Oral vaccine against carp spring viremia virus, its preparation method and application

Abstract

This invention discloses an oral vaccine against carp spring viremia virus, its preparation method, and its application. The oral vaccine is constructed by linking a carp spring viremia virus antigen protein and a Yellow River carp defensin protein via a flexible linker to form a fusion protein, which is then expressed in *E. coli* and enriched using a biological vector. The gene sequence encoding this fusion protein includes a truncated fragment of the carp spring viremia virus antigen protein gene (64-1389 bp), a fragment of the Yellow River carp defensin protein gene (70-201 bp without the signal peptide), and a flexible linker fusion gene connecting the two gene fragments. Compared with previously reported anti-SVCV vaccines, the oral vaccine against carp spring viremia virus prepared in this invention does not harm the fish during the entire immunization process, is simple to operate, safe and stable, and has a higher protection rate. Therefore, this oral vaccine provides broad prospects for the prevention and control of carp spring viremia.

Description

Technical Field

[0001] This invention belongs to the technical field of fish vaccines against spring viremia of carp, specifically relating to an oral vaccine against spring viremia of carp, its preparation method, and its application. Background Technology

[0002] Spring viraemia of carp (SVC) is an acute hemorrhagic and epidemic septicemia caused by Spring viraemia of carp virus (SVCV). It is prevalent globally and is one of the most prominent problems in freshwater aquaculture in my country. This disease primarily infects carp and its variants, with a mortality rate exceeding 70%. Given its widespread prevalence and high pathogenicity, SVCV remains one of the fish viruses that must be reported, according to the latest 2021 list of aquatic animal diseases published by the World Organisation for Animal Health. SVCV is a single-stranded negative-sense RNA virus with a genome size of approximately 11 kb, encoding five structural proteins: nuclear protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L). The surface G protein is the most important antigenic protein, possessing strong immunogenicity and capable of inducing an immune response. Therefore, G proteins are currently the main proteins used in SVCV virus detection, antibody preparation, and vaccine development. Developing effective prevention and control measures against SVCV will benefit the healthy development of aquaculture in my country and the world.

[0003] For the prevention and control of viral diseases, vaccines are the most effective measure. Fish vaccines can be divided into two types according to their development technology: traditional vaccines and novel vaccines. Traditional vaccines are mainly inactivated vaccines and live attenuated vaccines; novel vaccines are developed using modern biotechnology, including gene-deleted live vaccines, recombinant vaccines, genetically engineered subunit vaccines, genetically engineered live vector vaccines, and nucleic acid vaccines. Genetically engineered live vector vaccines, also known as biological vector vaccines, are vaccines that express exogenous antigens in microbial or animal / plant vectors through genetic engineering technology. They have advantages such as high immunogenicity, low cost, and good safety, and represent the most promising direction for future vaccine research and development. Fish vaccines can be divided into three types according to the method of administration: injection, immersion, and oral administration. Among them, oral immunization is a relatively convenient method of immunization, not limited by the size of the recipient, the type of vaccine, the timing and frequency of administration; in addition, oral immunization can avoid the stress response and damage to the body caused by injection, and can also reduce manpower and material resources, making it easy to promote and having broad application prospects. With the development of modern biological technology, fish vaccines have transitioned from traditional vaccines to novel oral vaccines.

[0004] Currently, vaccine development for SVCV is still in the exploratory stage. Reported inactivated SVCV vaccines offer only limited protection, while attenuated vaccines have risks such as improper attenuation methods and virulence reversion. Early DNA vaccines against SVCV showed low protection rates. Researchers conducted multiple experiments targeting the SVCV G protein, with relative survival rates ranging from 11% to 33%; the treatment group injected with the full-length G gene showed better results, with a protection rate of 48%. Furthermore, the handling pressure on fish due to direct injection of DNA vaccines, as well as the high labor and production costs, make large-scale immunization difficult to achieve. An oral subunit vaccine expressing SVCV G and koi herpesvirus ORF81 genes via Lactobacillus fusion can achieve a 71% immunoprotective rate in carp, while the relative protection rate of carp immunized by immersion in an SVCV subunit vaccine expressed using insect rod cells is only 34%. Based on current research, developing efficient vaccine antigen vectors for oral immunization may be an effective route for SVCV vaccination.

[0005] In vaccine development, the addition of fusion proteins offers advantages such as enhanced antigen activity, extended antigen effectiveness time, and the generation of bifunctional or even multifunctional proteins. Defensins are effector molecules of innate immunity and can serve as natural immune adjuvants. In this invention, SVCV G protein is fused with Yellow River carp defensin (β-defensin3, BD3) via a flexible linker, and the resulting protein is enriched in rotifers and fed to Yellow River carp to prepare a simple and effective novel oral vaccine. Summary of the Invention

[0006] The technical problem solved by this invention is to provide an oral vaccine for carp against spring viremia virus that is simple to prepare, safe and stable, and has good immunoprotective effect, as well as the preparation method thereof. The oral vaccine for carp against spring viremia virus prepared by this method can be used as an oral vaccine for the prevention or treatment of spring viremia in carp.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: an oral vaccine for carp against spring viremia virus, characterized in that: the oral vaccine is constructed by connecting the spring viremia virus antigen protein and the Yellow River carp defensin protein through a flexible linker to form a fusion protein, which is then expressed in Escherichia coli and enriched by a biological vector. The gene sequence encoding the fusion protein includes a truncated fragment of the spring viremia virus antigen protein gene (64-1389 bp) and a fragment of the Yellow River carp defensin protein gene (70-201 bp without the signal peptide), as well as a flexible linker fusion gene connecting the two gene fragments.

[0008] The nucleotide sequence of the gene encoding the antigen protein of carp spring viremia virus is shown in SEQ ID NO:1;

[0009] The nucleotide sequence of the defensin protein encoding gene of Yellow River carp is shown in SEQ ID NO:2;

[0010] The nucleotide sequence of the flexible linker fusion gene is shown in SEQ ID NO:3.

[0011] Further specifying, the prokaryotic expression plasmid pET-28a used to express the fusion protein in Escherichia coli contains a green fluorescent protein (GFP) tag.

[0012] Further specifying, the biological carrier is the freshwater Brachiopoda calycifolia, which is a favorite food of carp.

[0013] Further specifying, the C-terminus of the truncated fragment 64-1389bp of the carp spring viremia virus antigen protein gene is fused with the N-terminus of the designal peptide portion of the Yellow River carp defensin protein gene fragment 70-201bp via a flexible adapter to form a fusion protein encoding gene.

[0014] The method for preparing the oral vaccine against carp spring viremia virus of the present invention is characterized by the following specific process;

[0015] Step S1: The gene sequences encoding the antigen protein of carp spring viremia virus and the defensin protein of Yellow River carp are amplified and obtained respectively. The sequence of the gene sequence encoding the antigen protein of carp spring viremia virus is analyzed and its N-terminal signal peptide is removed. Based on the hydrophobicity and antigenic determinants of the amino acids corresponding to the antigen protein of carp spring viremia virus, the amino acid fragments with good antigenicity and easy expression are selected. The corresponding gene sequence is the truncated fragment of the gene of carp spring viremia virus antigen protein, 64-1389bp. The sequence of the gene sequence encoding the defensin protein of Yellow River carp is analyzed and its N-terminal signal peptide is removed. The remaining amino acid sequences are selected. The corresponding gene sequence is the gene fragment of Yellow River carp defensin protein with the signal peptide removed, 70-201bp.

[0016] Step S2: The truncated fragment of the carp spring viremia virus antigen protein gene (64-1389 bp) and the fragment of the Yellow River carp defensin protein gene (70-201 bp) were fused together using a flexible adapter and inserted into the MCS region of the pET-28a-GFP plasmid to construct a recombinant plasmid.

[0017] Step S3: Transform the recombinant plasmid into Rosetta (DE3) competent cells. Positive clones were screened by bacterial PCR and verified by sequencing before inducing expression: When the OD600 reached 0.5-0.6, different concentrations of IPTG were added and the cells were cultured overnight at 28°C. 1 mL of bacterial culture was collected by centrifugation, washed with PBS, and 40 μL of protein lysis buffer was added. Protein loading buffer was added and the cells were boiled to denature them. After cooling, SDS-PAGE electrophoresis was performed to separate the cells. Coomassie brilliant blue staining and destaining were performed to identify protein expression.

[0018] Step S4: Centrifuge and collect the induced bacterial culture, wash it three times with PBS, and finally prepare it with PBS to the same concentration. Observe the expression of fluorescent protein under a fluorescence microscope. The rest are inactivated by formalin, centrifuged and washed three times with PBS to remove formalin, and stored for later use.

[0019] Step S5: Feed the prokaryotic expression bacteria of the fluorescent fusion protein to rotifers. After 1 hour, detect the fluorescence intensity under a fluorescence microscope and take a picture. Feed the prokaryotic expression bacteria to rotifers separately. Determine the degree of protein enrichment based on the fluorescence intensity. Then collect the rotifers with a 300-mesh sieve. Use immediately or store at -80℃ for later use.

[0020] The oral vaccine against carp spring viremia virus described in this invention is used as an oral vaccine for the prevention or treatment of carp spring viremia.

[0021] Compared with existing technologies, this invention has the following advantages and beneficial effects: This invention develops a novel oral vaccine for carp against spring viremia virus. It utilizes the fusion expression of carp spring viremia virus antigen protein and Yellow River carp defensin protein through a flexible linker in *E. coli*, and enriches the fusion protein using rotifers, a food source favored by carp, as a biological carrier to obtain the oral vaccine. This vaccine is then administered orally to Yellow River carp mixed with their feed. This oral vaccine exhibits a high immunoprotective efficacy (77.28%). Compared with previously reported anti-SVCV vaccines, the oral vaccine for carp against spring viremia virus prepared in this invention does not harm the fish during the entire immunization process, is simple to operate, safe and stable, and has a higher protection rate. Therefore, this oral vaccine provides broad prospects for the prevention and control of spring viremia in carp. Attached Figure Description

[0022] Figure 1 This is a diagram showing the expression of proteins from different recombinant plasmids.

[0023] Figure 2 These are fluorescence analysis images of different expression bacteria under a fluorescence microscope.

[0024] Figure 3 The enrichment of each protein group in rotifers was observed using a fluorescence microscope.

[0025] Figure 4 These are images showing the clinical symptoms of different experimental fish.

[0026] Figure 5 These are the survival curves of different experimental fish. Detailed Implementation

[0027] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0028] Example

[0029] Materials and methods:

[0030] 1. Laboratory animals

[0031] The Yellow River carp used in the experiment had the following specifications: body length 8.0±1.0cm, weight 19.0±1.5g, and were raised at the aquaculture base of Henan Normal University. During the rearing process, continuous aeration was maintained, and the water temperature was kept between 15-20℃. The Yellow River carp were fed twice a day, morning and evening, and the water was changed and wastewater removed every other day.

[0032] The Brachionus calyx used in the experiment was purchased from a live bait shop in Shanghai and cultured with Chlorella at 25-30℃.

[0033] 2. Carrier and Recipient Bacteria

[0034] To visualize the results of feeding rotifers with E. coli, a GFP tag was inserted into the MCS region of the prokaryotic expression vector pET-28a; both E. coli DH5α and Rosetta(DE3) were products of Novizan.

[0035] 3. Reagents

[0036] LATaq DNA polymerase, endonuclease, T4 ligase, etc. were purchased from Takara Bio Inc.; IPTG was purchased from Solarbio Biotechnology Co., Ltd.; and the SDS-PAGE gel kit was purchased from Yage Biotechnology Co., Ltd.

[0037] 4. Cells, viruses

[0038] Carp epithelioma cells (EPC) were cultured in L15 medium containing 10 wt% fetal bovine serum (FBS). Serum, medium, and trypsin were all Gibco products. Carp spring viremia virus (SVCV) was prepared and stored in our laboratory.

[0039] 5. Construction of fusion proteins

[0040] The gene sequences of SVCV G and Yellow River carp defensin (BD3) were amplified, and their N-terminal signal peptides were removed. Based on the hydrophobicity and antigenic determinants of the amino acids encoding the G protein, suitable gene fragments were selected and fused with the selected Yellow River carp defensin protein gene fragment using a flexible linker. The fusion gene sequences were synthesized by Kinkairui Pharmaceutical Co., Ltd. (Wuhan, China). They were synthesized in the order of G first, BD3 last, and BD3 first, G last, and inserted into the MCS region of the pET-28a-GFP plasmid, respectively, to construct three recombinant plasmids: G, G-BD3, and BD3-G.

[0041] 6. Induction and identification of fusion proteins

[0042] The three recombinant plasmids and the control plasmid pET-28a-GFP were transformed into Rosetta(DE3) competent cells. Positive clones were screened by bacterial culture PCR and verified by sequencing before inducing expression: when the OD600 reached 0.5-0.6, different concentrations of IPTG were added, and the cells were incubated overnight at 28°C. 1 mL of bacterial culture was collected by centrifugation, washed with PBS, and 40 μL of protein lysis buffer was added. Protein loading buffer was added, and the cells were boiled to denature them. After cooling, SDS-PAGE electrophoresis was performed for separation, and Coomassie brilliant blue staining and destaining were used to identify protein expression.

[0043] 7. Feeding rotifers with prokaryotic bacteria expressing fusion protein

[0044] The fusion proteins of each group were induced in large quantities under the above conditions. The bacterial culture was then collected by centrifugation and washed three times with PBS. Finally, the culture was prepared with PBS to the same concentration. A small amount was taken to observe the expression of fluorescent proteins under a fluorescence microscope. The rest was inactivated by formalin, centrifuged and washed three times with PBS to remove formalin, and then aliquoted and stored for later use.

[0045] 8. Rotifer enriched fusion protein

[0046] The prokaryotic bacteria expressing the fluorescent fusion protein were fed to rotifers. After 1 hour, the fluorescence intensity was detected under a fluorescence microscope and photographed. Four groups of prokaryotic expression bacteria were fed to four groups of rotifers respectively. The degree of protein enrichment was determined based on the fluorescence intensity. The rotifers were then collected using a 300-mesh sieve and either used immediately or stored at -80℃ for later use.

[0047] 9. Administration of oral vaccines

[0048] Yellow River carp were divided into 4 groups of 100 fish each. Rotifers from groups 1 to 4 were mixed with feed (Tongwei Fish Health) and fed to the experimental fish in each group. Each group was fed once in the morning and once in the evening for 30 days.

[0049] 10. Immunoprotective trials of vaccines

[0050] After oral vaccination, each group of Yellow River carp was intraperitoneally injected with SVCV at a dose equal to the median lethal dose (LD50) of SVCV for Yellow River carp. Each carp was injected with 100 μL of virus solution, with three replicates per group, for a total of 45 carp. The same method was used to challenge the carp. The fish were observed and recorded for 28 consecutive days. The relative percent survival (RPS) of the vaccine was calculated as follows: RPS = [1 - (mortality rate of the immunized group / mortality rate of the control group)] × 100%.

[0051] Results and Analysis:

[0052] 1. Gene amplification and fusion protein construction

[0053] This invention first amplified the gene sequences of SVCV G and Yellow River carp defensin (BD3). The G gene ORF is 1530 bp in length, encoding 509 amino acids, as shown in SEQ ID NO:1; the D gene ORF is 201 bp in length, encoding 66 amino acids, as shown in SEQ ID NO:2. SignlP 6.0 analysis revealed that both proteins contain a signal peptide, which needs to be removed for prokaryotic expression. Furthermore, based on Protean analysis of the hydrophobicity and antigenic determinants of the G protein, we ultimately selected a truncated fragment (64-1389 bp) of the G protein encoding gene and a fragment of the Yellow River carp defensin gene (70-201 bp) without the signal peptide, linking them together using a flexible linker to create two fusion protein encoding sequences: G-BD3 and BD3-G. The G, G-BD3, and BD3-G gene sequences were then introduced into the previously modified empty vector plasmid pET-28a-GFP, successfully constructing three recombinant plasmids.

[0054] 2. Fusion protein induced expression

[0055] The control group (pET-28a-GFP) and *E. coli* containing recombinant plasmids of G, G-BD3, and BD3-G proteins were induced to express the protein. The induction conditions were 28℃ for 12 hours. The results are as follows: Figure 1 As shown, all proteins were successfully expressed. Since a good induction effect was obtained with an IPTG concentration of 0.5 mM, this concentration was used for large-scale induction. Green fluorescence was observed in all collected bacterial cells under a fluorescence microscope, which can serve as a good indicator. Figure 2 ).

[0056] 3. Rotifer enriched fusion protein

[0057] The *E. coli* strains obtained from the above four groups of induced expression were inactivated and fed to each group of rotifers. The proteins were then collected and observed under a fluorescence microscope to determine their enrichment within the rotifers. The results are as follows: Figure 3As shown, fluorescent E. coli were clearly observed to be ingested by rotifers in each group. This indicates that a good enrichment effect can be achieved using rotifers as a biological carrier. The rotifers from each group were collected and administered as an oral vaccine mixed with feed or stored at -80℃ for later use.

[0058] 4. Vaccine immunization protection effect

[0059] Table 1. Determination of the median lethal dose of SVCV in Yellow River carp.

[0060]

[0061] Note: 0.1 mL SVCV was injected intraperitoneally into each tail.

[0062] To determine the LD50 of SVCV in Yellow River carp, a viral infection experiment was conducted on the fish. As shown in Table 1, each group was injected with 100 μL of virus solution or L15 culture medium. One week after infection, the experimental fish began to die, exhibiting clinical symptoms such as abdominal swelling, bulging eyes, and hemorrhage on the body surface and base of the fins. Figure 4 The mortality rate of Yellow River carp in each group was statistically analyzed (Table 1). Using SPSS, the median lethal dose (LD50) of SVCV for Yellow River carp was calculated to be 2.03 × 10⁻⁶. 7 TCID 50 The four groups of Yellow River carp were fed oral rotifer vaccines containing empty vector (Control), G, G-BD3, and BD3-G, respectively. Thirty days later, the LD50 values ​​were used to inject each group of experimental fish. Symptoms were observed, and mortality rates were recorded. Survival curves for the experimental fish were plotted using R language. The results are as follows: Figure 5 As shown, the survival rates of experimental fish fed with oral vaccines (Control, G, G-BD3, and BD3-G) after SVCV infection were 51.11%, 77.78%, 88.89%, and 60.00%, respectively. There was a significant difference between G and Control, a highly significant difference between G-BD3 and Control, but no significant difference between BD3-G and Control. The results indicate that after oral vaccination, both the G and G-BD3 groups of Yellow River carp developed some immunity, with relative immune protection rates of 54.56% and 77.28%, respectively. The G-BD3 group showed the best immune protection effect.

[0063] This invention develops an oral vaccine against spring viremia virus in carp. The study utilized a fusion expression of viral and host proteins, enriching the fusion protein using rotifers, a preferred food of carp, as a biological carrier. The vaccine was then administered orally to Yellow River carp mixed with their feed. This oral vaccine demonstrated a high level of immunoprotective efficacy (77.28%). Therefore, this oral vaccine offers promising prospects for the prevention and control of spring viremia in carp.

[0064] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. An oral vaccine against carp spring viremia virus, characterized in that: The oral vaccine is constructed by linking the carp spring viremia virus antigen protein and the Yellow River carp defensin protein through a flexible adapter to form a fusion protein, which is then expressed in Escherichia coli and enriched through a biological vector. The gene sequence encoding the fusion protein includes a truncated fragment of the carp spring viremia virus antigen protein gene (64-1389 bp) and a fragment of the Yellow River carp defensin protein gene (70-201 bp without the signal peptide), as well as a flexible adapter fusion gene connecting the two gene fragments. The C-terminus of the truncated fragment of the carp spring viremia virus antigen protein gene (64-1389 bp) and the N-terminus of the fragment of the Yellow River carp defensin protein gene (70-201 bp without the signal peptide) are linked by a flexible adapter fusion gene to form the fusion protein encoding gene. The nucleotide sequence of the gene encoding the antigen protein of carp spring viremia virus is shown in SEQ ID NO:1; The nucleotide sequence of the defensin protein encoding gene of Yellow River carp is shown in SEQ ID NO:2; The nucleotide sequence of the flexible linker fusion gene is shown in SEQ ID NO:3; The specific preparation process of the oral vaccine against carp spring viremia virus is as follows: Step S1: The gene sequences encoding the antigen protein of carp spring viremia virus and the defensin protein of Yellow River carp are amplified and obtained respectively. The sequence of the gene sequence encoding the antigen protein of carp spring viremia virus is analyzed and its N-terminal signal peptide is removed. Based on the hydrophobicity and antigenic determinants of the amino acids corresponding to the antigen protein of carp spring viremia virus, the amino acid fragments with good antigenicity and easy expression are selected. The corresponding gene sequence is the truncated fragment of the gene of carp spring viremia virus antigen protein, 64-1389bp. The sequence of the gene sequence encoding the defensin protein of Yellow River carp is analyzed and its N-terminal signal peptide is removed. The remaining amino acid sequences are selected. The corresponding gene sequence is the gene fragment of Yellow River carp defensin protein with the signal peptide removed, 70-201bp. Step S2: The truncated fragment of the carp spring viremia virus antigen protein gene (64-1389 bp) and the fragment of the Yellow River carp defensin protein gene (70-201 bp) were fused together using a flexible adapter and inserted into the MCS region of the pET-28a-GFP plasmid to construct a recombinant plasmid. Step S3: Transform the recombinant plasmid into Rosetta (DE3) competent cells. Positive clones were screened by bacterial PCR and verified by sequencing before inducing expression: When the OD600 reached 0.5-0.6, different concentrations of IPTG were added and the cells were cultured overnight at 28°C. 1 mL of bacterial culture was collected by centrifugation, washed with PBS, and 40 μL of protein lysis buffer was added. Protein loading buffer was added and the cells were boiled to denature them. After cooling, SDS-PAGE electrophoresis was performed to separate the cells. Coomassie brilliant blue staining and destaining were performed to identify protein expression. Step S4: Centrifuge and collect the bacterial cultures induced in each group, wash them three times with PBS, and finally prepare them with PBS to the same concentration. Observe the expression of fluorescent proteins under a fluorescence microscope. The rest are inactivated by formalin, centrifuged and washed three times with PBS to remove formalin, and stored for later use. Step S5: Feed the prokaryotic bacteria expressing the fluorescent fusion protein to rotifers. After 1 hour, detect the fluorescence intensity under a fluorescence microscope and take a picture. Determine the degree of protein enrichment based on the fluorescence intensity. Then collect the rotifers with a 300-mesh sieve. Use them immediately or store them at -80℃ for later use.

2. The oral vaccine against carp spring viremia virus according to claim 1, characterized in that: The rotifer in question is the freshwater Brachycephala rotifer, a species favored by carp.

3. The use of the oral vaccine against carp spring viremia virus according to any one of claims 1-2 in the preparation of an oral vaccine for the prevention or treatment of carp spring viremia.

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