An mRNA based on the CP protein gene of fish neuronecrosis virus, a vaccine, and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses an mRNA based on the CP protein gene of fish nervous necrosis virus, a vaccine, and a preparation method and application thereof. The present invention provides an mRNA of fish nervous necrosis virus, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the structure includes a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and polyA. The mRNA vaccine contains the ORF sequence of the capsid protein CP gene that is resistant to NNV virus infection. The results of animal safety tests on the fish nervous necrosis virus mRNA vaccine prepared by the present invention show that the vaccine is safe and effective, and is convenient and simple to use clinically, and has broad application prospects in the prevention and control of viral nervous necrosis in marine cultured fish.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an mRNA based on the fish neuronecrosis virus CP protein gene, a vaccine, and a preparation method and application thereof. Background Art

[0002] Fish Nervous Necrosis Virus (NNV), belonging to the genus Betanodavirus in the family Nodaviridae, is the primary pathogen causing viral nervous necrosis (VNN). Since its first discovery in Australia in the 1980s, NNV has become a serious threat to the global aquaculture industry. It causes extremely high mortality in larvae and juveniles, with severe cases reaching 100% mortality within a week. The number of infected fish species and the extent of the damage have increased rapidly in recent years. The NNV virion is a non-enveloped icosahedral structure approximately 25 nanometers in diameter. Its genetic material consists of two positive-sense single-stranded RNAs: RNA1 (2970 base pairs (bp)) and RNA2 (1440 bp). The 3' end of the RNA molecules lacks a poly(A) sequence. Despite the long history of NNV, prevention and control of VNN remains challenging. In theory, vertical transmission can be prevented by screening broodstock for virus-free broodstock. However, due to the limitations of detection technology, it is difficult to ensure that the selected broodstock are completely virus-free. In addition, although disinfection with ozone and iodine reagents can reduce horizontal transmission of the virus, the virus may be present both on the surface of fertilized eggs and within the egg membrane, which means that surface disinfection alone cannot completely block virus transmission.

[0003] In recent years, mRNA vaccine technology has received widespread attention as a new vaccine development method. mRNA vaccines have the advantages of rapid production, no need to enter the host cell nucleus, and no integration of exogenous genes into the host genome, and have been widely used in vaccine development. mRNA vaccines can effectively deliver target gene information to host cells and stimulate the body to produce effective and long-lasting specific humoral and cellular immune responses. mRNA vaccines have extremely high infection efficiency and can induce the body to produce effective mucosal immunity and T cell immune responses. In addition, the immune response induced by mRNA vaccines in vivo is highly specific and persistent, providing new ideas and methods for the development of NNV vaccines. In a variety of animal models, the mucosal immune response induced by mRNA vaccines has been shown to be more effective than traditional parenteral immunization, which provides a new strategy for the prevention and control of NNV. Summary of the Invention

[0004] The present invention provides an mRNA based on the fish neurological necrosis virus CP protein gene, a vaccine, and a preparation method and application thereof, which solve the problem that fish are susceptible to NNV infection in aquaculture.

[0005] The present invention adopts the following technical solution: an mRNA based on the fish nervous necrosis virus CP protein gene, the mRNA consists of the following structure: a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and a polyA nucleic acid sequence, the nucleotide sequence of the mRNA is shown in SEQ ID NO:1, the NNV virus antigen coding region contains the capsid protein CP gene infected by the NNV virus, and the nucleotide sequence of the NNV virus antigen coding region is shown in SEQ ID NO:5.

[0006] Furthermore, the nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 3.

[0007] Furthermore, the amino acid sequence of the signal peptide sequence is shown in SEQ ID NO: 4.

[0008] Furthermore, the nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 6.

[0009] Furthermore, the nucleotide sequence of polyA is shown in SEQ ID NO: 7

[0010] A fish neuronecrosis virus mRNA vaccine, comprising the mRNA of the fish neuronecrosis virus CP protein gene.

[0011] A method for preparing an mRNA vaccine for fish neuronecrosis virus comprises the following steps:

[0012] (1) The mRNA template DNA consists of a T7 promoter, a 5' untranslated region, a signal peptide sequence, an NNV viral antigen coding region, a 3' untranslated region, and a polyA linker. The DNA template is synthesized by chemical synthesis;

[0013] (2) T7 RNA polymerase and cap analogs were used to perform T7 RNA transcription and capping using the DNA chemically synthesized in the previous step as a template, and the mRNA of fish neural necrosis virus was obtained by purification using ethanol precipitation.

[0014] Furthermore, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 2.

[0015] The invention relates to an application of an mRNA vaccine for fish nervous necrosis virus and its application in the preparation of a drug against fish nervous necrosis virus.

[0016] The CP gene of NNV is crucial for the viral infection cycle and the induction of host immune responses. As a unique structural protein that constitutes the virus particles, CP is not only responsible for the construction of virus particles, but also carries multiple antigenic sites that can activate the host immune system. The NNV-CP protein also has a variety of strategies to inhibit the host's defense mechanism, including using the ubiquitination pathway to promote the degradation of key immune proteins, thereby reducing the synthesis of type I interferon. Given the core role of the NNV-CP gene in the viral life cycle and immune evasion. By synthesizing the CP gene of NNV in vitro, recombinant proteins with immune activation potential can be produced, which is crucial for the development of highly effective vaccines.

[0017] By expressing the CP gene of NNV in Escherichia coli, recombinant CP protein can be successfully synthesized, and it has been shown that it can stimulate the host's protective immune response. However, the antigenic properties of the NNV-CP gene in the host body and in vitro may be different, which suggests that the antigenic properties of the virus in the host body must be considered when designing the vaccine. In addition, the stability of the NNV-CP gene and the antigenic sites displayed on the surface of the virus particles are extremely critical to the effectiveness of the vaccine. The surface antigenic sites of the NNV virus particles may change under certain conditions, such as heat treatment or specific solutions, affecting their antigenicity and infectivity. Therefore, the NNV-CP gene plays a decisive role in viral infection, immune escape and vaccine development. In the present invention, an mRNA vaccine containing the NNV-CP gene was constructed by cloning the NNV-CP gene.

[0018] Beneficial effects of the present invention:

[0019] (1) The viral neuronecrosis mRNA vaccine prepared by the present invention selects the 5' untranslated region, signal peptide sequence, NNV virus antigen coding region, 3' untranslated region and polyA nucleic acid sequence, which is obtained by nucleotide modification during the transcription process. After immunization of grouper, high-level antibodies are produced, and the antibodies have a high titer;

[0020] (2) The viral neuronecrosis mRNA vaccine prepared by the present invention has been proven to be safe, effective, and non-toxic through animal safety tests. mRNA vaccines do not contain live viral components and therefore pose no risk of infection. They are degraded into nucleotides in the body and do not integrate into the host genome. mRNA vaccines can stimulate a strong immune response in the body, including humoral and cellular immunity, and can effectively prevent viral infection. They have good market application prospects in the marine aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a structural diagram of the mRNA of the fish nervous necrosis virus NNV-CP of the present invention.

[0022] Figure 2 This is the detection of fish nervous necrosis virus NNV-CP mRNA of the present invention: lane: mRNA vaccine.

[0023] Figure 3 This is the expression of the fish neuronecrosis virus NNV-CP mRNA vaccine of the present invention in HEK293 cells.

[0024] Figure 4 It is the survival rate of grouper 7 days after injection of the fish nervous necrosis virus NNV-CP mRNA vaccine of the present invention.

[0025] Figure 5 It is the antibody titer against the CP protein of NNV virus in the serum of grouper 28 days after immunization with the fish nervous necrosis virus NNV-CP mRNA vaccine of the present invention.

[0026] Figure 6 This is a diagram showing the effect of the fish nervous necrosis virus NNV-CP mRNA vaccine of the present invention in preventing and treating NNV virus in grouper. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0028] Human embryonic kidney 293 cells (HEK293 cells) were grown adherently in a cell culture medium in a 5% CO2, 37°C incubator. HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 mg / ml streptomycin.

[0029] The Nervous Necrosis Virus (NNV) in the present invention is stored in the National Aquatic Animal Pathogen Bank.

[0030] Example 1: Preparation of mRNA based on NNV-CP gene:

[0031] (1) Synthesis of DNA template based on mRNA of NNV-CP gene: The template DNA of the mRNA consists of T7 promoter, 5' untranslated region, signal peptide sequence, NNV virus antigen coding region, 3' untranslated region and poly A linker. The DNA template is synthesized by chemical synthesis according to the above sequence;

[0032] The nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 2;

[0033] The nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 3;

[0034] The amino acid sequence of the signal peptide sequence is shown in SEQ ID NO: 4;

[0035] The amino acid sequence of the NNV virus antigen coding region (NNV-CP gene amino acid sequence) is shown in SEQ ID NO: 5;

[0036] The nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO: 6;

[0037] and polyA nucleotide sequence is shown in SEQ ID NO: 7;

[0038] (2) T7 RNA transcription of NNV-CP gene mRNA: T7 RNA transcription and capping were performed using the synthesized DNA as a template;

[0039] The specific steps are as follows: 1 μg of DNA template, 2 μL of T7 transcription buffer, 4 μL of NTP mixture (10 mM), 0.1 μL of T7 RNA polymerase (1 kU / μl), Cap Analog (40 mM), add nuclease-free pure water to 20 μL, and incubate at 37°C for 120 minutes;

[0040] (3) mRNA purification: After the reaction is completed, the transcribed linear RNA is purified using ethanol precipitation;

[0041] The specific steps are as follows: first, add 160 μL of nuclease-free pure water to expand the reaction volume to 180 μL;

[0042] Then add 20 μL of 3M sodium acetate (pH 5.2) or 20 μL of 5M ammonium acetate and mix thoroughly; add an equal volume of phenol / chloroform mixture (1:1) and extract once (vortex mix for 20-30 seconds, then centrifuge at 14,000 g for 5-10 minutes and collect the supernatant), then extract 1-2 times with chloroform (vortex mix for 20-30 seconds each time, then centrifuge at 14,000 g for 5-10 minutes and collect the supernatant);

[0043] Precipitate RNA with double volume of anhydrous ethanol and incubate at -20°C for at least 30 minutes. Centrifuge at 14,000g for 5-10 minutes at 4°C to precipitate RNA. Discard the supernatant and wash the precipitate with 500 μL of pre-cooled 70% ethanol. Resuspend and dissolve the RNA in 20 μL of nuclease-free water.

[0044] Finally, the synthesized mRNA was detected by agarose gel electrophoresis; the results were as follows Figure 2 shown.

[0045] Depend on Figure 2 It can be seen that mRNA based on the NNV-CP gene has been successfully synthesized using the above method.

[0046] (4) Detection of the effect of synthesized NNV-CP gene mRNA on the production of target protein:

[0047] HEK293 cells in the logarithmic growth phase were obtained and digested with trypsin solution. The cells were then diluted with cell culture medium to a cell density of 2.5 × 10 5 cells / ml to form a cell suspension, add the cell suspension to a 12-well cell culture plate at 1 ml / well, and culture in a cell culture incubator with a CO2 volume content of 5% and a temperature of 37°C;

[0048] After 24 hours of incubation, the supernatant was discarded, the cells were rinsed with sterile PBS, and 1 ml of cell culture medium was added per well. The synthesized mRNA carrying the NNV-CP gene was then transfected into the HEK293 cells using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific). 48 hours after transfection, the supernatant was removed and the cells were lysed using RIPA lysis buffer. Following lysis, the protein sample was ultrasonically disrupted and centrifuged at 12,000 rpm for 10 minutes at 4°C to separate the supernatant. The supernatant was mixed with 6× Loading Buffer and then heated at 95°C for 10 minutes to denature the proteins.

[0049] Next, the treated protein sample was loaded into the SDS-PAGE electrophoresis gel well for electrophoresis separation. The electrophoresis conditions were set to 80V for 1 hour, and then the voltage was increased to 120V until the end of electrophoresis. Tubulin was used as an internal reference. According to the protein molecular weight standard, the protein bands to be tested in the gel were cut out and transferred to the PVDF membrane. The transfer conditions were electrophoresis at a current of 200mA for 2 hours. After the membrane transfer was completed, the PVDF membrane was placed in a 5% skim milk powder solution for 1 hour to reduce nonspecific binding. After blocking, the membrane was incubated with a 1:1000 diluted NNV-CP antibody overnight to specifically bind to the target protein; the next day, the membrane was washed 3 times with PBST buffer for 10 minutes each time to remove unbound antibodies. After that, the membrane was incubated with a 1:2000 diluted goat anti-rabbit secondary antibody for 2 hours, and then washed 3 times with PBST buffer for 10 minutes each time to remove excess secondary antibody.

[0050] Finally, the developer was added and the chemiluminescence reaction on the membrane was detected to evaluate the efficiency of mRNA expression of NNV-CP; the results were as follows: Figure 3 It is known.

[0051] like Figure 3 The results showed that HEK293 cells transfected with mRNA carrying the NNV-CP gene were able to efficiently express NNV-CP protein, while HEK293 cells in the control group that were not transfected with anything did not express NNV-CP protein, indicating that the mRNA synthesized using the scheme of the present invention can effectively produce the target antigen NNV-CP protein.

[0052] Example 2: Verification of mRNA vaccine safety and efficacy:

[0053] In order to comprehensively evaluate the safety of the mRNA vaccine of the present invention in preventing viral neuronecrosis and ensure the safety of its clinical application, a series of safety tests were conducted on the vaccine in accordance with the provisions of the "Chinese Veterinary Pharmacopoeia".

[0054] The experiment selected grouper weighing about 5g as experimental animals, and the mRNA vaccine injection dose used was 10μg.

[0055] The experimental design involved randomized grouping of fish into two groups, each consisting of six fish: a control group and an mRNA vaccine group. Groupers in the control group were injected intraperitoneally with PBS at a rate of 0.1 ml per grouper, while groupers in the vaccine group were injected intraperitoneally with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) at a rate of 0.1 ml / 10 μg per grouper.

[0056] During the trial, all experimental groups of zebrafish were closely monitored for clinical adverse reactions, and the number of dead fish was recorded daily for 7 days after immunization. In addition, the length and weight changes of fish in each group were regularly measured and recorded for 60 days after vaccination. Figure 4 It is known.

[0057] according to Figure 4 The results show that while the grouper fish in the control group were growing well, no fish mortality or adverse clinical reactions were observed in the first two groups vaccinated within seven days. Furthermore, there were no significant differences in fish length and weight after 60 days in the vaccinated groups compared to the control group. These results demonstrate the high safety of this mRNA vaccine and provide a scientific basis for its clinical application.

[0058] Example 3: Efficiency of mRNA vaccines in producing antibodies in grouper:

[0059] Groupers weighing approximately 25 grams were selected and divided into two groups, a control group and an mRNA vaccine group, according to the random block method, with 3 fish in each group.

[0060] Grouper in the control group were injected intraperitoneally with PBS solution at a rate of 0.1 ml / fish, while grouper in the vaccine group were injected intraperitoneally with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) at a rate of 0.1 ml / 10 μg / fish. Immunizations were repeated two weeks apart for two consecutive times. After a 28-day immunization period, blood was collected from the tail vein of all grouper and serum was isolated. Antibody titers against the NNV virus CP protein in the serum on day 28 were measured by ELISA. The results are shown in the figure below. Figure 5 shown.

[0061] according to Figure 5 The experimental results shown show that after a 28-day immunization period, the binding antibody titer in the serum of grouper vaccinated with mRNA vaccine was approximately 103-104, and the antibody positive conversion rate of this group was 100%.

[0062] Example 4: Application of mRNA vaccine in inhibiting NNV virus:

[0063] Groupers weighing about 25 grams were selected and divided into a control group and an mRNA vaccine group according to the random block method, with a total of 2 groups, 40 fish in each group.

[0064] The control group groupers were injected intraperitoneally with PBS solution at 0.1 ml / fish, and the vaccine group group groupers were injected intraperitoneally with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) at 0.1 ml / 10 μg / fish. The groupers were immunized once every two weeks for two consecutive times. After the 28-day immunization period, all groupers were injected intraperitoneally with 0.1 ml of 1×10 5 TCID50 / mL of NNV virus. During the subsequent observation period, the survival and death of groupers in each group were recorded every day, and survival curves were drawn based on these data to evaluate the protective effect of the vaccine. Figure 6 shown.

[0065] according to Figure 6 The experimental results shown in the figure show that grouper vaccinated with the NNV mRNA vaccine experienced a mortality rate of approximately 15% over a 20-day observation period. In contrast, the mortality rate of grouper in the unvaccinated negative control group reached 100% over the same period. This significant difference indicates that the NNV mRNA vaccine administered by injection has an approximately 85% protective efficacy against NNV infection in grouper.

[0066] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An mRNA based on the fish nervous necrosis virus CP protein gene, characterized in that: The mRNA consists of the following structure: a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and a polyA nucleic acid sequence. The nucleotide sequence of the mRNA is shown in SEQ ID NO:

1. The NNV virus antigen coding region contains the capsid protein CP gene of NNV virus infection. The amino acid sequence of the NNV virus antigen coding region is shown in SEQ ID NO:

5. The nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO: 3; The amino acid sequence of the signal peptide sequence is shown in SEQ ID NO: 4; The nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO:

6.

2. An mRNA vaccine for fish neuronecrosis virus, characterized by: The vaccine comprises The mRNA of the fish nervous necrosis virus CP protein gene according to claim 1.

3. The method for preparing the mRNA vaccine of fish nervous necrosis virus as claimed in claim 2, characterized in that: The steps include: (1) The mRNA template DNA consists of a T7 promoter, a 5' untranslated region, a signal peptide sequence, an NNV viral antigen coding region, a 3' untranslated region, and a polyA linker. The DNA template is synthesized by chemical synthesis; (2) T7 RNA polymerase and cap analogs were used to transcribe and cap the DNA chemically synthesized in the previous step as a template, and the mRNA of fish neural necrosis virus was obtained by purification by ethanol precipitation; The nucleotide sequence of the T7 promoter is shown in SEQ ID NO:

2.

4. Use of the mRNA vaccine of fish nervous necrosis virus as claimed in claim 2 in the preparation of anti-fish nervous necrosis virus drugs.

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