Avian hepatitis e virus orf3 subunit vaccine and method of making same

Abstract

The application discloses an avian hepatitis E virus ORF3 subunit vaccine and a preparation method thereof, and belongs to the technical field of genetic engineering. The avian hepatitis E virus ORF3 subunit vaccine is composed of an antigen composition and a vaccine adjuvant; the antigen composition is composed of VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein; the amino acid sequence of the VaHEV ORF3 recombinant protein is shown as SEQ ID NO. 1, and the amino acid sequence of the YT-aHEV ORF3 recombinant protein is shown as SEQ ID NO. 2. The avian hepatitis E virus ORF3 subunit vaccine has good immunization effect and can reduce the adverse influence of aHEV on the production performance of chickens.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to an avian hepatitis E virus ORF3 subunit vaccine and its preparation method. Background Technology

[0002] Avian hepatitis E virus (aHEV) is considered a major pathogen of Big Liver and Spleen Disease (BLS) and Hepatic Rupture and Hemorrhage Syndrome (HRHS) in chickens. Avian HEV has caused severe economic losses to the poultry industry in various countries. As early as 2010, Zhao et al. found that the genome sequence of Chinese avian HEV isolates shared 98.3% homology with European avian HEV (Zhao et al., 2010). Epidemiological surveys in recent years have shown that the detection rate of aHEV in China is continuously increasing (Su et al., 2018; Su et al., 2020; Zhao et al., 2017; Zhang et al., 2022; Liu et al., 2018; Li et al., 2020). To date, there is no commercially available vaccine against aHEV globally.

[0003] Currently, there is a lack of efficient in vitro culture systems for large-scale culture of aHEV, which poses a significant challenge to the research of traditional inactivated vaccines and live attenuated vaccines. Therefore, most research focuses on aHEV genetically engineered subunit vaccines. The avian HEV genome includes three open reading frames (ORFs): ORF1, ORF2, and ORF3 (Kabrane-Lazizi et al., 1999). ORF2 is closely related to viral infection and invasion of host cells, playing a role in capsid protein assembly. Existing research on aHEV genetically engineered subunit vaccines also largely focuses on ORF2.

[0004] ORF3 encodes the smallest phosphorylated protein. Recent studies have found that ORF3 contains antigenic epitopes that can stimulate a strong immune response in animals and can influence viral particle assembly and release, immunosuppression, and cellular signaling (Jiang Fenglin, 2014), making it a potential candidate target protein for developing aHEV subunit vaccines. Zhao et al., through epitope identification of the Chinese isolate CaHEV-ORF3, found three antigenic domains on CaHEV-ORF3: (aa)1-28, 55-74, and 75-88. Among these, (aa)75-88 is the dominant antigenic domain. Specific monoclonal antibody identification revealed that this domain contains at least two antigenic epitopes and is a clearly dominant domain (Zhao et al., 2002).

[0005] Therefore, in-depth research on ORF3 can open up new avenues for the design of vaccines against hepatitis E virus. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide an avian hepatitis E virus ORF3 subunit vaccine and its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an avian hepatitis E virus ORF3 subunit vaccine, comprising an antigen composition and a vaccine adjuvant;

[0009] The antigen composition consists of VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein;

[0010] The amino acid sequence of the VaHEV ORF3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the YT-aHEV ORF3 recombinant protein is shown in SEQ ID NO.2.

[0011] Preferably, the weight ratio of the VaHEV ORF3 recombinant protein to the YT-aHEV ORF3 recombinant protein is 1:1.

[0012] Preferably, the weight ratio of the antigen composition to the vaccine adjuvant is 1:1.

[0013] Preferably, the vaccine adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant.

[0014] A second aspect of the present invention provides a method for preparing the above-mentioned avian hepatitis E virus ORF3 subunit vaccine, comprising the following steps:

[0015] (1) Mix VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein at a weight ratio of 1:1 to obtain an antigen composition;

[0016] (2) Mix the antigen composition with the adjuvant and emulsify to prepare the avian hepatitis E virus ORF3 subunit vaccine.

[0017] A third aspect of the present invention provides the use of the above-described avian hepatitis E virus ORF3 subunit vaccine in the preparation of articles for the prevention or treatment of diseases caused by avian hepatitis E virus.

[0018] In the above applications, diseases caused by avian hepatitis E virus include, but are not limited to: chicken hepatomegaly and splenomegaly and liver rupture and hemorrhage syndrome.

[0019] The beneficial effects of this invention are:

[0020] This invention, through in-depth research on avian hepatitis E virus ORF3, developed and designed an avian hepatitis E virus ORF3 subunit vaccine using VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein as active ingredients. The avian hepatitis E virus ORF3 subunit vaccine of this invention has excellent immunogenicity and can mitigate the adverse effects of aHEV on chicken production performance. Attached Figure Description

[0021] Figure 1 Preparation of recombinant ORF3 protein from avian hepatitis E virus; In the figure, A: ORF3 amplification gel electrophoresis image. M: marker; 1: negative control; 2: YT-aHEV ORF3 amplification fragment; 3: VaHEV ORF3 amplification fragment;

[0022] B: Enzyme digestion fragments of the ORF3 recombinant plasmid. M: Marker; 1, 2: Enzyme-digested ORF3 recombinant plasmid and pMD. TM 18-T vector;

[0023] C: ORF3 protein gel SDS-PAGE results. M: marker; 1: YT-aHEV ORF3 inclusion bodies; 2: VaHEV ORF3 inclusion bodies;

[0024] D: ORF3 protein purification diagram. M: marker; 1: YT-aHEV ORF3 purified protein; 2: VaHEV ORF3 purified protein.

[0025] Figure 2 Indirect immunofluorescence of chicken ORF3 antibody serum; (a) YT-aHEV; (b) VaHEV; (c) YT+VaHEV; (d) Blank control.

[0026] Figure 3 The body weight of chickens in different treatment groups in Experiment Example 3. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0028] Because avian hepatitis E virus (HEV) is highly variable and lacks a stable in vitro culture system, there is currently no commercially available vaccine for this virus in clinical practice. Furthermore, the high degree of mutation in avian HEV results in low homology between different strains, making it difficult to achieve good protective effects with only a single vaccine.

[0029] To address current clinical challenges, this invention amplified the ORF3 gene of the VaHEV strain identified in Chinese laying hens and the YT strain identified in broilers, respectively. The prokaryotic expression protein of these strains was used as an immunogen to immunize SPF chickens, and their immunogenicity and protective effect against HEV infection were observed. This invention also prepared a subunit vaccine by mixing two viruses from different sources with significant differences. This experiment not only fills the gap in avian hepatitis E vaccines but also further expands the antibody protection range of vaccines.

[0030] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0031] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0032] Example 1: Preparation of recombinant protein

[0033] Primers for ORF3 amplification were designed for the VaHEV (laying hen origin) strain sequence (NCBI GenBank, accession number: MG976720.1) and the YT-aHEV (broiler origin) strain sequence (NCBI GenBank, accession number: MZ736614.1). Among them:

[0034] The upstream and downstream primer sequences corresponding to the VaHEV strain are as follows:

[0035] Va-ORF3-F: 5'-GGATCCATGCGCCTCGGCTGCCAGCAC-3'; (SEQ ID NO.3)

[0036] Va-ORF3-R: 5'-CTCGAGCTACATCTGGTACCGTGCG-3'. (SEQ ID NO.4)

[0037] The upstream and downstream primer sequences corresponding to YT-aHEV are as follows:

[0038] YT-ORF3-F: 5'-GGATCCATGTGTCTTAGTTGCCAGTT-3'; (SEQ ID NO.5)

[0039] YT-ORF3-R: 5'-CTCGAGCTACGTCTGGTACCGTGCGA-3'. (SEQ ID NO.6)

[0040] The amplification products of Va-ORF3-F and Va-ORF3-R primers are 261 bp in length, and their sequences are shown in SEQ ID NO.7; specifically as follows:

[0041] Va-ORF3(261bp)atgcgcctcggctgccagcactgtctgcagtgccaggagactccggtgggatgtcgtcgcgtggattgctgctcatgcttgcaatgtgctgcggggtgtcaaggggctcccaagcgctcccagc ccgagattggcgcggccaaccccgccgcgacaactcagcacagtggagcgctcaagaacgccctgaaggagccgtcggcccagctgcttccactgatgttgtcaccgcggcaggtactcgcacggtaccagatgtag.

[0042] The amplification products of YT-ORF3-F and YT-ORF3-R primers are 264 bp in length, and their sequences are shown in SEQ ID NO.8; specifically as follows:

[0043] YT-ORF3(264bp)atgtgtcttagttgccagttctggtgtttggagtgccaggatagtggggtgggatgtcgctgcgtggattgctgctcatgcttgctatgtgctgcggggtgtcaaggggctcccaaacgctccca gcaggaagcaggcgcggtcaacgccgccgtgacaacccagcccagtggagcgctcaacaacgccccgaaggagccgtcggccccgcccctcttaccgacgttgtcaccgcggcaggtactcgcacggtaccagacgtag.

[0044] The amplification products were subjected to agarose gel electrophoresis, and the results are as follows: Figure 1 As shown in Figure A, the band is single and matches the theoretical length. The electrophoresis product is then purified. Next, the recombinant plasmid is constructed, and the correct fragment is digested with enzymes (…). Figure 1 B) The recombinant plasmid was constructed by ligating it into the pEASY-Blunt expression vector and transformed into the host bacterium BL21 to obtain recombinant bacteria. The recombinant bacteria were amplified and cultured at 37℃, 220 rpm for 2 hours. Large-scale amplification of the recombinant bacterial culture was followed by induction expression. IPTG was added to the induction culture at a final concentration of 1 mmol / L, and the induction conditions were 30℃ for 6 hours. The results of low-level protein expression showed that the target protein was mainly present in protein inclusion bodies, with very little protein in the supernatant. The protein molecular weight was approximately 34 kDa. Figure 1 C). Protein purification was then performed according to the standard kit procedure (Ni-NTA affinity chromatography medium, Protein A affinity chromatography medium, and chromatography column were purchased from Genscript Biotech). Figure 1 D).

[0045] VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein were prepared using the above method. The amino acid sequence of VaHEV ORF3 recombinant protein is shown in SEQ ID NO.1; the amino acid sequence of YT-aHEV ORF3 recombinant protein is shown in SEQ ID NO.2. Details are as follows:

[0046] VaHEV ORF3: MRLGCQHCLQCQETPVGCRRVDCCSCLQCAAGCQGAPKRSQPEIGAANPAATTQHSGALKNALKEPSAQLLPLMLSPRQVLARYQM.

[0047] YT-aHEV ORF3: MCLSCQFWCLECQDSGVGCRCVDCCSCLLCAAGCQGAPKRSQQEAGAVNAAVTTQPSGALNNAPKEPSAPPLLPTLSPRQVLARYQT.

[0048] Example 2: Preparation of avian hepatitis E virus ORF3 subunit vaccine

[0049] The purified VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein from Example 1 were quantified using the BCA protein quantification method. The quantified VaHEV ORF3 recombinant protein (2454.41 μg / ml) and YT-aHEV ORF3 recombinant protein (2147.21 μg / ml) were mixed at a weight ratio of 1:1 to obtain the antigen composition. Then, the antigen composition was mixed with Freund's complete adjuvant at a weight ratio of 1:1 and emulsified at room temperature using a protein emulsifier. Complete emulsification was indicated by the emulsified protein forming a stable water-in-oil structure when dropped into water, thus preparing the avian hepatitis E virus ORF3 subunit vaccine.

[0050] Comparative Example 1:

[0051] The VaHEV ORF3 recombinant protein prepared in Example 1 was emulsified with Freund's complete adjuvant at a weight ratio of 1:1. The weight of the VaHEV ORF3 recombinant protein was the same as the weight of the antigen composition in Example 2, and subunit vaccine A was prepared.

[0052] Comparative Example 2:

[0053] The YT-aHEV ORF3 recombinant protein prepared in Example 1 was emulsified with Freund's complete adjuvant at a weight ratio of 1:1. The weight of the YT-aHEV ORF3 recombinant protein was the same as the weight of the antigen composition in Example 2, and subunit vaccine B was prepared.

[0054] Experimental example:

[0055] 1. Test method:

[0056] The chickens used were SPF chickens (breed: Laihang; purchased from Jinan Sais), which were randomly divided into four groups and immunized three times at 1, 7 and 14 days of age. The immunization test was carried out by subcutaneous injection in the neck of the chickens, with each chicken receiving 100 μg of immunized protein each time.

[0057] Subunit vaccine B prepared for comparison example 2 of YT-aHEV group immunization;

[0058] Subunit vaccine A prepared for VaHEV group immunization control example 1;

[0059] The avian hepatitis E virus ORF3 subunit vaccine prepared in Example 2 of the YT+VaHEV group immunization;

[0060] An equal volume of physiological saline was injected as a blank control.

[0061] Antibody detection was performed after three immunizations. The primary antibody was post-immunization chicken serum (1:50 dilution), and the secondary antibody was FITC-labeled rabbit anti-chicken IgG antibody. IFA detection was performed on LMH cells infected with aHEV (the strain was the domestically prevalent strain YT-aHEV (GenBank ID: MZ736614.1)).

[0062] After HEV antibodies in chickens tested positive, two aHEV challenge experiments were conducted at 35 and 42 days of age. Each chicken was inoculated with 600 TCID50 of the YT-aHEV strain (GenBank ID: MZ736614.1) via a combination of intravenous and intramuscular injection in the leg. On days 4 and 7 post-challenge, 6 chickens from each group were randomly selected to collect anal swabs. RNA was extracted and aHEV viral load was detected by real-time quantitative PCR. A CT value less than 30 was considered aHEV positive.

[0063] Two weeks after the second viral challenge, eight chickens were randomly selected from each group and weighed.

[0064] 2. Test Results:

[0065] The results showed that immunization successfully induced the production of antibodies in chickens. Figure 2 Chicken serum containing immune proteins was diluted at 1:50, 1:100, and 1:150, respectively. When all serums were diluted at 1:150, the YT+VaHEV group still showed good recognition performance.

[0066] After two challenges with the YT strain, cloacal swabs from each group were subjected to real-time quantitative PCR detection against HEV. The results (Table 1) showed that on days 2, 4, and 7 post-challenge, the positive rate of aHEV nucleic acid in the non-immunized control group was 100%. In the YT-aHEV protein immunization group, the positive rates on days 2, 4, and 7 were 4 / 8, 0 / 8, and 0 / 8, respectively; in the VaHEV protein immunization group, the positive rates were 5 / 8, 1 / 8, and 0 / 8, respectively; and in the mixed protein immunization group, the positive rates were 3 / 8, 0 / 8, and 0 / 8, respectively. These results indicate that the antibodies produced after immunization successfully blocked the proliferation and shedding of aHEV in vivo, with the mixed protein immunization group showing the best protective effect.

[0067] Table 1: Virus positivity rate on days post-immunization in different treatment groups

[0068] Number of days after infection VaHEV Group YT-aHEV group YT+VaHEV group control group 2 5 / 8 4 / 8 3 / 8 8 / 8 4 1 / 8 0 / 8 0 / 8 8 / 8 7 0 / 8 0 / 8 0 / 8 8 / 8

[0069] Two weeks after challenge with the virus, eight chickens were randomly selected from each group and weighed. The average weight, from highest to lowest, was as follows: YT+Va-ORF3 mixed protein immunization group (1001.875g), Va-ORF3 protein immunization group (949g), YT-ORF3 protein immunization group (879.25g), and unimmunized control group (793.625g). This indicates that aHEV infection inhibits chicken growth, and ORF3 subunit vaccine immunization can mitigate the adverse effects of aHEV on production performance. Figure 3 ).

[0070] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of avian hepatitis E virus ORF3 subunit vaccine in the preparation of products that alleviate growth inhibition caused by avian hepatitis E virus infection, characterized in that, The avian hepatitis E virus ORF3 subunit vaccine consists of an antigen composition and a vaccine adjuvant. The antigen composition consists of VaHEV ORF3 recombinant protein and YT-aHEV ORF3 recombinant protein; The amino acid sequence of the VaHEV ORF3 recombinant protein is shown in SEQ ID NO.1, and the amino acid sequence of the YT-aHEV ORF3 recombinant protein is shown in SEQ ID NO.2; The weight ratio of the VaHEV ORF3 recombinant protein to the YT-aHEV ORF3 recombinant protein is 1:1; The preparation method of the avian hepatitis E virus ORF3 subunit vaccine is as follows: The antigen composition and Fluoride complete adjuvant were mixed at a weight ratio of 1:1 and emulsified at room temperature using a protein emulsifier. The emulsified protein was emulsified completely when it was dropped into water and did not disperse, forming a stable water-in-oil structure. This process yielded the avian hepatitis E virus ORF3 subunit vaccine.

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