Hepatitis E virus targeting antigen binding fragment and application thereof in preparation of DNA vaccine

Abstract

The invention relates to the technical field of DNA vaccines, in particular to a hepatitis E virus targeting antigen binding fragment and application thereof in preparation of a DNA vaccine. The base sequence of the antigen binding fragment is shown as SEQ ID NO. 1, and the amino acid sequence of the antigen binding fragment is shown as SEQ ID NO. 2; and the molecular structure is 6 * His labelle-ORF2 antigen fragment-6 * His label. According to the scheme, the DNA vaccine with stable quality can be rapidly prepared, and cellular immunity and humoral immunity protection can be provided for an organism.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA vaccines, in particular to a hepatitis E virus targeted antigen binding fragment and application thereof in the preparation of DNA vaccines. Background Art

[0002] Hepatitis E is caused by the Hepatitis E Virus (HEV). Genotypes 3 and 4 of the Hepatitis E Virus can infect pigs and humans and transmit each other, causing infected patients to develop symptoms of hepatitis E. It can cause serious complications in special groups such as those with underlying liver disease, weakened immunity, and pregnant women. In particular, the mortality rate in pregnant women can reach 30%.

[0003] Due to the immaturity of in vitro HEV culture methods and experimental animal systems, there is still a lack of effective treatments for HEV. Ribavirin is primarily used clinically to eliminate HEV infection, but its teratogenic effects prevent its use in pregnant women. Currently, vaccination remains the optimal approach for HEV prevention. Currently, only one commercially available human subunit recombinant vaccine is available, but vaccination during pregnancy has been shown to increase the risk of miscarriage. DNA vaccines, a third-generation vaccine after subunit vaccines, are highly stable and can activate cytotoxic T lymphocytes, induce cellular immunity, strengthen effector T cells, and enhance immune memory.

[0004] Therefore, there is an urgent need to develop a DNA vaccine that can prevent hepatitis E, which can solve problems such as complex vaccine production process, unstable quality, slow immune response of the body, and inability to induce effective cellular immunity. The hepatitis E virus DNA vaccine provides a new method for the prevention of hepatitis E and provides new means and new ideas for the prevention and control of hepatitis E virus in my country. Summary of the Invention

[0005] In order to solve or partially solve the problems existing in the related art, the present application provides a DNA vaccine for preventing hepatitis E virus and a preparation method thereof. The vaccine selects the 153-641 amino acid region of the open reading frame 2 (ORF2) of genotype 4 hepatitis E virus Saba Swine-X001 as the antigenic fragment. The prepared vaccine has a simple production process, low production cost, stable quality, can induce cellular immunity and humoral immunity of the body, has good safety, and can effectively prevent hepatitis E virus.

[0006] In one aspect, the present application provides an antigen-binding fragment that binds to a hepatitis E virus antigen. The base sequence of the antigen-binding fragment is shown in SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2; and the molecular structure is 6×His tag-ORF2 antigen fragment-6×His tag; The antigen is the amino acid region 153-641 of ORF2 of genotype 4 hepatitis E virus strain.

[0007] On the other hand, the present application also provides a DNA vaccine for preventing hepatitis E virus, which comprises the above-mentioned antigen-binding fragment.

[0008] On the other hand, the present application also discloses a method for constructing a DNA vaccine for preventing hepatitis E virus, wherein the specific steps of the construction method are as follows: 1) The amino acid region 153-641 of the ORF2 region of the genotype 4 hepatitis E virus strain Saba Swine-X001 was used as the antigen fragment. Kpn I and EcoR I restriction sites, a Kozak sequence, and a 6× His tag were added to construct the antigen-binding fragment, which was inserted into the empty pcDNA3.1(+) vector to form a recombinant plasmid. 2) Transform the recombinant plasmid into competent E. coli, culture on ampicillin-resistant plates, select single colonies, and verify by PCR amplification; 3) After verification, the inserted single colony is selected as the DNA vaccine engineering strain and cultured in Luria Bertani lysate broth; 4) Centrifuging the DNA vaccine engineered strain cultured in 3), collecting the cells, and using SDS alkaline lysis to release the recombinant plasmid. After denaturation, filtration, binding, and elution, the endotoxin-free recombinant plasmid is obtained; after double enzyme digestion verification and endotoxin limit inspection, the concentration is adjusted to 500 mg / mL to obtain the above-mentioned hepatitis E virus DNA vaccine.

[0009] Furthermore, the antigen-binding fragment is prepared by the following steps: (1) The amino acid region 153-641 of ORF2 of the genotype 4 hepatitis E virus strain (Saba Swine-X001) was selected as the antigenic fragment, and the base sequence corresponding to this region was optimized using mice as the host; The optimization includes codon preference, GC content, and enzyme cutting sites that need to be avoided; (2) The optimized antigen fragment base sequence is inserted with a KpnⅠ restriction site, a Kozak sequence, and a 6×His tag base sequence in the front, and a stop codon, a 6×His tag base sequence, and an EcoR Ⅰ restriction site in the back to form the base sequence of the antigen binding fragment.

[0010] Furthermore, the base sequence of the antigen-binding fragment is shown in SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2; and the molecular structure is 6× His tag-ORF2 antigen fragment-6× His tag.

[0011] Furthermore, the specific operation of the endotoxin limit check is: according to the requirements of the National Pharmacopoeia, the gel method is used to detect whether the extracted plasmid meets the DNA vaccine endotoxin limit.

[0012] On the other hand, the present application also provides an application of a hepatitis E virus DNA vaccine in preventing hepatitis E, wherein the hepatitis E virus DNA vaccine is the above-mentioned hepatitis E virus DNA vaccine.

[0013] Beneficial effects 1. This application is based on the genotype 4 hepatitis E virus strain (Saba Swine-X001) isolated from pigs, and selects the 153-641 amino acid sites of the ORF2 region as the antigen protein. After constructing recombinant plasmids, screening DNA vaccine engineered bacteria, plasmid extraction, double enzyme digestion verification, and endotoxin limit testing, the hepatitis E virus DNA vaccine was finally obtained.

[0014] 2. The DNA vaccine prepared in this application has excellent immunogenicity and can rapidly induce strong humoral and cellular immune responses to hepatitis E virus infection. Furthermore, this vaccine can be prepared under laboratory conditions without the use of live virus. In actual production, it requires only a few steps, has stable quality, and is easy to control. This provides a new approach and new ideas for the prevention and control of hepatitis E virus in my country.

[0015] 3. The antigenic fragment of the ORF2 153-641 amino acid region screened from the genotype 4 hepatitis E virus strain (Saba Swine-X001) has good immunogenicity and can be used to prepare hepatitis E virus DNA vaccine. It can effectively prevent hepatitis E virus and lay the foundation for the development of hepatitis E virus DNA vaccine.

[0016] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0018] Figure 1 Schematic diagram of the design of hepatitis E virus DNA vaccine.

[0019] Figure 2 Gel electrophoresis diagram for screening of DNA vaccine engineering strains.

[0020] Figure 3 This is a diagram showing the verification of DNA vaccine after double enzyme digestion with Kpn Ⅰ and EcoR Ⅰ.

[0021] Figure 4 These are the results of the DNA vaccine endotoxin limit test.

[0022] Figure 5 is the mRNA expression level of DNA vaccine in HepG2 cells.

[0023] Figure 6 is the protein expression level of DNA vaccine in HepG2 cells.

[0024] Figure 7 Immunofluorescence images of DNA vaccine in HepG2 cells (10 µm).

[0025] Figure 8 Flowchart for mouse immunization.

[0026] Figure 9 These are the organ indices of various organs in mice after immunization with DNA vaccine.

[0027] Figure 10 Schematic diagram of the gate for flow cytometric analysis of splenic T lymphocyte typing.

[0028] Figure 11 Typing of spleen T lymphocytes after DNA vaccine immunization.

[0029] Figure 12 Elispot analysis of IFN-γ secretion by splenic lymphocytes after DNA vaccine immunization. The top graph shows the spot-forming unit counts for each group, and the bottom graph shows the spot-forming unit scans for each group.

[0030] Figure 13 It is the specific stimulation index of spleen lymphocytes after DNA vaccine immunization.

[0031] Figure 14are the levels of anti-hepatitis E virus antibodies in mouse serum; A: the level of anti-hepatitis E virus IgM antibodies in mouse serum after DNA vaccine immunization; B: the level of anti-hepatitis E virus IgG antibodies in mouse serum after DNA vaccine immunization.

[0032] Figure 15 This is the gross image and HE staining results of various organs of mice after immunization. DETAILED DESCRIPTION

[0033] The following describes embodiments of the present application in more detail with reference to the accompanying drawings. Although the accompanying drawings illustrate embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0034] DNA vaccines offer advantages such as a short development cycle, low R&D costs, and the absence of live viruses. Production requires only simple fermentation and plasmid extraction, making quality control easier and production costs lower than attenuated, inactivated, subunit, and mRNA vaccines. DNA vaccines can simultaneously induce strong cellular and humoral immunity to rapidly achieve immune responses. Furthermore, DNA vaccines, being essentially circular DNA plasmids, are more stable than proteins and mRNA, making them easier to store and transport, effectively reducing immune failures caused by quality degradation during storage and transportation.

[0035] The selection of antigens is crucial in vaccine design and directly determines the immune effect of the vaccine. This application is based on the gene 4b subtype hepatitis E virus (Saba Swine-X001) isolated from Yunnan pigs (Saba pigs) (the virus is preserved in the laboratory of the College of Veterinary Medicine of Yunnan Agricultural University), and selects the 153-641 amino acids of the open reading frame 2 (OpenReading Frame 2, ORF2) of the hepatitis E virus as the antigen sequence to develop a DNA candidate vaccine. This vaccine can quickly induce good cellular immunity and effective humoral immunity after immunization, can be developed and produced in a relatively short time, and has good safety, providing a new means for the prevention and control of hepatitis E virus in my country.

[0036] The purpose of this application is to overcome the drawbacks of hepatitis E virus vaccine development, production, and use, such as long development cycles, high investment costs, and poor immune efficacy. This application provides a DNA vaccine expressing a fragment of the ORF2 antigen of genotype 4 hepatitis E virus, including methods for preparing and using the DNA vaccine. Furthermore, this application provides a DNA vaccine for preventing hepatitis E virus that is simple to prepare, exhibits excellent immune protection, and possesses a high safety profile.

[0037] The synthesis of the recombinant plasmids described in the following examples was performed by Sangon Biotechnology Co., Ltd.; the competent Escherichia coli was stored by the College of Veterinary Medicine of Yunnan Agricultural University; and the HepG2 cells were stored by the College of Veterinary Medicine of Yunnan Agricultural University.

[0038] Example 1 Construction and preparation of hepatitis E virus DNA vaccine 1.1 Construction of hepatitis E virus DNA vaccine DNA vaccine construction involves the selection of antigenic fragments and the construction of antigen-binding fragments. Antigenic fragments are key factors in triggering immune responses and determining immune efficacy. ORF2 is the only capsid protein of the hepatitis E virus and the primary region responsible for triggering immune responses. In this study, amino acids 153-641 of ORF2 were selected as the vaccine's antigenic site.

[0039] In order to ensure the large-scale expression and expression monitoring of the antigen fragment, the Kozak sequence GCCACCATGG to increase antigen expression and the 6×His tag CACCATCACCATCACCAT to monitor antigen expression were added near the antigen fragment. The specific DNA vaccine structure is as follows Figure 1 .

[0040] 1.2 Screening of DNA vaccine engineered strains The synthesized recombinant plasmid was transformed into competent Escherichia coli by heat shock and cultured at 37°C for 16 hours on a resistance plate containing 100 μg / mL ampicillin. Single colonies were selected for PCR. The PCR products were subjected to 2% agarose gel electrophoresis. The expected bands were sequenced correctly. The strains with correct sequencing were the DNA vaccine engineering strains. Figure 2 Wells 1-4 are selected plasmid-transformed colonies, and wells 2-4 all have antigen-binding fragments of the expected size.

[0041] 1.3 Preparation of DNA vaccines The DNA vaccine engineered strain was cultured in LB (Luria Bertani lysis broth) liquid medium containing 100 μg / mL ampicillin at 37°C for 16 hours, and the cells were collected by centrifugation. The plasmid was extracted using the GeneJET Endotoxin-Free Plasmid Extraction Kit from Thermo Fisher Scientific through the steps of cell lysis, denaturation, filtration, column binding, and elution. The extracted plasmid was double-digested with Kpn Ⅰ and EcoR Ⅰ enzymes. The results are shown in Figure 3 After enzyme digestion, the empty vector of pcDNA3.1 only showed a band at about 5400 bp. After enzyme digestion, the plasmid with the antigen fragment successfully inserted showed two bands at about 5400 bp and 1530 bp respectively.

[0042] According to the Chinese Pharmacopoeia endotoxin limit formula, the endotoxin limit of this vaccine should be less than 1 EU / mL, assuming a mouse weight of 40 g and a 0.2 mL injection. The DNA vaccine extracted and diluted to 500 µg / mL was tested using a kit with a detection limit of 0.125 EU / mL. The results are as follows: Figure 4 The negative control sample did not coagulate and the positive control sample formed a solid gel, indicating that the sensitivity of the horseshoe crab reagent met the requirements; the DNA vaccine positive control sample formed a solid gel, indicating that there was no interference in the test sample; the DNA vaccine sample did not coagulate, indicating that the endotoxin content in the sample was lower than the reagent detection limit of 0.125EU / mL, which is in line with the requirement of the national pharmacopoeia that the endotoxin limit should be lower than 1 EU / mL.

[0043] Example 2 Transcription and expression of hepatitis E virus DNA vaccine in in vitro cell lines 2.1 Transcriptional levels of HEV DNA vaccine in HepG2 cells 10 μL of 500 mg / mL DNA vaccine was mixed with 135 μL of 1.74×10 7 Add 125 µL of electroporation buffer to 100 cells / mL HepG2 cells and mix thoroughly. Electroporation of the DNA vaccine into HepG2 cells was performed at 250 V and 950 µF. The cells were incubated for 36 hours. Total RNA was extracted and reverse transcribed. Real-time quantitative PCR was performed to detect the transcriptional level of the DNA vaccine in HepG2 cells. A standard curve was established using serial dilutions of the DNA vaccine as a standard.

[0044] The linear regression equations of the antigen fragment standard curves were established as y = -3.61x + 35.799 (R 2 =0.999), the copy number of the antigen fragment after being transferred into HepG2 cells was approximately 2.29x10 7 Copies / mL, e.g. Figure 5.

[0045] 2.2 Expression levels of HEV DNA vaccine in HepG2 cells 10 μL of 500 mg / mL DNA vaccine was mixed with 135 μL of 1.74×10 7 Add 125 µL of electroporation buffer to 100 cells / mL HepG2 cells and mix thoroughly. Electroporation of the DNA vaccine into HepG2 cells was performed at 250 V and 950 µF. The cells were incubated for 36 hours. Total protein was extracted and the protein concentration in the samples was determined. Western blotting was used to detect the expression of the antigen protein in HepG2 cells. No anti-ORF2 band was observed in the control group, while a band around 53 kDa was observed in the DNA vaccine group, consistent with the expected protein molecular weight. Figure 6 .

[0046] 2.3 Colocalization of HEV DNA Vaccine in HepG2 Cells 10 μL of 500 mg / mL DNA vaccine was mixed with 135 μL of 1.74×10 7 Add 125 µL of electroporation solution to 100 cells / mL HepG2 cells and mix thoroughly. Electroporation was used to transfer the DNA vaccine into HepG2 cells at a voltage of 250 V and a capacitance of 950 µF. The cells were then fixed with 4% neutral paraformaldehyde fixative and then permeabilized, blocked, and incubated with antibodies for fluorescent staining. DAPI was used to label the cell nuclei, which displayed blue fluorescence under a 360 nm fluorescence microscope. Immunofluorescence staining with a FITC-conjugated antibody revealed green fluorescence in the cytoplasm of the antigen fragment, indicating that the DNA vaccine was primarily expressed in the cytoplasm 36 hours after transfer into HepG2 cells. Figure 7 .

[0047] Example 3 DNA vaccine can induce strong humoral and cellular immune responses in mice after immunization 3.1 Animal grouping Twenty-four female Kunming mice were randomly divided into a control group and a DNA vaccine immunization group, with 12 mice in each group. The immunization procedure was as follows Figure 8 Blood was collected from the infraorbital venous plexus on days 0, 7, 14, 21, and 28. 0.1 mL of 500 mg / mL DNA vaccine was injected intramuscularly into each hind limb of the mice on days 7, 14, and 21. Each mouse received 100 mg of DNA vaccine each time. Three mice were sacrificed on days 7, 14, 21, and 28, and the tissues were fixed with 4% neutral paraformaldehyde. Splenic lymphocytes were aseptically isolated and extracted. The immunization procedure was as follows: Figure 8 .

[0048] 3.2 Indexes of various organs in mice after DNA vaccine immunization There was no significant difference in the organ indexes of mice compared with the control group on days 7, 14, 21, and 28 after DNA vaccine immunization. Figure 9 .

[0049] 3.3 Flow cytometry analysis of T cell typing in the spleen of mice after DNA vaccine immunization Sterile extracted spleen lymphocytes from immunized mice were diluted to 4×10 6 cell / mL, aspirate 1mL of cell suspension and centrifuge at 250× g for 15 min. Resuspend the cell pellet in 100 μL of CD3+CD4+CD8 antibody mixture and incubate at 4°C in the dark for 40 min. In addition, spleen lymphocytes from the control group were used to prepare samples for single staining, CD3 single staining, CD4 single staining, and CD8 single staining. The prepared samples were tested on a flow cytometer. Circle CD3 according to the flow cytometry results. + cell population, and then CD3 + The cell population is divided into CD4 + CD8 - Group, CD4 + CD8 + Group, CD4 - CD8 - Group, CD4 - CD8 + Group, such as Figure 10 , count CD4 + CD8 - Group, CD4 - CD8 + CD3 occupied by the group + The proportion of cells. On the 7th and 14th days after immunization, the DNA vaccine immunization group showed a cellular immune response trend, and CD3 + CD8 + The proportion of cells was significantly higher than that in the control group, and CD3 + CD4 + There was no significant difference in the proportion of cells compared with the control group. + CD4 + The cell ratios were significantly higher than those in the control group. Figure 11 .

[0050] 3.4 Elispot detection of IFN-γ secretion in mouse spleen lymphocytes after DNA vaccine immunization Negative control wells, experimental wells, and positive control wells were set for each sample test. Splenic lymphocytes from immunized mice were extracted and diluted to 4×10 6cell / mL, 25 µl of cell suspension was added to the negative control well, followed by 85 µl of cell culture medium; 100 µl of cell suspension was added to the experimental well, followed by 10 µl of hepatitis E virus-specific stimulator; 100 µl of cell suspension was added to the positive control well, followed by 10 µl of positive stimulator Concanavalin A. After 70 hours of static culture in a cell culture incubator, the spots were developed and the number of spots formed in each well was counted. 14 days after the first dose of DNA vaccine immunization, the number of live cells in the spleen lymphocytes of mice that could secrete IFN-γ after hepatitis E virus antigen-specific stimulation was significantly higher than that in the control group, and increased again on the 28th day. Figure 12 .

[0051] 3.5 Specific stimulation index of splenic lymphocytes in mice after DNA vaccine immunization The aseptically extracted spleen lymphocytes of mice on day 28 after immunization were diluted to 4×10 6 Cells were cultured at a concentration of 100 µL per well of a 96-well plate. Nine wells were added for each sample. 10 µL of the positive stimulus concanavalin A at a concentration of 50 µg / mL was added to three wells, 10 µL of the virus suspension was added to three wells, and 10 µL of 1640 complete medium was added to the remaining three wells. After 60 hours of culture, CCK-8 reagent was added and cultured for another 4 hours. The absorbance was measured on a microplate reader to calculate the specific stimulation index. After immunization, mouse spleen lymphocytes showed a strong proliferation effect when stimulated by hepatitis E virus antigens. The proliferation activity of the DNA vaccine-immunized group was significantly higher than that of the control group. Figure 13 .

[0052] 3.6 Elisa assay for HEV IgG and IgM antibody levels in mouse serum after DNA vaccine immunization The mouse hepatitis E antibody IgM and IgG enzyme-linked immunosorbent assay kits provided by Jiangsu Jingmei Biotechnology Co., Ltd. were used to detect the levels of hepatitis E virus IgG and IgM antibodies after DNA vaccine immunization. On the 7th day after immunization, the anti-hepatitis E virus IgM and IgG antibodies in the mouse serum increased rapidly, IgM reached a peak on the 14th day, and then gradually decreased, while IgG continued to increase within 14 days after immunization, and showed a brief decline on the 21st day, and returned to a high level again on the 28th day. Figure 14 .

[0053] Example 4 DNA vaccine has good safety in mice 28 days after immunization, no obvious pathological changes such as hemorrhage, edema, infarction, necrosis, hyperplasia, and fibrosis were observed in the heart, liver, spleen, lung, kidney, intestine, and brain of mice. HE staining showed that myocardial fibers were arranged regularly and stained evenly, the myocardial interstitium was not obvious, and no inflammatory cell infiltration, hemorrhage, or edema was observed in the myocardial tissue; hepatocytes were stained evenly, the hepatocyte nuclei were located in the center, and no abnormal nuclear division was observed. The liver tissue structure was normal and complete, without cell degeneration, necrosis, or inflammatory reaction; the red and white pulp structures in the spleen were intact, with abundant red blood cells in the red pulp and regular distribution of lymphocytes in the white pulp. The number and area of ​​the centers increased after DNA vaccine immunization; the alveolar walls in the lungs were thin and uniform, and no inflammatory cells were observed. There was no inflammatory cell infiltration in the kidney tissue, and the alveolar cavity was clear and clean, without effusion, bleeding, or desquamated cells. There was no inflammatory cell infiltration in the kidney tissue, and the structures of the glomeruli, renal tubules, and renal interstitium were clear. There was no abnormal deposition in the renal tubules, and the thickness of the glomerular capsule was normal, with no abnormal proliferation. In the intestinal tissue, the muscle layer was arranged regularly, the villi were of normal height, the intestinal epithelial cells were arranged regularly, and the glandular structure was intact. There was no increase in goblet cells, edema, bleeding, fibrosis, necrosis, or other pathological changes. There was no bleeding or neurodegenerative lesions in the brain tissue, and no significant proliferation of glial cells and nerve fibers was observed. Figure 15 .

[0054] The sequence listing is as follows: SEQ ID NO. 1 SEQ ID NO. 2 HHHHHHSTSPLTSTIATGTNLVLYAAPLSPLLPLQDGTNTHIMATEASNYAQYRVVRATIRYRPLVPNAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQG WRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYTGALGLLDFALELEFRNLTPGNTNTRVSRYSSSARHKLRRGPDGTAELTTTAATRFMKDLHFTGTNGVGEVGRGIALTLFNLADTLLGG LPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRPFSVLRANDVLWLSLTAAEYDQTTYGSSTNPMYVSDTVTFVNVATGA QGVARSLDWSKVTLDGRPLTIQQYSKTFFVLPLRGKLSFWEAGTTKAGYPYNYNTTASDQILIENAAGHRVCISTYTTNLGSGPVSISAVGVLAPHSALAVLEDTVDYPARAHTFDDFCHHHHHH The embodiments of the present application have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A hepatitis E virus targeting antigen binding fragment, characterized in that: The base sequence of the antigen-binding fragment is shown in SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2; the molecular structure is 6× His tag-ORF2 antigen fragment-6× His tag; The antigen is the amino acid region 153-641 of ORF2 of genotype 4 hepatitis E virus strain.

2. A DNA vaccine for preventing hepatitis E virus, characterized in that: The hepatitis E virus DNA vaccine comprises the antigen-binding fragment of claim 1.

3. A method for constructing a DNA vaccine for preventing hepatitis E virus, characterized in that: The specific steps of the construction method are: 1) The amino acid region 153-641 of the ORF2 region of the genotype 4 hepatitis E virus strain Saba Swine-X001 was used as the antigen fragment. Kpn I and EcoR I restriction sites, a Kozak sequence, and a 6× His tag were added to construct the antigen-binding fragment, which was inserted into the empty pcDNA3.1(+) vector to form a recombinant plasmid. 2) Transform the recombinant plasmid into competent E. coli, culture on ampicillin-resistant plates, select single colonies, and verify by PCR amplification; 3) After verification, the inserted single colony is selected as the DNA vaccine engineering strain and cultured in Luria Bertani lysate broth; 4) Centrifuging the DNA vaccine engineered strain cultured in 3), taking the cells, and using SDS alkaline lysis to release the recombinant plasmid. After denaturation, filtration, column binding, and elution, the endotoxin-free recombinant plasmid is obtained; after double enzyme digestion verification and endotoxin limit inspection, the concentration is adjusted to 500 mg / mL to obtain the hepatitis E virus DNA vaccine according to claim 2.

4. The construction method according to claim 3, characterized in that The antigen-binding fragment is prepared by the following steps: (1) The amino acid region 153-641 of ORF2 of the genotype 4 hepatitis E virus strain Saba Swine-X001 was selected as the antigenic fragment, and the base sequence corresponding to this region was optimized using mice as the host; The optimization includes codon preference, GC content, and enzyme cutting sites that need to be avoided; (2) The optimized antigen fragment base sequence is inserted with a KpnⅠ restriction site, a Kozak sequence, and a 6× His tag base sequence in the front, and a stop codon, a 6× His tag base sequence, and an EcoR Ⅰ restriction site in the back to form the base sequence of the antigen binding fragment.

5. The construction method according to claim 3, characterized in that The base sequence of the antigen-binding fragment is shown in SEQ ID NO. 1; the amino acid sequence is shown in SEQ ID NO. 2; and the molecular structure is 6× His tag-ORF2 antigen fragment-6× His tag.

6. The construction method according to claim 3, characterized in that: The specific operation of the endotoxin limit check is: according to the requirements of the National Pharmacopoeia, the gel method is used to detect whether the extracted plasmid meets the DNA vaccine endotoxin limit.

7. Use of a hepatitis E virus DNA vaccine for preventing hepatitis E, characterized in that: The hepatitis E virus DNA vaccine is the hepatitis E virus prevention DNA vaccine according to claim 2.

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