 2-5 minutes, add 100 μL of stop solution 1 mol / L H2SO4 to each well, mix well, and immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0110] (6) Result analysis: The OD values ​​of the plates coated with 400ng and 200ng ORF3 protein were higher, while the OD values ​​of the plates coated with 100ng and 50ng ORF3 decreased with the decrease of the coating amount ( Figure 6 Therefore, 200 ng / well is the optimal coating amount.

[0111] 3. Detection of antibody levels after ORF3 immunization

[0112] (1) Antigen coating: Dilute the purified ORF3 protein to a final concentration of 2 μg / mL using coating solution (0.1 M Na2CO3 / NaHCO3 pH = 9.0) and add 100 μL per well to the ELISA plate, with 200 ng of coating protein per well. Place the coated ELISA plate in a 4°C refrigerator overnight. The next day, remove the plate and incubate it at 37°C for 1 hour. After discarding the liquid, wash the plate three times with 200 μL per well of PBS for 3 minutes each time.

[0113] (2) Blocking: Use 5% skim milk powder in PBS as the blocking solution, add 100 μL to each well, incubate at 37°C for 1 hour, and then discard the liquid.

[0114] (3) Add rabbit serum to test the serum titer after immunization: dilute the New Zealand white rabbit serum collected before immunization and after the third, fourth and sixth immunizations with PBS to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 After 2 h of incubation at 37°C, 50 μL of the solution was added to each well. The plate was then shaken off and washed three times with PBST containing 0.05% Tween.

[0115] (4) Adding anti-rabbit IgG: Add 100 μL of enzyme-labeled antibody to each well (dilute the anti-rabbit IgG antibody 1:2500 with 1% skim milk powder PBS), incubate at 37°C for 1 hour, shake off the liquid, and wash the plate three times with 0.05% PBST.

[0116] (5) Color development: Add 100 μL of substrate color developer TMB to each well, incubate at room temperature in the dark, and observe the color development. After about 2-5 minutes, add 100 μL of stop solution 1 mol / L H2SO4 to each well, mix well, and immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0117] (6) Antibody level detection: After the third immunization, the serum was diluted 1000 times and still had good binding with the antigen; after the fourth and sixth immunizations, the serum was diluted 10,000 times and still had good binding with the antigen ( Figure 7 ).

[0118] Example 5: Polyclonal antibodies produced after immunization with ORF3 protein can be used as a laboratory test for HEV

[0119] 1. Construction of ORF3 eukaryotic expression plasmid pcDNA3.1-ORF3-flag:

[0120] 1.1 The ORF3 gene sequence is 340 bp long. Using pcDNA3.1-Flag as the vector, XhoⅠ and EcoRI restriction sites were added upstream and downstream, respectively, and primers were designed for amplifying the gene sequence.

[0121] Upstream primer: AACCTCGAGATGGGATCACCATGTGCCCTAG

[0122] Downstream primer: AACGAATTCACGGCGCAGCCCCAGCTGG

[0123] 1.2 PCR amplification of the full-length ORF3 fragment (NCBI sequence number: JQ679013.1)

[0124] (1) Add the following reaction mixture to a 0.2 mL EP tube. The reaction system is shown in Table 1:

[0125] Table 6. PCR reaction system

[0126] Components volume Primer STAR Max DNA polymerase 25 μL Forward Primer (10μM) 1 μL Reverse Primer (10μM) 1 μL Template 2μL Sterile distilled water 21 μL Total volume 50 μL

[0127] (2) Place the sample tube in the PCR instrument. The reaction program is shown in Table 2. The number of cycles is 35. The ORF3 protein PCR results ( Figure 8 ).

[0128] Table 7. PCR amplification reaction program

[0129] temperature time 98℃ 10s 63.9℃ 30s 72℃ 40s

[0130] 1.3 Gel recovery of PCR products

[0131] (1) After electrophoresis of the PCR product on a 2% agarose gel, carefully cut out the desired gel block under ultraviolet light.

[0132] (2) Weigh the empty 1.5 mL EP tube after high pressure and record the weight. Then take the target gel block and put it into the EP tube and weigh it again. The difference between the two weights is the weight of the target gel block.

[0133] (3) Add the correct amount of Binding Buffer into the EP tube according to the requirement of 1ug gel + 1μL Binding Buffer.

[0134] (4) Place the tube in a 55°C water bath for 10 minutes, remove the EP tube, and carefully observe whether the rubber block is completely dissolved. If not, heat it again briefly until it is completely dissolved.

[0135] (5) Transfer the DNA agarose solution to the adsorption column, let it stand for 2 minutes, and centrifuge at 12000 rpm / min for 75 seconds. (The adsorption column can only accommodate 700 μL of solution at a time. If the entire solution cannot be added to the adsorption column at one time, the remaining solution can be added to the adsorption column after centrifugation.)

[0136] (6) Discard the waste liquid, add 700 μL of Washing Buffer to the adsorption column, and centrifuge at 10,000 rpm / min for 75 seconds.

[0137] (7) Repeat the previous step.

[0138] (8) Discard the waste liquid and run at 10,000 rpm / min for 2 minutes.

[0139] (9) Transfer the column to a new high-pressure 1.5 mL EP tube, open the lid, and let it stand for 2 minutes.

[0140] (10) Add 30 μL of Elution Buffer to the center of the column, let it stand for 2 min, and then centrifuge at 13,000 rpm / min for 1 min.

[0141] (11) The concentration of the recovered product was measured and recorded on the wall of the EP tube and stored at -20 °C.

[0142] 1.4 Enzyme digestion of target fragment and vector

[0143] (1) Add the following reaction mixture to a 0.2 mL EP tube.

[0144] Table 8. Enzyme digestion system

[0145] Components volume enzyme 1 1 μL Enzyme 2 1 μL 10x Buffer 2μL DNA ≤1ug Sterile distilled water Supplement Total volume 20 μL

[0146] (2) Mix the sample according to the reaction system, centrifuge briefly, and place in a 37°C incubator for 3 hours.

[0147] 1.5 Ligation of target fragment and vector fragment

[0148] (1) The ratio of the target fragment (340 bp) to the vector fragment (approximately 5500 bp) is 3 to 10. Based on this, the required amount of the target fragment is calculated (see Table 4).

[0149] (2) Place the sample in a connector at 16°C and connect overnight.

[0150] Table 9. Connection system

[0151] Components volume T4 DNA Ligase 1 μL 10x T4 DNA Ligase Buffer 2μL 10x Buffer 2μL Destination fragment Vector fragment 80ng Sterile distilled water Supplement Total volume 20 μL

[0152] 1.6 Transformation (DH5α)

[0153] (1) Take 50 μL of E. coli DH5α competent cells and thaw them quickly on ice. Add 10 μL of the ligation product, swirl gently to mix, and place on ice for 30 minutes.

[0154] (2) Immediately place the tube in a 42°C water bath and heat shock for 90 seconds without shaking until it reaches the correct heat shock temperature.

[0155] (3) Quickly transfer to ice and place in an ice bath for 2 minutes.

[0156] (4) Add 650 μL of LB medium to the sample tube in a clean bench and mix gently.

[0157] (5) Place the sample tube in a shaker at 37°C, 220 rpm / min, and shake for 1 h.

[0158] (6) Centrifuge at 5000 rpm / min for 1 min at room temperature.

[0159] (7) Discard 550 μL of the supernatant and mix the remaining 150 μL. Add all of it to the antibiotic plate and spread it until no liquid flows on the plate. Place the plate upright for 10 minutes, then invert the plate and incubate at 37°C for 12 to 16 hours.

[0160] 1.7 The recombinant plasmid was sequenced and confirmed to be pcDNA3.1-ORF3-FLAG.

[0161] 2. Detection of ORF3 protein eukaryotic expression-positive cells using polyclonal antibodies produced from ORF3

[0162] 2.1 Transfect HEK 293T cells with the pcDNA3.1-ORF3-flag plasmid constructed in 1

[0163] (1) 6×10 5 cells, add 2 mL of DMEM high-glucose medium containing 10% fetal bovine serum, mix thoroughly and culture in a 37°C, 5% CO2 cell culture incubator for 12 to 24 hours until the cell coverage reaches 80%.

[0164] (2) Dissolve 4 μg of plasmid and 12 μL of liposome PEI in 100 μL of DMEM high-glucose medium respectively, and let it stand at room temperature for 5 minutes. Add the plasmid solution to the liposome solution, mix gently, and let it stand at room temperature for 20 minutes.

[0165] (3) Add 200 μL / well of the DNA-liposome mixture to the cells in the six-well plate, mix thoroughly, and then culture in a 37°C, 5% CO2 cell culture incubator. After 4 to 6 hours, replace it with DMEM high-glucose medium containing 10% fetal bovine serum to complete cell transfection.

[0166] 2.2 Detection of ORF3 expression in cells using Western blot:

[0167] (1) Extraction of total protein: discard the culture medium, wash the cells three times with PBS, add 500 μL of trypsin cell digestion solution, digest at room temperature for 2 minutes, discard the trypsin, blow down the cells with 1 mL of DMEM high glucose medium containing 10% fetal bovine serum, and transfer them to a 1.5 mL EP tube. Pre-cool the centrifuge at 4°C in advance, centrifuge at 1000 rpm / min for 5 minutes. Discard the supernatant, add 1 mL of PBS to wash the cells, and centrifuge until only the cell pellet remains. Prepare cell lysis buffer, mix RIPA, phosphatase inhibitors, and protease inhibitors (volume ratio 100:1:1), add 200 μL of cell lysis buffer / well, gently blow to mix, transfer to a 1.5 mL EP tube, and lyse on ice for 30 minutes. Turn on the ultrasonic disruptor, ultrasonically lyse each tube of cells three times, centrifuge at 4°C, 13000 rpm / min for 10 minutes, and remove the supernatant to a 1.5 mL EP tube. Add the corresponding amount of 5× protein loading buffer, mix well and boil the sample for 10 minutes. After cooling, store the sample at -20℃.

[0168] (2) SDS-PAGE electrophoresis: Prepare 12.5% ​​separation gel and 4% stacking gel. Prepare the separation gel first, mix thoroughly and pour it into the gel plate. Use anhydrous ethanol as the pressure line liquid. Wait for about 40 minutes, pour out the upper layer of anhydrous ethanol and absorb the residual liquid with absorbent paper. Prepare the stacking gel again, mix thoroughly and pour the upper layer. Insert the molding comb and let it stand at room temperature for about 40 minutes. After the gel is completely solidified, put it in a 4℃ refrigerator and store it for future use. Prepare the electrophoresis solution as shown in Table 1. Place the gel plate in the electrophoresis rack. After adding the electrophoresis solution, remove the molding comb. Add 10-20μL of protein sample to each gel well and add protein marker to one well. Place the electrophoresis rack in the electrophoresis tank. After filling the inner tank and outer tank with electrophoresis solution, start electrophoresis. Press the 70V electrophoresis line for about 30 minutes. When the sample is electrophoresed to the boundary between the stacking gel and the separation gel, change the voltage to 120V. Stop the electrophoresis when the bromophenol blue runs to the bottom of the separation gel.

[0169] (3) Western blot protein immunoblotting experiment: Cut a PVDF membrane of the same size as the target gel block, soak it in methanol for about 30 seconds to fully activate the PVDF membrane, and then put the PVDF membrane into the recycled wet transfer solution and soak it for 3 to 5 minutes. Cut a white filter pad of the same size as the gel block, soak it fully and place it on the wet transfer clamp. The membrane is transferred in a wet transfer electroporator in the following order: white board - sponge pad - white filter paper - PVDF membrane - gel - white filter paper - sponge pad - black board. Turn on the power, stabilize the voltage at 100V, and wet transfer for 100 minutes. Soak the membrane in blocking solution (5% skim milk powder) and block it on a shaker at room temperature for 2 hours. After blocking, wash the PVDF membrane briefly and cut it to the same size as the target protein. Place it in the antibody incubation box and dilute the ORF3 polyclonal antibody serum with 1% BSA to 1000, 2000, 3000, and 4000 times respectively and add it to the incubation box. Incubate overnight at 4°C on a shaker. Use a flag antibody (1:2000) to detect the presence of a flag tag on the protein. The next day, remove the antibody incubation box and rewarm on a shaker at room temperature for 30 minutes. Then, aspirate the primary antibody and wash three times with TBST wash buffer (10 minutes each). Place the PVDF membrane in the antibody incubation box and add a diluted anti-rabbit IgG antibody at a dilution of 1:5000 (in 1% BSA). Incubate on a shaker at room temperature for 2 hours. Aspirate the secondary antibody and wash three times with TBST wash buffer (10 minutes each).

[0170] (4) Western blot was developed using ECL, and the analysis results showed that after dilution 4000 times, the pan-genomic ORF3 could detect ORF3 protein expressed in eukaryotic cells ( Figure 9 and Figure 10 ).

[0171] 3. Detect the expression of ORF3 in cells using immunofluorescence:

[0172] (1) After transfecting pcDNA3.1-ORF3-flag plasmid into 293T cells for 48 h, discard the cell culture medium and wash twice with PBS.

[0173] (2) Add 4% paraformaldehyde to fix the cells and let them stand at room temperature for 30 minutes.

[0174] (3) Discard the paraformaldehyde, add PBS along the side wall, and wash twice to wash away the remaining paraformaldehyde.

[0175] (4) Add 0.1-0.3% Triton to perforate the cells, cover the cells, and let them stand at room temperature for 15 minutes.

[0176] (5) Discard the Triton and add PBS along the side wall to wash twice.

[0177] (6) Add blocking solution (5% skim milk powder) to the cell wells and let it stand at room temperature for 45 to 60 minutes.

[0178] (7) Dilute the pan-genotypic ORF3 polyclonal antibody at 1:500, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, and 1:4000, respectively, and incubate at 37°C for 1 hour.

[0179] (8) Add PBS along the side wall, place the plate on a shaker and shake slowly, and wash three times, 5 minutes each time.

[0180] (9) Protect from light and stain the cells with Cora Lite 488 / 594-conjugated anti-rabbit IgG (green / red) (dilution 1:500) and Hochest (blue) (dilution 1:500) and incubate at 37°C for 2 hours.

[0181] (10) Add PBS along the side wall, place the plate on a shaker and shake slowly, and wash three times, 5 minutes each time.

[0182] (11) Observe and photograph under an inverted fluorescence microscope.

[0183] (12) The results are as follows Figure 11 As shown, even a 4000-fold dilution of the pan-ORF3 polyclonal antibody can still detect ORF3 protein expression. Since the pcDNA-flag vector does not contain the ORF3 gene and there is no start codon before the flag tag, this plasmid cannot express ORF3 and flag and serves as a negative control.

[0184] Example 6: Serum antibodies produced after immunization with ORF2 protein, the main component of HEV vaccine, cannot interact with ORF3

[0185] The p239 protein (aa368-aa606), encoded by a recombinantly expressed truncated ORF2 gene, is the primary component of the existing HEV vaccine, Yikoning, and exhibits excellent immunogenicity and antigenicity. Immunization with HEV ORF2 produces antibodies specific to ORF2. Consequently, current HEV serological assays (ELISA kits coated with ORF2 antigen) cannot distinguish between vaccine-immunized and naturally infected individuals when screening for anti-HEV ORF2 antibodies in the general population.

[0186] To verify whether the pan-genomic ORF3 protein of Hepavirus A binds to antibodies induced by ORF2, New Zealand white rabbits were immunized with the HEV gene p239 protein (aa368-aa606). Serum was obtained and assayed for binding to both ORF2 and ORF3 using ELISA. The serum reacted with ORF2 but not ORF3. These results indicate that the pan-genomic ORF3 protein of Hepavirus A does not interact with antibodies produced after immunization with ORF2. Combined with the patient testing results (Example 3), this ORF3 protein can be used to distinguish between natural HEV infection and immunization with an HEV vaccine.

[0187] The specific experimental operations are as follows:

[0188] 1. Immunization of New Zealand White Rabbits with ORF2 Protein

[0189] (1) Immunization process: 1 mg of ORF2 protein was diluted to 1 mL with normal saline and emulsified with an equal volume of 1 mL of Freund's complete adjuvant. After spraying alcohol on multiple sites on the rabbit's back for disinfection, the rabbit was subcutaneously injected. The sites were generally divided into four, and 250 μL was injected at each site. Two weeks later, 10 mL of blood was collected from the ear vein for the second time to detect the titer of the antibodies produced by the first injection. 1 mg of ORF2 protein was diluted to 1 mL with normal saline and emulsified with an equal volume of 1 mL of Freund's incomplete adjuvant. The second injection was performed at multiple sites on the back. The immunization process required a total of 5 injections of emulsified ORF3 protein. Before each immunization, blood was collected from the ear vein to detect the titer of the antibodies produced after the previous immunization. One week after the last immunization, blood was collected from the ear vein to detect the titer. Afterwards, the rabbit was anesthetized with 10 mL of 10% chloral hydrate, and all blood was collected from the rabbit's heart.

[0190] (2) Preparation of serum antibodies against ORF2 protein:

[0191] After collecting rabbit blood, place it at 37°C without adding anticoagulant. Allow the blood to coagulate for 1 to 2 hours, then place it in a 4°C refrigerator overnight to allow the blood clot to solidify. After the serum naturally precipitates, centrifuge it at 3000 rpm at 4°C for 10 minutes to separate the serum and discard the insoluble matter. Transfer the serum to a clean test tube, divide it into small portions, and store it at -80°C.

[0192] 2. Detection of ORF2-immunized serum using ORF2 protein and pan-genomic ORF3 protein as antigens

[0193] (1) Antigen coating: Dilute ORF2 and ORF3 proteins to a final concentration of 2 μg / mL using coating solution (0.1 M Na2CO3 / NaHCO3 pH = 9.0). Add 100 μL per well to the ELISA plate. The amount of coated protein in each well is 200 ng. Use coating solution as a negative control (0 ng). Place the coated ELISA plate in a 4°C refrigerator overnight. The next day, remove the plate and incubate it at 37°C for 1 hour. After discarding the liquid, wash the plate three times with 200 μL per well of PBS for 3 minutes each time.

[0194] (2) Blocking: Use 5% skim milk powder in PBS as the blocking solution, add 100 μL to each well, incubate at 37°C for 1 hour, and then discard the liquid.

[0195] (3) Incubation with rabbit serum: New Zealand white rabbit serum collected before and after immunization was diluted 100-fold with PBS and 50 μL was added to each well. After incubation at 37°C for 2 h, the liquid was discarded and the plate was washed three times with PBST containing 0.05% Tween.

[0196] (4) Adding anti-rabbit IgG: Add 100 μL of enzyme-labeled antibody to each well (dilute the anti-rabbit IgG antibody 1:2500 with 1% skim milk powder PBS), incubate at 37°C for 1 hour, shake off the liquid, and wash the plate three times with 0.05% PBST.

[0197] (5) Color development: Add 100 μL of substrate color developer TMB to each well, incubate at room temperature in the dark, and observe the color development. After about 2-5 minutes, add 100 μL of stop solution 1 mol / L H2SO4 to each well, mix well, and immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0198] (6) Result analysis Figure 12 ): The ELISA coated with ORF2 was able to react with the serum of rabbits immunized with ORF2 (OD value ~1.2), while the ELISA coated with ORF3 was unable to react with the serum of rabbits immunized with ORF2 (OD value ~0.1). Sequence Listing <110> Xuzhou Medical University <120> A pan-genotype ORF3 protein of Hepatitis E virus genus A and its application <130> 1 <160> 1 <170> SIPOSequenceListing 1.0 <210> 2 <211> 113 <212> PRT <213> Orthohepevirus <400> 2 Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys 1 5 10 15 Phe Cys Leu Cys Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala Ala 20 25 30 Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr 35 40 45 Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr 50 55 60 Pro Ser Pro Pro Thr Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu 65 70 75 80 Gly Ser Arg Pro Ala His Ser Ala Pro Leu Gly Val Thr Ser Pro Ser 85 90 95 Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg 100 105 110 Silver

Claims

1. A pan-genotype A ORF3 protein of Hepatitis E virus, characterized by: The amino acid sequence of the pan-genotype ORF3 protein of the Hepacivirus genus A is shown in SEQ ID NO:

1.

2. A kit for detecting hepatitis E virus, characterized in that: Contains the pan-genotype ORF3 protein of the Hepatovirus genus A; The amino acid sequence of the pan-genotype ORF3 protein of the Hepacivirus genus A is shown in SEQ ID NO: 1.

Images