Fish nervous necrosis virus CP protein gene-based mRNA (messenger ribonucleic acid) and vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses mRNA based on a fish nervous necrosis virus CP protein gene, a vaccine and a preparation method and application of the mRNA. The invention provides mRNA (messenger Ribonucleic Acid) of a fish nervous necrosis virus, which has a nucleotide sequence as shown in SEQ ID NO: 1 and structurally comprises a 5'end untranslated region, a signal peptide sequence, an NNV (Neuronecrosis Virus) antigen coding region, a 3 'end untranslated region and polyA. The mRNA vaccine contains an ORF sequence of a capsid protein CP gene capable of resisting NNV virus infection. Animal safety test results of the fish nervous necrosis virus mRNA vaccine prepared by the invention show that the vaccine is safe, effective and convenient and simple in clinical use, and has a wide application prospect in the aspect of prevention and control of viral nervous necrosis of mariculture fishes.

Description

Technical Field

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

[0002] Fish Nervous Necrosis Virus (NNV) belongs to the genus Betanodavirus of the family Nodaviridae and is the main pathogen causing viral nervous necrosis (VNN). Since it was first discovered in Australia in the 1980s, NNV has become a serious threat to the global aquaculture industry. It has a very high mortality rate for fry and young fish, with a mortality rate of up to 100% within a week in severe cases. In recent years, the number of infected fish species and the degree of damage have increased rapidly. The virus particle of NNV is a non-enveloped icosahedral structure with a diameter of about 25 nanometers. Its genetic material consists of two positive single-stranded RNAs, namely RNA1 of 2970 base pairs (bp) and RNA2 of 1440 bp, and the 3' end of the RNA molecule does not contain a poly (A) sequence. Although NNV has been discovered for many years, the prevention and control measures for viral nervous necrosis still face challenges. In theory, vertical transmission can be prevented by screening broodstock that do not carry the virus, but due to the limitations of detection technology, it is difficult to ensure that the screened broodstock are completely virus-free. In addition, although disinfection with ozone and iodine reagents can reduce the horizontal transmission of the virus, the virus may exist both on the surface of the fertilized egg and inside the egg membrane, which means that surface disinfection alone cannot completely block the spread of the virus.

[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 transmit target gene information to host cells and stimulate the body to produce effective and 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 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 CP protein gene of fish neuronecrosis virus, a vaccine and a preparation method and application thereof, so as to solve the problem that fish are susceptible to NNV infection in aquaculture.

[0005] The present invention adopts the following technical scheme: an mRNA based on the CP protein gene of fish neuronecrosis virus, 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 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 A mRNA vaccine for fish nervous necrosis virus, comprising the mRNA of the CP protein gene of the fish nervous necrosis virus.

[0010] A method for preparing an mRNA vaccine of fish neuronecrosis virus comprises the following steps: (1) The template DNA of the mRNA is composed of a T7 promoter, a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and a polyA linker, and the DNA template is synthesized by chemical synthesis; (2) Using T7 RNA polymerase and cap analogs, the DNA chemically synthesized in the previous step was used as a template for T7 RNA transcription and capping, and purified by ethanol precipitation to obtain fish neuronecrosis virus mRNA.

[0011] Furthermore, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO:2.

[0012] The invention discloses an application of an mRNA vaccine of fish nervous necrosis virus and its application in the preparation of a drug against fish nervous necrosis virus.

[0013] The CP gene of NNV is essential for the viral infection cycle and the induction of host immune response. 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. NNV-CP protein also has a variety of strategies to inhibit the host's defense mechanism, including promoting the degradation of key immune proteins through the ubiquitination pathway, 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.

[0014] By expressing the CP gene of NNV in Escherichia coli, recombinant CP protein can be successfully synthesized, and it has been proven that it can stimulate the host's protective immune response. However, the antigenic properties of the NNV-CP gene in the host and in vitro may be different, which suggests that the antigenic properties of the virus in the host 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 virion are extremely critical to the vaccine effect. The surface antigenic sites of the NNV virion may change under certain conditions, such as heat treatment or in a specific solution, affecting its antigenicity and infectivity. Therefore, the NNV-CP gene plays a decisive role in viral infection, immune escape and vaccine development. In the present invention, the mRNA vaccine containing the NNV-CP gene was constructed by cloning the NNV-CP gene.

[0015] Beneficial effects of the present invention: (1) The mRNA vaccine for viral neuronecrosis 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 immunizing grouper, high-level antibodies are produced, and the antibodies have a high titer; (2) The mRNA vaccine for viral neuronecrosis prepared by the present invention has been proven to be safe, effective, and non-toxic through animal safety tests. The mRNA vaccine does not contain live virus components, so there is no risk of infection. It is degraded into nucleotides in the body and will not be integrated into the host genome. The mRNA vaccine can stimulate a strong immune response in the body, including humoral immunity and cellular immunity, and can effectively prevent viral infection. It has a good market application prospect in the marine aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural diagram of the mRNA of the fish nervous necrosis virus NNV-CP of the present invention.

[0017] Figure 2This is the detection of fish neuronecrosis virus NNV-CP mRNA of the present invention: Lane: mRNA vaccine.

[0018] Figure 3 The figure shows the expression of the fish neuronecrosis virus NNV-CP mRNA vaccine of the present invention in HEK293 cells.

[0019] Figure 4 It is the survival rate of grouper 7 days after injection of the fish neuronecrosis virus NNV-CP mRNA vaccine of the present invention.

[0020] Figure 5 It is the antibody titer against the CP protein of the NNV virus in the serum of grouper 28 days after immunization with the fish neuronecrosis virus NNV-CP mRNA vaccine of the present invention.

[0021] Figure 6 This is a diagram showing the effect of the fish neuronecrosis virus NNV-CP mRNA vaccine of the present invention in preventing and treating NNV virus in grouper. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment 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 creative work should fall within the scope of protection of the present invention.

[0023] Human embryonic kidney 293 cells (HEK293 cells) were grown adherently in a cell culture medium in a constant temperature incubator at 5% CO2 and 37°C. DMEM medium (Gibco) containing 10% fetal bovine serum, 100 U / ml penicillin and 100 mg / ml streptomycin was used as the cell culture medium for HEK293 cells.

[0024] The Nervous Necrosis Virus (NNV) in the present invention is stored in the National Aquatic Animal Pathogen Bank.

[0025] Example 1: Preparation of mRNA based on NNV-CP gene: (1) Synthesis of DNA template of mRNA based on 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 polyA linker. The DNA template is synthesized by chemical synthesis in the above order; The nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 2; 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 amino acid sequence of the NNV virus antigen coding region (NNV-CP gene amino acid sequence) is shown in SEQ ID NO:5; The nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO:6; and polyA nucleotide sequence is shown in SEQ ID NO:7; (2) T7 RNA transcription of NNV-CP gene mRNA: T7 RNA transcription and capping were performed using the synthesized DNA as a template; The specific steps are as follows: add 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; (3) mRNA purification: After the reaction is completed, the transcribed linear RNA is purified using ethanol precipitation; The specific steps are as follows: first, add 160 μL of nuclease-free pure water to expand the reaction volume to 180 μL; Then add 20 μL 3M sodium acetate (pH 5.2) or 20 μL 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 14000g for 5-10 minutes to obtain the supernatant), and then extract with chloroform 1-2 times (vortex mix for 20-30 seconds each time, then centrifuge at 14000g for 5-10 minutes to obtain the supernatant); Precipitate RNA with double volume of anhydrous ethanol, incubate at -20℃ for at least 30 minutes, then centrifuge at 14000g at 4℃ for 5-10 minutes to precipitate RNA, discard the supernatant, wash the precipitate with 500μL pre-cooled 70% ethanol, and resuspend and dissolve the RNA with 20μL of nuclease-free pure water.

[0026] Finally, the synthesized mRNA was detected by agarose gel electrophoresis; the results were as follows Figure 2 shown.

[0027] Depend on Figure 2 It can be seen that mRNA based on the NNV-CP gene has been successfully synthesized using the above method.

[0028] (4) Detection of the effect of synthesized NNV-CP gene mRNA on the production of target protein: HEK293 cells in the logarithmic growth phase were obtained and digested with trypsin solution, and then diluted with cell culture medium to a cell density of 2.5×10 5 cells / ml, forming a cell suspension, adding the cell suspension into a 12-well cell culture plate at 1 ml / well, and culturing in a cell culture incubator, wherein the CO2 volume content in the cell culture incubator is 5% and the temperature is 37°C; After 24 hours of culture, the cells were discarded, the supernatant was washed with sterile PBS, and then 1 ml / well of cell culture medium was added; then the synthesized mRNA with NNV-CP gene was transfected into the HEK293 cells using Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific). After 48 hours of transfection, the cell supernatant was removed and the cells were lysed using RIPA lysis buffer. After lysis, the protein samples were broken by ultrasound and centrifuged at 12000 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 protein.

[0029] 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 and transferred to the PVDF membrane. The transfer conditions were electrophoresis at 200mA for 2 hours. After the membrane transfer was completed, the PVDF membrane was blocked in a 5% skim milk powder solution for 1 hour to reduce nonspecific binding. After blocking, the NNV-CP antibody diluted 1:1000 was incubated with the membrane 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, and then the membrane was incubated with a 1:2000 dilution of 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; Finally, the developer was added and the chemiluminescent reaction on the membrane was detected to evaluate the efficiency of mRNA expression of NNV-CP; the results were shown in Figure 3 It is knowable.

[0030] 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.

[0031] Example 2: Verification of mRNA vaccine safety and efficacy: 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.

[0032] The experiment selected grouper weighing about 5g as experimental animals, and the mRNA vaccine injection dose used was 10μg.

[0033] In the experimental design, the fish were divided into two groups, the control group and the mRNA vaccine group, according to the random block method, with 6 fish in each group. The grouper in the control group was injected with PBS solution at 0.1 ml / fish, and the grouper in the vaccine group was injected with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher) at 0.1 ml / 10 μg / fish.

[0034] During the trial, zebrafish in all experimental groups 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 knowable.

[0035] according to Figure 4 The results shown in the figure show that while the grouper in the control group grew well, no fish deaths were observed in the first two groups vaccinated within 7 days, and no clinical adverse reactions were found. Moreover, there was no significant difference in fish length and weight after 60 days in the vaccinated group compared with the control group. These results show that the mRNA vaccine has a high safety profile and provides a scientific basis for its clinical application.

[0036] Example 3: Efficiency of mRNA vaccines in producing antibodies in grouper: Groupers weighing about 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.

[0037] The grouper in the control group was injected with PBS solution at 0.1 ml / fish, and the grouper in the vaccine group was injected with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher) at 0.1 ml / 10 μg / fish. The grouper was immunized once every two weeks, and immunized twice in succession. After the 28-day immunization period, blood was collected from the tail vein of all groupers and serum was separated. The antibody titer against NNV virus CP protein in the serum on the 28th day was detected by ELISA. The results are shown in Figure 5 shown.

[0038] according to Figure 5The 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%.

[0039] Example 4: Application of mRNA vaccine in inhibiting NNV virus: 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.

[0040] The groupers in the control group were injected with PBS solution at 0.1 ml / fish, and the groupers in the vaccine group were injected with a mixture of mRNA and Lipofectamine™ 3000 transfection reagent (Thermo Fisher) at 0.1 ml / 10 μg / fish. The groupers were immunized once every two weeks, and immunized twice in succession. 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.

[0041] according to Figure 6 The experimental results shown in the figure show that the mortality rate of grouper vaccinated with NNV virus mRNA vaccine was about 15% during the 20-day observation period. In contrast, the mortality rate of grouper in the negative control group that was not vaccinated reached 100% during the same period of time. This significant difference indicates that the NNV virus mRNA vaccine administered by injection has a protective efficacy of about 85% in preventing NNV virus infection in grouper.

[0042] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An mRNA based on the CP protein gene of fish neuronecrosis virus, characterized in that: The mRNA consists of the following structures: 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. The amino acid sequence of the NNV virus antigen coding region is shown in SEQ ID NO:

5.

2. The mRNA based on the fish nervous necrosis virus CP protein gene as claimed in claim 1, characterized in that: The nucleotide sequence of the 5' untranslated region is shown in SEQ ID NO:

3.

3. The mRNA based on the fish nervous necrosis virus CP protein gene as claimed in claim 1, characterized in that: The amino acid sequence of the signal peptide sequence is shown in SEQ ID NO:

4.

4. The mRNA based on the fish nervous necrosis virus CP protein gene as claimed in claim 1, characterized in that: The nucleotide sequence of the 3' untranslated region is shown in SEQ ID NO:

6.

5. An mRNA vaccine for fish neuronecrosis virus, characterized in that: The vaccine comprises The mRNA of the fish neuronecrosis virus CP protein gene according to claim 1.

6. The method for preparing the mRNA vaccine of fish neuronecrosis virus as claimed in claim 5, characterized in that: The steps include: (1) The template DNA of the mRNA is composed of a T7 promoter, a 5' untranslated region, a signal peptide sequence, an NNV virus antigen coding region, a 3' untranslated region and a polyA linker, and the DNA template is synthesized by chemical synthesis; (2) Using T7 RNA polymerase and cap analogs, the DNA chemically synthesized in the previous step was used as a template for T7 RNA transcription and capping, and purified by ethanol precipitation to obtain fish neuronecrosis virus mRNA.

7. The method for preparing the mRNA vaccine of fish neuronecrosis virus as claimed in claim 6, characterized in that: The nucleotide sequence of the T7 promoter is shown in SEQ ID NO:

2.

8. Use of the mRNA vaccine of fish nervous necrosis virus as claimed in claim 5 in the preparation of anti-fish nervous necrosis virus drugs.

Images