Grass carp reovirus type Ⅱ mRNA vaccine, preparation method and application thereof

Abstract

The present invention discloses a grass carp reovirus type II mRNA vaccine, its preparation method, and application, belonging to the field of vaccines. The technical solution comprises a grass carp reovirus type II mRNA vaccine, comprising: a modified mRNA chain; the modified mRNA chain sequence comprises: a 5' untranslated region (UTR) sequence, a Kozak sequence, a grass carp immunoglobulin μ heavy chain signal peptide coding sequence, a GCRV II capsid protein VP35 coding sequence, three stop codons, a 3' untranslated region (UTR) sequence, and a polyA nucleic acid sequence. The present invention is used to enhance fish resistance to GCRV infection and reduce mortality, filling the current gap in grass carp reovirus type II mRNA vaccines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of vaccines, and particularly relates to a grass carp reovirus type II mRNA vaccine, a preparation method and application thereof. BACKGROUND

[0002] As one of the four major carp in China, grass carp (Ctenopharyngodon idella) is the highest yield of fish in the country, and is an important part of China's agricultural economy. Although grass carp grows fast, it has poor disease resistance and is easily infected with diseases, which brings huge economic losses to the aquaculture industry. Common grass carp disease infections include grass carp enteritis disease, red skin disease, gill rot disease and grass carp hemorrhagic disease. Among them, grass carp hemorrhagic disease is the most serious, has a very wide prevalence, high incidence and mortality, and seriously threatens the development of grass carp aquaculture.

[0003] Grass carp reovirus (GCRV) is the pathogen of grass carp hemorrhagic disease. GCRV mainly infects grass carp within one year, and two-year-old grass carp is also occasionally infected, which has a great impact on the grass carp aquaculture industry. The infected fish generally die within one day, and the disease develops very rapidly. In view of the characteristics of GCRV, from the aspects of effectiveness, safety, environment and ethics, vaccine is absolutely the most appropriate and concentrated method to control grass carp hemorrhagic disease. At present, the vaccine types mainly include: inactivated vaccine, attenuated vaccine, protein subunit vaccine, nucleic acid vaccine and genetically engineered live vector vaccine.

[0004] mRNA vaccine immunization belongs to nucleic acid vaccine, which is one of the important and effective methods for preventing and treating viral diseases. The mRNA containing the coding antigen protein can be introduced into the body to directly translate and form the corresponding antigen protein, so as to induce the body to produce specific immune response and achieve the effect of preventive immunity. The mRNA vaccine has simple production process, does not need cell culture or animal source matrix, is more specific and effective, has shorter research and development cycle and lower production cost. However, the mRNA vaccine involves knowledge of biological information, biochemistry, material chemistry and immunology, and has great research and development difficulty. Therefore, there are few reports on the mRNA vaccine for fish, and the development of grass carp GCRV mRNA vaccine is still blank. SUMMARY

[0005] In view of the deficiencies in the prior art, the technical problem to be solved by the present application is to fill the blank of the grass carp reovirus type II mRNA vaccine, and to provide a grass carp reovirus type II mRNA vaccine which can enhance the fish body's resistance to GCRV virus infection and reduce the mortality, a preparation method and application thereof.

[0006] To solve the technical problem, the present application adopts the technical scheme of a grass carp reovirus type II mRNA vaccine, comprising: a modified mRNA chain; the sequence of the modified mRNA chain comprises: a 5'UTR sequence, a Kozak sequence, a grass carp immunoglobulin mu heavy chain signal peptide coding sequence, a GCRV II outer coat protein VP35 coding sequence, three stop codons, a 3'UTR sequence and a polyA nucleic acid sequence; the 5'UTR sequence is shown as SEQ ID No. 1, the Kozak sequence is shown as SEQ ID No. 2, the grass carp immunoglobulin mu heavy chain signal peptide sequence is shown as SEQ ID No. 3, the GCRV II outer coat protein VP35 coding sequence is shown as SEQ ID No. 4, the three stop codon sequences are shown as SEQ ID No. 5, the 3'UTR sequence is shown as SEQ ID No. 6, and the polyA nucleic acid sequence is shown as SEQ ID No. 7.

[0007] Preferably, the 5'UTR sequence is the 5' untranslated region of carp glycerol-3-phosphate dehydrogenase, and the 3'UTR sequence is the 3' untranslated region of goldfish Ywhaz-like protein b.

[0008] Preferably, the modified mRNA chain is wrapped by a nanoliposome to form VP35-mRNA-LNP, and the wrapping rate of the modified mRNA chain in the VP35-mRNA-LNP is >90%.

[0009] Preferably, the VP35-mRNA-LNP has a particle size of 60-120nm, and the particle size distribution PDI is <0.2.

[0010] Preferably, the nanoliposome preparation raw materials comprise: ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids.

[0011] Preferably, the ratio of ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids is 50:38.5:10:1.5; the ionizable cationic lipids are 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl; the neutral lipids are distearoyl phosphatidylcholine; and the polyethylene glycol lipids are 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0012] The application further provides a preparation method of any one of the grass carp reovirus type II mRNA vaccines, comprising: constructing a plasmid; the plasmid construction comprises: selecting GCRV II coat protein VP35 as a targeted antigen; performing codon optimization on the GCRV II coat protein VP35 coding sequence with fish as a host to obtain an mRNA chain sequence; inserting the mRNA chain sequence into an E. coli plasmid vector pOK12 containing a T7 promoter to obtain a complete plasmid; performing codon optimization with fish as a host, comprising: adding a carp glycerol-3-phosphate dehydrogenase 5'UTR sequence, a Kozak sequence, a grass carp immunoglobulin mu heavy chain signal peptide coding sequence, three stop codons, a Sparus aurata Ywhaz-like protein b 3'UTR sequence and a polyA nucleic acid sequence to the GCRV II coat protein VP35 coding sequence; and the polyA nucleic acid sequence is used as a tail of the mRNA chain sequence.

[0013] Preferably, the preparation method of the grass carp reovirus type II mRNA vaccine comprises preparing a modified mRNA chain; the preparation of the modified mRNA chain comprises: linearizing the complete plasmid by using a restriction endonuclease BspQ I; and performing in vitro transcription on the linearized DNA product by using an in vitro transcription kit, wherein a cap analog is added during the in vitro transcription, and uridine triphosphate is replaced by N1-methyl-pseudouridine triphosphate.

[0014] Preferably, the preparation method of the grass carp reovirus type II mRNA vaccine comprises preparing VP35-mRNA-LNP; the preparation of the VP35-mRNA-LNP comprises: dissolving ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids by using anhydrous ethanol, and preparing an organic phase according to a molar ratio of nitrogen atoms to phosphorus atoms of 7:1; diluting the modified mRNA chain by using a sodium citrate solution to obtain an aqueous phase; mixing the organic phase and the aqueous phase by using microfluidics, performing dialysis, concentrating and replacing a buffer to obtain the VP35-mRNA-LNP.

[0015] The application further provides an application method of any one of the grass carp reovirus type II mRNA vaccines, which is used for performing injection immunization on grass carp by using a dorsal fin base muscle injection method, the injection immunization volume is 50 muL, and the immunization dose is 10 mu g / tail.

[0016] Compared with the prior art, the application has the beneficial effects that:

[0017] The application provides a grass carp reovirus type II mRNA vaccine for enhancing the anti-GCRV virus infection ability of fish and reducing the mortality rate; after intraperitoneal injection of GCRV II virus into the grass carp body for one day, the mortality rate of the blank control group of grass carp is 100%, while the mortality rate of the immune group of grass carp injected with the grass carp reovirus type II mRNA vaccine is 6.9%, and the relative protection rate is 93.1%, which greatly improves the anti-GCRV virus infection ability of the grass carp and reduces the mortality rate of the grass carp. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The plasmid electrophoretogram provided by the embodiment of the application;

[0019] Figure 2 The Western blotting detection VP35-mRNA-LNP in vitro expression result graph provided by the embodiment of the application;

[0020] Figure 3 The quantitative PCR analysis VP35 mRNA-LPN vaccine immune grass carp immune related gene expression change graph provided by the embodiment of the application. DETAILED DESCRIPTION

[0021] The technical solutions in the specific embodiments of the application will be described in detail below. Obviously, the described embodiments are only part of the specific embodiments of the general technical solution of the application, but not all the embodiments. Based on the general concept of the application, all other embodiments obtained by those skilled in the art fall within the scope of protection of the application.

[0022] The application provides a grass carp reovirus type II mRNA vaccine, which comprises a modified mRNA chain; the sequence of the modified mRNA chain comprises a 5'UTR sequence, a Kozak sequence, a grass carp immunoglobulin mu heavy chain signal peptide coding sequence, a GCRV II outer capsid protein VP35 coding sequence, three stop codons, a 3'UTR sequence and a polyA nucleic acid sequence; the 5'UTR sequence is shown as SEQ ID No. 1, the Kozak sequence is shown as SEQ ID No. 2, the grass carp immunoglobulin mu heavy chain signal peptide sequence is shown as SEQ ID No. 3, the GCRV II outer capsid protein VP35 coding sequence is shown as SEQ ID No. 4, the three stop codon sequences are shown as SEQ ID No. 5, the 3'UTR sequence is shown as SEQ ID No. 6, and the polyA nucleic acid sequence is shown as SEQ ID No. 7.

[0023] The specific sequence is shown in Table 1.

[0024] Table 1 mRNA vaccine sequence list of grass carp reovirus type II

[0025]

[0026]

[0027] The grass carp reovirus type II mRNA vaccine disclosed in the scheme can significantly enhance the ability of grass carp to resist GCRV virus infection and reduce mortality.

[0028] In a preferred embodiment, the 5'UTR sequence is the 5' untranslated region of carp glyceraldehyde-3-phosphate dehydrogenase, and the 3'UTR sequence is the 3' untranslated region of Sparus aurata Ywhaz-like protein b.

[0029] The scheme can enhance translation efficiency, improve mRNA stability, and enhance immunogenicity. The grass carp immunoglobulin mu heavy chain signal peptide coding sequence can guide the translated protein of mRNA to target specific cell locations. In a vaccine, it can guide antigen proteins to reach the appropriate cell sites for expression and presentation, which is beneficial for the immune system to recognize. At the same time, it can avoid protein mislocalization or degradation in cells, ensuring that antigen proteins effectively play an immunogenic role. The 5'UTR sequence of carp glyceraldehyde-3-phosphate dehydrogenase contains specific elements that bind to translation initiation factors, which can improve the binding efficiency of ribosomes and mRNA, precisely initiate the translation process, and help increase the expression amount of antigen proteins, so that the vaccine can stimulate a stronger immune response. The 3'UTR sequence of Sparus aurata Ywhaz-like protein b contains some cis-acting elements that can interact with RNA-binding proteins in cells, protect mRNA from degradation by nucleases, extend the half-life of mRNA in cells, and enable mRNA to continuously express antigen proteins for a longer period of time, continuously stimulate the immune system, and thus enhance immune memory and immune effect. Through the above sequence optimization, the mRNA vaccine can more efficiently and stably express antigen proteins in grass carp cells, and the expressed antigen proteins can be correctly positioned and folded, maintaining good immunogenicity, thereby more effectively activating the immune system of grass carp, including inducing the production of specific antibodies, activating cellular immunity, and providing more effective immune protection for grass carp.

[0030] In a preferred embodiment, the modified mRNA chain is wrapped by a nanoliposome to form VP35-mRNA-LNP, and the wrapping rate of the modified mRNA chain in the VP35-mRNA-LNP is >90%.

[0031] The mRNA can be protected by the nanoliposome: mRNA is easily degraded by nucleases in the extracellular environment, the nanoliposome can serve as a protective shell to encapsulate mRNA inside, so that it is protected from nucleases, thereby improving the stability of mRNA and ensuring that it is not destroyed during transportation and can reach the target cells smoothly; the nanoliposome can enhance cell uptake: the structure of the nanoliposome has some similarity with the cell membrane, which can more easily fuse with the cell or be taken up by the cell through endocytosis, and the nanoliposome encapsulating mRNA can use this property to help mRNA enter the cell more efficiently, so that mRNA can function in the cell to translate the target antigen protein; the nanoliposome can enhance immunogenicity: the nanoliposome can change the distribution and metabolism of mRNA vaccine in the body, making it easier for antigen-presenting cells to take up and process, thereby enhancing the recognition and presentation ability of antigen-presenting cells to mRNA vaccine, activating immune cells, and improving the strength and effect of immune response.

[0032] In a preferred embodiment, the VP35-mRNA-LNP has a particle size of 60-120 nm and a particle size distribution PDI <0.2. A suitable particle size can further enhance cell uptake. In this scheme, the VP35-mRNA-LNP has a particle size of 60-120 nm, and such sized nanoliposomes are more easily passed through the cell membrane, further enhancing the immune effect of VP35-mRNA-LNP; the particle size distribution PDI <0.2, most of the particles have a relatively close particle size, and the size difference is small, which can more uniformly encapsulate mRNA in the mRNA vaccine and more stably enter the cell, having better stability and consistency.

[0033] In a preferred embodiment, the nanoliposome preparation raw materials include: ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids.

[0034] In a preferred embodiment, the ratio of ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids is 50:38.5:10:1.5; the ionizable cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28, 31-tetraen-19-yl; the neutral lipid is distearoyl phosphatidylcholine; and the polyethylene glycol lipid is 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0035] The application further provides a preparation method of any one of the grass carp reovirus type II mRNA vaccines, comprising: constructing a plasmid; the construction of the plasmid comprises: selecting GCRV II coat protein VP35 as a targeted antigen; performing codon optimization on the GCRV II coat protein VP35 coding sequence with fish as a host to obtain an mRNA chain sequence; inserting the mRNA chain sequence into an E. coli plasmid vector pOK12 containing a T7 promoter to obtain a complete plasmid; performing codon optimization with fish as a host, comprising: adding a carp glycerol-3-phosphate dehydrogenase 5'UTR sequence, a Kozak sequence, a grass carp immunoglobulin mu heavy chain signal peptide coding sequence, three stop codons, a Sparus aurata Ywhaz-like protein b 3'UTR sequence and a polyA nucleic acid sequence to the GCRV II coat protein VP35 coding sequence; and the polyA nucleic acid sequence is used as a tail of the mRNA chain sequence.

[0036] In a preferred embodiment, the preparation method of the grass carp reovirus type II mRNA vaccine comprises preparing a modified mRNA chain; the preparation of the modified mRNA chain comprises: linearizing the complete plasmid by using a restriction endonuclease BspQ I; and performing in vitro transcription on the linearized DNA product by using an in vitro transcription kit, wherein a cap analog is added during the in vitro transcription, and uridine triphosphate is replaced by N1-methyl-pseudouridine triphosphate. Replacing uridine triphosphate with N1-methyl-pseudouridine triphosphate can reduce immunogenicity, improve translation efficiency and enhance mRNA stability. Natural uridine exists in various RNAs in cells, and the immune system can recognize foreign RNA containing uridine and trigger an immune response, while N1-methyl-pseudouridine is a modified nucleoside, and its incorporation into mRNA can reduce the possibility of the mRNA being recognized as a foreign object by the immune system, reduce non-specific immune activation, reduce immune-related side effects, and make the mRNA vaccine safer; N1-methyl-pseudouridine modification can change the secondary structure of mRNA, making it more stable and flexible, which is conducive to the movement of ribosomes on mRNA and the translation process, at the same time, this modification can also enhance the interaction between mRNA and translation-related factors, thereby improving translation efficiency, increasing the expression amount of antigen proteins, and helping to improve the immune effect of the vaccine; N1-methyl-pseudouridine modification can improve the tolerance of mRNA to nucleases, reduce the degradation of mRNA by nucleases, enable mRNA to exist more stably in cells, continuously play a role, provide a more persistent template for the synthesis of antigen proteins, and further enhance the immune response.

[0037] The above technical solution limits the linearization of the intact plasmid to the restriction endonuclease BspQⅠ. The reason is that after the restriction endonuclease BspQⅠ cuts the DNA, the terminal conditions produced are special. Except for base A, no other types of bases will appear at the end. This special terminal property enables specific connection sites to be provided in subsequent operations, reducing nonspecific connection and improving accuracy and efficiency. Therefore, the restriction endonuclease BspQⅠ is chosen.

[0038] In a preferred embodiment, a method for preparing a grass carp reovirus type II mRNA vaccine includes preparing VP35-mRNA-LNP. The preparation of VP35-mRNA-LNP includes: dissolving an ionizable cationic lipid, cholesterol, a neutral lipid, and a polyethylene glycol lipid in anhydrous ethanol, preparing an organic phase at a nitrogen to phosphorus molar ratio of 7:1; diluting the modified mRNA strand with a sodium citrate solution to form an aqueous phase; mixing the organic and aqueous phases via microfluidics, dialysis, concentration, and buffer exchange to obtain VP35-mRNA-LNP. A nitrogen to phosphorus molar ratio of 7:1 results in an ideal nanoparticle size and PDI value, resulting in good vaccine uniformity. This ratio also demonstrates improved immunogenicity when immunized animals.

[0039] The present invention also provides an application method of any of the above grass carp reovirus type II mRNA vaccines, wherein the grass carp is immunized by intramuscular injection at the base of the dorsal fin, the injection volume is 50 μL, and the immunization dose is 10 μg / tail.

[0040] In order to more clearly and in detail introduce the grass carp reovirus type II mRNA vaccine, its preparation method and application provided by the embodiments of the present invention, the following description will be made in conjunction with specific examples.

[0041] Example 1

[0042] 1. Preparation

[0043] 1.1 Sample

[0044] 1.1.1 Viruses and cell lines:

[0045] The grass carp reovirus strain (GCRV097) was provided by the laboratory of the College of Fisheries, Huazhong Agricultural University, and the grass carp kidney cell line (CIK) was purchased from the Shenzhen Import and Export Inspection and Quarantine Bureau and preserved in this laboratory.

[0046] 1.1.2 Peptides:

[0047] GCRV II coat protein VP35 has been proven to be highly immunogenic as a GCRV vaccine candidate antigen. There are 36 polypeptides (Table 2) covering GCRV II VP35 protein, each polypeptide has 20 amino acids, and the overlapping region between adjacent polypeptides is 10 amino acids. These polypeptides are synthesized by Shanghai Jier Biochemical Company.

[0048] Table 2 Peptide sequence

[0049]

[0050]

[0051] 1.2 Preparation method

[0052] GCRV II coat protein VP35 has been proven to be highly immunogenic as a GCRV vaccine candidate antigen. GCRV II VP35 protein was selected as the target antigen, and the sequence-optimized antigen expression gene was added with 5'UTR, signal peptide coding sequence, 3'UTR and polyA sequence, and inserted into pOK12 plasmid containing T7 promoter; then the protein expression effect of in vitro transcribed mRNA was verified at the cell level; then four kinds of lipids (neutral lipid, ionizable cationic lipid, PEG-modified lipid and cholesterol) were used to prepare liposome nanoparticles, and the liposome nanoparticles were used to encapsulate VP35-mRNA to form VP35-mRNA-LNP vaccine with immunization effect.

[0053] 1.2.1 Plasmid construction

[0054] TM T7 HighYield RNA Synthesis Kit in vitro transcription kit, in vitro transcription, add cap analogs and replace uridine triphosphate with N1-methyl-pseudouridine triphosphate during in vitro transcription, further remove proteins and salts and other impurities in RNA by LiCl precipitation method, obtain high purity RNA, and place in -80°C refrigerator for standby.

[0055] 1.2.2 Preparation of VP35-mRNA-LNP

[0056] Liquid nanoparticles (LNP) were prepared by ethanol injection method. In this method, first, the ionizable cationic lipid (DLin-MC3-DMA), cholesterol, neutral lipid (DSPC) and PEG lipid (DMG-PEG 2000) were dissolved with anhydrous ethanol, mixed in a ratio of 50:38.5:10:1.5, and the organic phase was prepared according to the ratio of N:P = 7; at the same time, the mRNA was diluted to 0.1 mg / mL with a sodium citrate solution at pH 4.0 as the aqueous phase, and then the organic phase and the aqueous phase were mixed by microfluidization to prepare VP35-mRNA-LNP; after dialysis, the buffer was replaced with 10% sucrose (10 mM Tris-HCl, pH 8.0) to obtain the final product of VP35-mRNA-LNP. The final product can be stored at -20°C for one year. The Ribogreen (Quant-iT RiboGreen RNA kit, Invitrogen, USA) was used to detect the encapsulation rate of VP35-mRNA-LNP and the concentration of VP35-mRNA, the encapsulation rate was > 90%, and the concentration of the vaccine was adjusted to 0.2 mg / mL using 10% sucrose. The Malvern laser particle size analyzer (Zetasizer Pro) was used to detect the particle size and particle size distribution of the sample, the particle size was 60 nm to 120 nM, and the particle size distribution PDI < 0.2. The preparation of VP35-mRNA-LNP vaccine mainly includes the following steps: preparation of organic phase containing four-component lipids and aqueous phase containing mRNA, preparation of VP35-mRNA-LNP by microfluidization technology, dialysis of VP35-mRNA-LNP, concentration of VP35-mRNA-LNP, filtration of VP35-mRNA-LNP, replacement of VP35-mRNA-LNP buffer, and quality detection of VP35-mRNA-LNP.

[0057] 2. Detection

[0058] 2.1 Plasmid construction detection

[0059] The VP35 protein encoded by the S11 gene segment of grass carp reovirus type II (GCRV) was selected as the antigen target. The VP35 antigen gene sequence was inserted into the pok12 plasmid containing 5'UTR, signal peptide coding sequence, and 3'UTR sequence to construct an mRNA vaccine. Figure 1

[0060] 2.2 Detection of mRNA-expressed proteins

[0061] In this experiment, mRNA was transfected into 293T cells using the transfection reagent lipofectamine 2000. After 24 hours of culture, the cells were collected and analyzed for the level of mRNA-expressed proteins by Western-blot and Fluorescence activated cell sorting (FACS).

[0062] 2.3 In vitro expression detection of VP35-mRNA-LNP

[0063] To verify whether VP35-mRNA-LNP can be normally expressed in vitro, the above VP35-mRNA-LNP was transfected into grass carp kidney cells (CIK), and cells transfected with mRNA were used as negative controls. The cells were stained with VP35 protein polyclonal antibody 24 hours after transfection, and the results showed that VP35-mRNA-LNP successfully expressed specific proteins in 48% of grass carp kidney cells, as shown in Figure 2

[0064] 2.4 Detection of immunogenicity in grass carp

[0065] In this experiment, 8-month-old grass carp were selected, and all immunization groups were injected intramuscularly. The grass carp was anesthetized with 100 mg / L MS-222 (Sigma, United States), and then injected intramuscularly at the base of the dorsal fin. The injection volume was 50 μL, and the immunization doses were 0 μg / fish (control group), 5 μg / fish (low-dose group), 10 μg / fish (medium-dose group), and 20 μg / fish (high-dose group), respectively. The blank control group used Empty-LNP without nucleic acid wrapping. Immunization was performed twice, with a two-week interval between the first and second immunizations. Each time, 0 μg / fish (control group), 5 μg / fish (low-dose group), 10 μg / fish (medium-dose group), and 20 μg / fish (high-dose group) were immunized, respectively.

[0066] ​​The grass carp blood and kidney tissue 28 days after immunization were taken for immunogenicity detection, which included the following aspects: binding antibody level, neutralizing antibody titer, and cytokine secretion level. The binding antibody titer was detected by enzyme linked immunosorbent assay (ELISA), the neutralizing antibody titer was detected by plaque reduction neutralization test (PRNT), and the cytokine expression was detected by enzyme linked immunospot assay (ELISpot).

[0067] 2.4.1 Binding antibody titer and neutralizing antibody titer detection

[0068] ELISA experiment was performed using VP35 protein as coating antigen to detect specific antibody reaction. The vaccine produced high level of VP35 binding antibody, and the serum of the VP35-mRNA-LNP immunization group had strong binding force to VP35 protein, with a serum end dilution of 78,000. PRNT experiment was used to detect the inhibition ability of immune serum to GCRV II infection, and grass carp serum was collected two weeks after the last immunization. The experimental results showed that the immune serum of grass carp in the VP35-mRNA-LNP vaccine group had strong neutralizing ability to the virus, with an average PRNT50 of 12,000.

[0069] 2.4.2 Cytokine expression detection

[0070] We used Elispot method to detect T cell response induced by GCRV II mRNA vaccine, and took kidney cells two weeks after the last immunization (28 days after the first immunization) for experiment. The kidney lymphocytes were specifically stimulated by four peptide libraries of VP35 and PBS, respectively, and Empty-LNP was used as negative control. The results showed that VP35-mRNA-LNP vaccine induced strong T cell response. Under the stimulation of P2 and P4 peptide libraries of VP35 protein, 300-1000 lymphocytes in VP35-mRNA immunization group PBMC secreted IFN-γ cytokine / 106 spleen lymphocytes. 6 Spleen lymphocytes.

[0071] 2.5 Immunization dose optimization of VP35 mRNA vaccine

[0072] To further optimize the immune dose of mRNA vaccine, grass carp were vaccinated with VP35-mRNA-LNP (20, 10 and 5 pg) at day 0 and day 14, respectively, and serum and kidney were collected from grass carp at day 28 after the first immunization to detect the antibody response and cytokine secretion level. The experimental results showed that the different dose immunization groups of VP35-mRNA-LNP produced obvious antigen-specific antibody response, and the serum binding antibody end-point dilution to the virus was 760,000, with no significant difference between groups. Notably, the serum of VP35-mRNA-LNP vaccine immunization group could induce high-titer neutralizing antibody response, with PRNT50 of 12,600. The above results showed that low-dose mRNA-LNP vaccine could induce high levels of antibody response.

[0073] The Elispot experiment was used to detect the T cell response of mouse kidney two weeks after the last immunization. The kidney lymphocytes were specifically stimulated by two peptide libraries P2 and P4 of VP35, and GCRV II-specific T cell response was detected. The results showed that the three groups of VP35-mRNA-LNP vaccines all produced strong specific T cell response, and IFN-γ cytokines were produced under specific stimulation. In the VP35-mRNA-LNP immunization group, 200-600 kidney cells produced IFN-γ / 10 6 PBMC under the stimulation of P2 peptide library of VP35 protein, and 800-2000 PBMC produced IFN-γ / 10 6 kidney lymphocytes under the stimulation of P4 peptide library. Notably, the induced T cell response was enhanced with the decrease of the dose, and more IFN-γ cytokines were secreted in the 10 pg immunization group under the same conditions. In summary, 10 pg of VP35-mRNA-LNP vaccine was selected for the subsequent immunoprotection experiment.

[0074] 2.6 Immune antibody titer determination

[0075] In this experiment, 8-month-old grass carp were selected, and all immunization groups were injected intramuscularly. The grass carp was anesthetized with 100 mg / L MS-222 (Sigma, United States), and then injected and immunized by the dorsal fin base muscle injection method. The injection volume was 50 pL, and the immunization dose of the experimental group was 10 pg per tail, and the blank control group used Empty-LNP without nucleic acid wrapping. Immunization was performed twice, and the second immunization was performed two weeks after the first immunization.

[0076] At the fourth week after the first immunization (day 28), 6 fish from each of the experimental and control groups were used for antibody titer determination. The sample collection was based on the methods established by Lin et al. (2007) and Yang et al. (2013). The caudal peduncle of the immunized grass carp was used for blood collection. The collected blood samples were allowed to stand at room temperature for 2 h, and then were placed at 4°C overnight for natural coagulation. Finally, the blood samples were centrifuged at 750 g for 10 min using a low-temperature centrifuge, and the supernatant was collected and stored at -20°C for subsequent antibody titer determination.

[0077] During the antibody titer determination, the purified VP35 protein was used as the antigen, the serum of the sampled grass carp was used as the antibody, and the artificially prepared rabbit anti-grass carp IgM antibody was used as the primary antibody. The horseradish peroxidase-labeled goat anti-rabbit IgG was used as the secondary antibody, and the dilution ratio of the primary and secondary antibodies was 1:1000. The color development reaction was performed using the DAB horseradish peroxidase color reagent kit. After color development, the absorbance at a wavelength of 450 nm was determined using a Thermo Multiskan MK3 enzyme label instrument (Molecular Devices Corp., Palo Alto, CA).

[0078] The antibody titer of the immune group was 1.2 ± 0.25 mg / mL, and the antibody titer of the control group was 0.26 ± 0.12 mg / mL. Through calculation, it was found that P < 0.05, and the antibody titer of the immune group was significantly higher than that of the control group.

[0079] 2.7 Determination of immune-related physiological indicators

[0080] In this experiment, 8-month-old grass carp were selected, and all the immune groups were injected intramuscularly. The grass carp was anesthetized with 100 mg / L MS-222 (Sigma, United States), and then was injected intramuscularly at the base of the dorsal fin. The injection volume was 50 μL, and the immune dose of the experimental group was 10 μg per fish. The blank control group used Empty-LNP without nucleic acid wrapping. The immunization was performed twice, with an interval of two weeks between the first and second immunizations.

[0081] The grass carp blood and kidney tissue 28 days after the first immunization were taken for the determination of immune-related physiological indicators. Each group took 6 grass carps, 3 parallel, 2 fish per parallel. The determination indexes included: blood leukocyte respiratory burst activity, serum lysozyme activity, complement activity and superoxide dismutase activity. Among them, the blood leukocyte separation technology used the method established by Yeh et al. (2008), and the blood leukocyte respiratory burst activity determination used the method established by Dan et al. (2013). Serum lysozyme activity and complement activity used the method established by Yeh et al. (2008) and Sunyer et al. (1995), and were determined by turbidimetry using 96-well plates. Superoxide dismutase activity (SOD) and alkaline phosphatase activity were determined by superoxide dismutase assay kit and alkaline phosphatase activity detection kit.

[0082] (1) Respiratory burst activity

[0083] The serum respiratory burst activity of the immune group was 1.38±0.25, and the serum respiratory burst activity of the control group was 0.56±0.24, and the calculation showed P<0.05, the serum respiratory burst activity of the immune group was significantly improved.

[0084] (2) Serum lysozyme activity

[0085] The serum lysozyme activity of the immune group was 2.36±0.15 g L -1 , and the serum lysozyme activity of the control group was 1.6±0.25 g L -1 , and the calculation showed P<0.05, the serum lysozyme activity of the immune group was significantly improved.

[0086] (3) Complement activity

[0087] The complement activity of the immune group was 1.28±0.28 g L -1 , and the complement activity of the control group was 0.76±0.16 g L -1 , and the calculation showed P<0.05, the complement activity of the immune group was significantly improved.

[0088] (4) SOD activity

[0089] The SOD activity of the immune group was 158±5.2 U mL -1 , and the SOD activity of the control group was 129±9.6 U mL -1 , and the calculation showed P<0.05, the SOD activity of the immune group was significantly improved.

[0090] (5) Alkaline phosphatase activity

[0091] The alkaline phosphatase activity of the immune group was 2.56±0.36 U g -1The alkaline phosphatase activity of the control group was 1.92±0.19 U / g -1 By calculation, P<0.05, the alkaline phosphatase activity of the immune group was significantly increased.

[0092] 2.8 Determination of immune-related gene expression

[0093] In this experiment, 8-month-old grass carp were selected, and all the immune groups were injected intramuscularly. The grass carp was anesthetized with 100 mg / L MS-222 (Sigma, United States), and then injected intramuscularly at the base of the dorsal fin. The injection volume was 50 μL, and the immune dose of the experimental group was 10 μg per tail. The blank control group used Empty-LNP without nucleic acid wrapping. The immunization was performed twice, and after the first immunization, the second immunization was performed two weeks later.

[0094] The kidney tissue of the grass carp 28 days after the first immunization was used for immune-related gene expression determination. Six grass carps were sampled from each of the experimental and control groups, with three parallels and two fish per parallel. The total RNA of the kidney samples was extracted by Trizol method (Invitrogen, USA). The extracted total RNA sample was treated with DNase I (RNase Free) to remove genomic DNA contamination, and then detected for RNA integrity by agarose gel electrophoresis and measured for sample RNA concentration and quality by micro-nucleic acid analyzer. The extracted RNA sample was reversely transcribed into cDNA by reverse transcription kit. The gene primers were designed by referring to the gene sequence in NCBI using Prime 5 software, and the primer synthesis was completed by Shanghai Genechem Co., Ltd. During the determination, 18S gene was used as the internal reference, and the specific primer sequence is shown in Table 3. The quantitative expression analysis of immune-related genes was determined by CFX96 real-time quantitative PCR instrument system (Bio-Rad Company, USA). The determination data was analyzed by ΔΔCt (Livak and Schmittgen 2001).

[0095] Table 3 Primer sequence for quantitative PCR detection

[0096]

[0097]

[0098] We selected the C-reactive protein (CRP) gene, the type I interferon (IFN-I) gene, the interferon-induced Mx-2 protein gene (Mx-2), the tumor necrosis factor alpha (TNF-a) gene, the interleukin 1 beta gene (IL-1 beta), the interleukin 8 gene (IL-8), the complement C3 gene (C3), the immunoglobulin M (IgM) and D genes (IgD), the CD8 alpha receptor gene (CD8a), the major histocompatibility complex I (MHC-I) and IIB genes (MHC-IIB) related to immunity. Our study showed that the VP35-mRNA immunization group was significantly up-regulated (P<0.05), as shown in Table 2. Figure 3

[0099] 2.9 Detection of the protective effect provided by the vaccine

[0100] In this experiment, 8-month-old grass carp were selected, and all the immunization groups were injected intramuscularly. The grass carp was anesthetized with 100 mg / L MS-222 (Sigma, United States), and then injected intramuscularly at the base of the dorsal fin. The injection volume was 50 μL, and the immunization dose of the immunization group was 10 μg per fish. The blank control group used Empty-LNP without nucleic acid wrapping. Immunization was performed twice, with a two-week interval between the first and second immunizations. At 4 weeks after the first immunization, GCRV II virus was injected into the grass carp through intraperitoneal injection, the water temperature was controlled at (25±0.5) °C, and the incidence was checked and recorded regularly. Finally, the mortality rate of grass carp and the immune protection were calculated, and the virus content in the blood and kidney of grass carp was detected 1 day after the challenge. The infected virus in the blood of the control group could be detected after the challenge, and the average viral load was 103.8 copies / ml. No GCRV II virus was detected in the blood of the immunization group, indicating that the grass carp reovirus type II mRNA vaccine could completely eliminate the virus in the blood. One day after the challenge, there was no virus residue in the kidney of the immunization group, while the average viral load in the control group was 106.5 copies / ml. One day after the challenge, the grass carp began to die. The mortality rate of the control group of grass carp was 100%, while the mortality rate of the immunization group of grass carp was 6.9%, and the relative protection rate was 93.1%.​

Claims

1. A grass carp reovirus type II mRNA vaccine, characterized in that: include: modified mRNA strand; The modified mRNA chain sequence includes: 5'UTR sequence, Kozak sequence, grass carp immunoglobulin μ heavy chain signal peptide coding sequence, GCRV II outer capsid protein VP35 coding sequence, three stop codons, 3'UTR sequence and polyA nucleic acid sequence; The 5'UTR sequence is shown as SEQ ID No. 1, the Kozak sequence is shown as SEQ ID No. 2, the grass carp immunoglobulin μ heavy chain signal peptide sequence is shown as SEQ ID No. 3, the GCRV II outer capsid protein VP35 coding sequence is shown as SEQ ID No. 4, the three termination codon sequences are shown as SEQ ID No. 5, the 3'UTR sequence is shown as SEQ ID No. 6, and the polyA nucleic acid sequence is shown as SEQ ID No.

7.

2. The grass carp reovirus type II mRNA vaccine according to claim 1, characterized in that The 5'UTR sequence is the 5'untranslated region of carp glyceraldehyde-3-phosphate dehydrogenase, and the 3'UTR sequence is the 3'untranslated region of gilthead seabream Ywhaz-like protein b.

3. The grass carp reovirus type II mRNA vaccine according to claim 1, characterized in that The modified mRNA chain is encapsulated by nanoliposomes to form VP35-mRNA-LNP, and the encapsulation rate of the modified mRNA chain in VP35-mRNA-LNP is greater than 90%.

4. The grass carp reovirus type II mRNA vaccine according to claim 3, characterized in that The particle size of the VP35-mRNA-LNP is 60-120 nm, and the particle size distribution PDI < 0.

2.

5. The grass carp reovirus type II mRNA vaccine according to claim 3, characterized in that The raw materials for preparing the nano liposome include: ionizable cationic lipid, cholesterol, neutral lipid and polyethylene glycol lipid.

6. The grass carp reovirus type II mRNA vaccine according to claim 5, characterized in that The molar ratio of the ionizable cationic lipid, the cholesterol, the neutral lipid and the polyethylene glycol lipid is 50:38.5:10:1.5; The ionizable cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid; the neutral lipid is distearoylphosphatidylcholine; and the polyethylene glycol lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

7. The method for preparing the grass carp reovirus type II mRNA vaccine according to any one of claims 1 to 6, characterized in that: include: construct plasmids; The constructed plasmid comprises: GCRV II outer capsid protein VP35 was selected as the target antigen; The coding sequence of GCRV II outer capsid protein VP35 was codon-optimized using fish as the host to obtain the mRNA chain sequence; The mRNA chain sequence was inserted into the E. coli plasmid vector pOK12 containing the T7 promoter to obtain a complete plasmid; The codon optimization using fish as a host comprises: adding a 5'UTR sequence of carp glyceraldehyde-3-phosphate dehydrogenase, a Kozak sequence, a grass carp immunoglobulin μ heavy chain signal peptide coding sequence, three stop codons, a gilthead seabream Ywhaz-like protein b 3'UTR sequence and a polyA nucleic acid sequence to a GCRV II outer capsid protein VP35 coding sequence; the polyA nucleic acid sequence serves as the tail of the mRNA chain sequence.

8. The method for preparing the grass carp reovirus type II mRNA vaccine according to claim 7, characterized in that: including preparing a modified mRNA chain; The preparation of the modified mRNA chain comprises: The complete plasmid was linearized using restriction endonuclease BspQⅠ; The linearized DNA product was in vitro transcribed using an in vitro transcription kit, and a cap analog was added during in vitro transcription, and uridine triphosphate was replaced with N1-methyl-pseudouridine triphosphate.

9. The method for preparing the grass carp reovirus type II mRNA vaccine according to claim 7, characterized in that: including preparing VP35-mRNA-LNP; The preparation of VP35-mRNA-LNP comprises: Ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids are dissolved in anhydrous ethanol, and an organic phase is prepared in a molar ratio of nitrogen atoms to phosphorus atoms of 7:1; The modified mRNA chain was diluted with sodium citrate solution as the aqueous phase; The organic phase and aqueous phase were mixed by microfluidics, dialyzed, concentrated and the buffer was replaced to obtain VP35-mRNA-LNP.

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