Grass carp reovirus type II mRNA (messenger ribonucleic acid) vaccine as well as preparation method and application thereof

Abstract

The invention discloses a grass carp reovirus type II mRNA (messenger Ribonucleic Acid) vaccine as well as a preparation method and application thereof, and belongs to the field of vaccines. According to the technical scheme, the grass carp reovirus type II mRNA vaccine comprises a modified mRNA chain, the modified mRNA chain sequence 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 termination codons, a 3' UTR sequence and a polyA nucleotide sequence. The GCRV II mRNA vaccine is applied to enhancing the GCRV infection resistance of a fish body, the death rate is reduced, and the blank of the current grass carp reovirus II mRNA vaccine is filled.

Description

Technical Field

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

[0002] As one of the "four major Chinese carps", grass carp (Ctenopharyngodon idella) is the fish with the highest aquaculture production in the country and an important part of China's agricultural economy. Although grass carp grows fast, its disease resistance is poor and it is easily infected by diseases, and various diseases cause huge economic losses to the aquaculture industry. Common grass carp diseases include grass carp enteritis, erythroderma, gill rot, and grass carp hemorrhage disease, etc. Among them, grass carp hemorrhage disease is the most harmful, with a very wide epidemic area, high incidence and mortality, seriously threatening the development of grass carp aquaculture.

[0003] Grass carp reovirus (GCRV) is the pathogen of grass carp hemorrhage disease. GCRV mainly infects grass carp within one year old, and occasionally infects two-year-old grass carp, which has a great impact on grass carp aquaculture. Diseased fish infected with GCRV will generally all die within about one day, and the disease develops very rapidly. In view of the characteristics of GCRV, from the perspectives of effectiveness, safety, environment, and ethics, vaccines are definitely the most suitable and concentrated method to control grass carp hemorrhage disease. Currently, the main types of vaccines include: inactivated vaccines, attenuated vaccines, protein subunit vaccines, nucleic acid vaccines, and genetically engineered live vector vaccines.

[0004] mRNA vaccine immunization belongs to nucleic acid vaccines and is one of the important and effective methods for preventing and treating viral diseases. It can introduce mRNA containing the encoded antigen protein into the body, directly translate it, form the corresponding antigen protein, thereby inducing the body to produce a specific immune response and achieving the effect of preventive immunization. The production process of mRNA vaccines is simple, without the need for cell culture or animal-derived matrices, with stronger specificity, higher effectiveness, shorter R & D cycles, and lower production costs. However, mRNA vaccines involve knowledge in many fields such as bioinformatics, biochemistry, material chemistry, and immunology, and the R & D difficulty is relatively large. Therefore, there are few reports on mRNA vaccines for fish at present, and the development of grass carp GCRV mRNA vaccines is still blank. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the technical problem to be solved by the present invention is to fill the blank of the current type II mRNA vaccine against grass carp reovirus, and to provide a type II mRNA vaccine against grass carp reovirus that enhances the ability of fish to resist GCRV virus infection and reduces the mortality rate, a preparation method thereof, and an application thereof.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A type II mRNA vaccine against grass carp reovirus, comprising: a modified mRNA strand; the sequence of the modified mRNA strand includes: a 5' UTR sequence, a Kozak sequence, a grass carp immunoglobulin μ 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 as shown in SEQ ID No.1, the Kozak sequence is as shown in SEQ ID No.2, the grass carp immunoglobulin μ heavy chain signal peptide sequence is as shown in SEQ ID No.3, the GCRV II outer capsid protein VP35 coding sequence is as shown in SEQ ID No.4, the sequence of the three stop codons is as shown in SEQ ID No.5, the 3' UTR sequence is as shown in SEQ ID No.6, and the polyA nucleic acid sequence is as shown in SEQ ID No.7.

[0007] Preferably, 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.

[0008] Preferably, the modified mRNA strand is encapsulated by nano-liposomes to form VP35-mRNA-LNP, and the encapsulation rate of the modified mRNA strand in VP35-mRNA-LNP > 90%.

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

[0010] Preferably, the raw materials for preparing nano-liposomes include: 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 lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester; the neutral lipid is distearoyl phosphatidylcholine; the polyethylene glycol lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0012] The present invention also provides a preparation method for any of the above-mentioned grass carp reovirus type II mRNA vaccines, including: constructing a plasmid; the construction of the plasmid includes: selecting the GCRV II outer capsid protein VP35 as the target antigen; codon-optimizing the coding sequence of the GCRV II outer capsid protein VP35 with fish as the host to obtain the mRNA strand sequence; inserting the mRNA strand sequence into the Escherichia coli plasmid vector pOK12 containing the T7 promoter to obtain a complete plasmid; codon-optimizing with fish as the host includes: adding the carp glyceraldehyde-3-phosphate dehydrogenase 5' UTR sequence, Kozak sequence, grass carp immunoglobulin μ heavy chain signal peptide coding sequence, 3 stop codons, gilthead seabream Ywhaz-like protein b 3' UTR sequence and polyA nucleic acid sequence to the coding sequence of the GCRV II outer capsid protein VP35; the polyA nucleic acid sequence serves as the tail of the mRNA strand sequence.

[0013] Preferably, the preparation method for the grass carp reovirus type II mRNA vaccine includes preparing a modified mRNA strand; preparing the modified mRNA strand includes: linearizing the complete plasmid with the restriction endonuclease BspQⅠ; in vitro transcribing the linearized DNA product with an in vitro transcription kit, adding a cap analog during in vitro transcription and replacing uridine triphosphate with N1-methyl-pseudouridine triphosphate.

[0014] Preferably, the preparation method for the grass carp reovirus type II mRNA vaccine includes preparing VP35-mRNA-LNP; preparing VP35-mRNA-LNP includes: dissolving the ionizable cationic lipid, cholesterol, neutral lipid and polyethylene glycol lipid with absolute ethanol, and preparing an organic phase according to the molar ratio of nitrogen atoms to phosphorus atoms of 7:1; diluting the modified mRNA strand with a sodium citrate solution as the aqueous phase; mixing the organic phase and the aqueous phase through microfluidics, and after dialysis, concentrating and replacing the buffer solution to obtain VP35-mRNA-LNP.

[0015] The present invention also provides an application method for any of the above-mentioned grass carp reovirus type II mRNA vaccines, and the grass carp is immunized by intramuscular injection at the base of the dorsal fin, the immunization injection volume is 50 μL, and the immunization dose is 10 μg / tail.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The present invention provides a grass carp reovirus type II mRNA vaccine that enhances the ability of fish to resist GCRV virus infection and reduces mortality. One day after injecting GCRV II virus into grass carp by intraperitoneal injection, the mortality rate of grass carp in the blank control group is 100%, while the mortality rate of grass carp in the immunized group injected with grass carp reovirus type II mRNA vaccine is 6.9%, and the relative protection rate is 93.1%. This greatly improves the ability of grass carp to resist GCRV virus infection and reduces the mortality rate of grass carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the plasmid electrophoresis diagram provided by the embodiment of the present invention;

[0019] Figure 2 It is the result diagram of detecting the in vitro expression of VP35-mRNA-LNP by Western blotting provided by the embodiment of the present invention;

[0020] Figure 3 It is the diagram of the change in the expression of immune-related genes in grass carp immunized with VP35 mRNA-LPN vaccine analyzed by quantitative PCR provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] Hereinafter, the technical solutions in the specific embodiments of the present invention will be described in detail and completely. Obviously, the described embodiments are only partial specific embodiments of the general technical solution of the present invention, rather than all embodiments. Based on the general concept of the present invention, all other embodiments obtained by those of ordinary skill in the art fall within the protection scope of the present invention.

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

[0023] The specific sequences are shown in Table 1.

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

[0025]

[0026]

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

[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] This solution can enhance translation efficiency, improve mRNA stability, and enhance immunogenicity. The coding sequence of the grass carp immunoglobulin μ heavy chain signal peptide can direct the protein translated from mRNA to a specific cellular location. In the vaccine, it can direct the antigen protein to reach the appropriate cellular site for expression and presentation, facilitating recognition by the immune system. At the same time, it can avoid mislocalization or degradation of the protein in the cell, ensuring the effective immunogenicity of the antigen protein; 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 to mRNA, precisely initiate the translation process, and help increase the expression level of the antigen protein, enabling the vaccine to 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 the cell to protect mRNA from degradation by nucleases, extend the half-life of mRNA in the cell, and enable mRNA to continuously express antigen proteins for a longer time, continuously stimulating the immune system, thereby enhancing immune memory and immune effects. Through the above sequence optimization, the mRNA vaccine can express antigen proteins more efficiently and stably in grass carp cells, and the expressed antigen proteins can be correctly localized and folded, maintaining good immunogenicity, so as to more effectively activate the immune system of grass carp, including inducing the production of specific antibodies and activating cellular immunity, providing more effective immune protection for grass carp.

[0030] In a preferred embodiment, the modified mRNA strand is encapsulated by nanoliposomes to form VP35-mRNA-LNP, and the encapsulation rate of the modified mRNA strand in VP35-mRNA-LNP > 90%.

[0031] It can protect mRNA after being encapsulated by nano-liposomes: mRNA is easily degraded by nucleases in the extracellular environment. Nano-liposomes can serve as a protective shell, encapsulating mRNA inside to protect it from nuclease attack, thereby enhancing the stability of mRNA, ensuring that it is not damaged during transportation and can reach the target cells smoothly; it can enhance cell uptake after being encapsulated by nano-liposomes: The structure of nano-liposomes is somewhat similar to that of cell membranes and can more easily fuse with cells or be taken up by cells through endocytosis. Nano-liposomes encapsulating mRNA can utilize this property to help mRNA enter the cell interior more efficiently, enabling mRNA to play a role inside the cell and translate the target antigen protein; it can enhance immunogenicity after being encapsulated by nano-liposomes: Nano-liposomes can change the distribution and metabolism of mRNA vaccines in the body, making them more easily taken up and processed by antigen-presenting cells, thereby enhancing the recognition and presentation ability of antigen-presenting cells to mRNA vaccines, activating immune cells, and improving the intensity and effect of the immune response.

[0032] In a preferred embodiment, the particle size of VP35-mRNA-LNP is 60 - 120 nm, and the particle size distribution PDI < 0.2. A suitable particle size can further enhance cell uptake. In this solution, the particle size of VP35-mRNA-LNP is 60 - 120 nm. Nano-liposomes of this size can more easily pass through the cell membrane, further enhancing the immune effect of VP35-mRNA-LNP; the particle size distribution PDI < 0.2, and the particle sizes of most particles are relatively close, with a small size difference. It can encapsulate mRNA more uniformly in the mRNA vaccine, enter the cell more stably, and has better stability and consistency.

[0033] In a preferred embodiment, the raw materials for preparing nano-liposomes 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 ester; the neutral lipid is distearoyl phosphatidylcholine; the polyethylene glycol lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

[0035] The present invention also provides a preparation method for any one of the above-mentioned grass carp reovirus type II mRNA vaccines, including: constructing a plasmid; constructing the plasmid includes: selecting the GCRV II outer capsid protein VP35 as the target antigen; codon-optimizing the GCRV II outer capsid protein VP35 coding sequence with fish as the host to obtain the mRNA strand sequence; inserting the mRNA strand sequence into the Escherichia coli plasmid vector pOK12 containing the T7 promoter to obtain a complete plasmid; codon-optimizing with fish as the host includes: adding the carp glyceraldehyde-3-phosphate dehydrogenase 5' UTR sequence, Kozak sequence, grass carp immunoglobulin μ heavy chain signal peptide coding sequence, 3 stop codons, gilthead seabream Ywhaz-like protein b 3' UTR sequence and polyA nucleic acid sequence to the GCRV II outer capsid protein VP35 coding sequence; the polyA nucleic acid sequence serves as the tail of the mRNA strand sequence.

[0036] In a preferred embodiment, the preparation method for the grass carp reovirus type II mRNA vaccine includes preparing a modified mRNA strand; preparing the modified mRNA strand includes: linearizing the complete plasmid with the restriction endonuclease BspQⅠ; in vitro transcribing the linearized DNA product with an in vitro transcription kit, adding a cap analog during in vitro transcription and replacing uridine triphosphate with 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 within cells, and the immune system can recognize foreign RNAs containing uridine and trigger an immune response. N1-methyl-pseudouridine is a modified nucleoside. Incorporating it into mRNA can reduce the likelihood of mRNA being recognized as a foreign substance 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, facilitating the movement of ribosomes on mRNA and the progress of 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 level of antigen proteins, and helping to improve the immune effect of the vaccine; N1-methyl-pseudouridine modification can improve the nuclease tolerance of mRNA, reduce the degradation of mRNA by nucleases, enable mRNA to exist more stably within 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 defines the linearization of the complete plasmid using the restriction endonuclease BspQⅠ. The reason is that after the restriction endonuclease BspQⅠ cuts DNA, the resulting ends have special properties. Except for base A, no other types of bases will appear at the ends. This special end property enables the provision of specific ligation sites in subsequent operations, reduces non-specific ligation, and improves accuracy and efficiency. Therefore, the restriction endonuclease BspQⅠ is selected for use.

[0038] In a preferred embodiment, the preparation method of the grass carp reovirus type Ⅱ mRNA vaccine includes preparing VP35-mRNA-LNP; preparing VP35-mRNA-LNP includes: dissolving ionizable cationic lipid, cholesterol, neutral lipid, and polyethylene glycol lipid with absolute ethanol, and preparing an organic phase according to a molar ratio of nitrogen atoms to phosphorus atoms of 7:1; diluting the modified mRNA strand with sodium citrate solution as the aqueous phase; mixing the organic phase and the aqueous phase through microfluidics, followed by dialysis, concentration, and buffer replacement to obtain VP35-mRNA-LNP. When the molar ratio of nitrogen atoms to phosphorus atoms is 7:1, the particle size and PDI value of the nanoparticles are ideal, resulting in good uniformity of the vaccine. At the same time, when immunizing animals, this ratio also shows better immunogenicity.

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

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

[0041] Example 1

[0042] 1. Preparation

[0043] 1.1 Samples

[0044] 1.1.1 Virus and cell line:

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

[0046] 1.1.2 Polypeptide:

[0047] The outer capsid protein VP35 of GCRV II, as a GCRV vaccine candidate antigen, has been previously demonstrated to have high immunogenicity. There are 36 polypeptides covering the GCRV II VP35 protein (Table 2). Each polypeptide has 20 amino acids, and the overlapping region between adjacent polypeptides is 10 amino acids. These polypeptides were all synthesized by Shanghai Genechem Co., Ltd.

[0048] Table 2 Peptide sequences

[0049]

[0050]

[0051] 1.2 Preparation method

[0052] The outer capsid protein VP35 of GCRV II, as a GCRV vaccine candidate antigen, has been previously demonstrated to have high immunogenicity. The antigen expression gene with optimized sequence was added with 5’UTR, signal peptide coding sequence, 3’UTR and polyA sequence, and then inserted into the pOK12 plasmid containing the T7 promoter. Then, the protein expression effect of the in vitro transcribed mRNA at the cellular level was verified. After that, liposome nanoparticles were prepared with four lipids (neutral lipid, ionizable cationic lipid, PEG-modified lipid, and cholesterol), and the VP35-mRNA was encapsulated by the liposome nanoparticles to form the VP35-mRNA-LNP vaccine with immunogenic effect.

[0053] 1.2.1 Plasmid construction

[0054] TM T7 HighYield RNA Synthesis Kit is an in vitro transcription kit. During in vitro transcription, a cap analog is added, and uridine triphosphate is replaced with N1-methyl-pseudouridine triphosphate. Further, the LiCl precipitation method is used to remove impurities such as proteins and salts in the RNA, obtaining high-purity RNA, which is placed in a -80°C refrigerator for standby.

[0055] 1.2.2 Preparation of VP35-mRNA-LNP

[0056] The ethanol injection method is adopted to prepare lipid nanoparticles (LNP). In this method, first, four lipids, namely ionizable cationic lipid (DLin-MC3-DMA), cholesterol, neutral lipid (DSPC), and PEG lipid (DMG-PEG 2000), are dissolved in absolute ethanol and mixed in a ratio of 50:38.5:10:1.5 to prepare the organic phase according to the ratio of N:P = 7; meanwhile, mRNA is 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 are mixed through microfluidics to prepare VP35-mRNA-LNP; after dialysis, concentration, and replacement of the buffer with 10% sucrose (10 mM Tris-HCl, pH 8.0), the final product of VP35-mRNA-LNP is obtained. This final product can be stored at -20°C for one year. Ribogreen (Quant-iT RiboGreen RNA kit, Invitrogen, USA) is used to detect the encapsulation rate of VP35-mRNA-LNP and the concentration of VP35-mRNA. The encapsulation rate > 90%, and the concentration of the vaccine is adjusted to 0.2 mg / mL with 10% sucrose. The Malvern laser particle size analyzer (Zetasizer Pro) is used to detect the particle size and particle size distribution of the sample. The particle size is between 60 nm and 120 nM, and the particle size distribution PDI < 0.2. The preparation of the VP35-mRNA-LNP vaccine mainly includes the following steps: preparing an organic phase containing four-component lipids and an aqueous phase containing mRNA, preparing VP35-mRNA-LNP through microfluidics technology, dialyzing VP35-mRNA-LNP, concentrating VP35-mRNA-LNP, filtering VP35-mRNA-LNP, replacing the buffer of VP35-mRNA-LNP, and performing quality detection on VP35-mRNA-LNP.

[0057] 2. Detection

[0058] 2.1 Plasmid construction detection

[0059] Select the outer capsid protein VP35 encoded by the GCRV gene fragment S11 of grass carp reovirus type II as the antigen target, insert the VP35 antigen gene sequence into the pok12 plasmid containing the 5'UTR, signal peptide coding sequence and 3'UTR sequence, and construct the mRNA vaccine. Pick monoclonal colonies and identify the recombinant plasmid using specific primers. The results show that the recombinant plasmid has been successfully constructed, as Figure 1 shown.

[0060] 2.2 Detection of mRNA-expressed proteins

[0061] In this experiment, the transfection reagent lipofectamine 2000 was used to transfect mRNA into 293T cells. After culturing for 24 h, the cells were collected, and the levels of mRNA-expressed proteins were analyzed 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 the cells not transfected with mRNA were used as negative controls. The cells transfected for 24 h were stained with a polyclonal antibody against VP35 protein. The results show that VP35-mRNA-LNP successfully expressed specific proteins in 48% of grass carp kidney cells, as Figure 2 shown.

[0064] 2.4 Immunogenicity detection of grass carp

[0065] In this experiment, 8-month-old grass carp were selected, and all immunization groups were immunized by intramuscular injection. The grass carp were anesthetized with 100 mg / L of MS-222 (Sigma, United States), and then immunized by intramuscular injection at the base of the dorsal fin. The immunization injection volume was 50 μL, and the immunization doses were 0 μg / tail (control group), 5 μg / tail (low-dose group), 10 μg / tail (medium-dose group), and 20 μg / tail (high-dose group) in sequence; the blank control group used Empty-LNP without encapsulating nucleic acid. The immunization was carried out in two times. After the first immunization, the second immunization was carried out again at an interval of two weeks. Each time, 0 μg / tail (control group), 5 μg / tail (low-dose group), 10 μg / tail (medium-dose group), and 20 μg / tail (high-dose group) were immunized respectively.

[0066] Take the blood and kidney tissues of grass carp after 28 days of the above immunization for immunogenicity detection. The immunogenicity detection includes the following aspects: binding antibody level, neutralizing antibody titer, and cytokine secretion level. The binding antibody titer is detected by enzyme-linked immunosorbent assay (ELISA), the neutralizing antibody titer is detected by plaque reduction neutralization test (PRNT), and the cytokine expression is detected by enzyme-linked immunospot assay (ELISpot).

[0067] 2.4.1 Detection of binding antibody titer and neutralizing antibody titer

[0068] Use VP35 protein as the coating antigen to detect the specific antibody response by ELISA. The vaccine produced a high level of VP35-binding antibodies. The serum of the VP35-mRNA-LNP immunized group had a strong binding ability to VP35 protein, and the serum end dilution was 78,000. Use the PRNT experiment to detect the inhibitory ability of the immune serum against GCRV II infection, and collect the serum of grass carp 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 a strong neutralizing ability against the virus, and the average PRNT50 was 12,000.

[0069] 2.4.2 Detection of cytokine expression

[0070] We used the Elispot method to detect the T cell response induced by the GCRV II mRNA vaccine, and used the kidney cells two weeks after the last immunization (28 days after the first immunization) for the experiment. Use 4 peptide libraries of VP35 and PBS to specifically stimulate the kidney lymphocytes, and Empty-LNP as the negative control. The results showed that the VP35-mRNA-LNP vaccine induced a strong T cell response. Under the stimulation of the P2 and P4 peptide libraries of VP35 protein, there were 300 - 1000 lymphocytes secreting IFN-γ cytokine / 10 6 Spleen lymphocytes.

[0071] 2.5 Immunodose optimization of VP35 mRNA vaccine

[0072] To further optimize the immunization dose of the mRNA vaccine, grass carp were inoculated with VP35-mRNA-LNP (20 μg, 10 μg, and 5 μg) on day 0 and day 14, respectively. Serum and kidneys of grass carp were collected on day 28 after the primary immunization to detect antibody responses and cytokine secretion levels. The experimental results showed that obvious antigen-specific antibody responses were generated in all immunization groups with different doses of VP35-mRNA-LNP. The end-point dilution of the binding antibody of the serum to the virus was 760,000, and there was no significant difference among the groups. It is worth noting that the serum of the VP35-mRNA-LNP vaccine immunization group could induce a high-titer neutralizing antibody response, with a PRNT50 of 12,600. The above results indicate that a low dose of the mRNA-LNP vaccine can induce a high level of antibody response.

[0073] The T cell response in the kidneys of mice was detected by the Elispot assay two weeks after the last immunization. Two peptide pools P2 and P4 of VP35 were used to specifically stimulate kidney lymphocytes to detect the GCRV II-specific T cell response. The results showed that strong specific T cell responses were generated in all three groups of the VP35-mRNA-LNP vaccine, and IFN-γ cytokines were produced under specific stimulation. In the VP35-mRNA-LNP immunization group, 200 - 600 cells of PBMC produced IFN-γ / 10 6 after renal cells, and 800 - 2000 PBMC produced IFN-γ / 10 under the stimulation of the P4 peptide pool 6 kidney lymphocytes. It is worth noting that the induced T cell response increased with the decrease in dose. Under the same conditions, the 10 μg immunization group secreted more IFN-γ cytokines. Based on the above results, it is advisable to select the 10 μg VP35-mRNA-LNP vaccine for subsequent immunoprotection experiments.

[0074] 2.6 Determination of immune antibody titer

[0075] In this experiment, 8-month-old grass carp were selected, and all immunization groups were immunized by intramuscular injection. Grass carp were anesthetized with 100 mg / L of MS-222 (Sigma, United States), and then immunized by intramuscular injection at the base of the dorsal fin. The immunization injection volume was 50 μL, and the immunization doses of the experimental groups were 10 μg / fish in sequence; the blank control group used Empty-LNP without encapsulating nucleic acid. Immunization was carried out in two times. After the first immunization, the fish were immunized again after a two-week interval.

[0076] At 4 weeks (day 28) after the first immunization, 6 fish were randomly selected from the experimental group and the control group for antibody titer determination. The sample collection method was referred to the methods established by Lin et al. (2007) and Yang et al. (2013). For the immunized grass carp, blood samples were collected from the caudal peduncle. The collected blood samples were left standing at room temperature for 2 h, and then placed at 4 °C overnight to allow natural coagulation. Finally, the samples were centrifuged at 750 g for 10 min using a cryogenic refrigerated centrifuge, and the upper serum was collected and stored at -20 °C for subsequent antibody titer determination.

[0077] During the determination of antibody titer, the purified VP35 protein was used as the antigen, the serum of the sampled grass carp mentioned above was used as the antibody, the artificially prepared rabbit anti-grass carp IgM antibody was used as the primary antibody, and the horseradish peroxidase-labeled goat anti-rabbit IgG was used as the secondary antibody. The dilution ratios of the primary antibody and the secondary antibody were both 1:1000. The color reaction was processed using a DAB horseradish peroxidase color development kit. After color development, the absorbance at a wavelength of 450 nm was measured using a Thermo Multiskan MK3 microplate reader (Molecular Devices Corp., Palo Alto, CA).

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

[0079] 2.7 Determination of immune-related physiological indexes

[0080] In this experiment, 8-month-old grass carp were selected, and all immunized groups were injected intramuscularly. The grass carp were anesthetized with 100 mg / L MS-222 (Sigma, United States), and then immunized by intramuscular injection at the base of the dorsal fin. The immunization injection volume was 50 μL. The immunization doses of the experimental groups were 10 μg / fish in sequence; the blank control group was injected with Empty-LNP without encapsulating nucleic acid. The immunization was carried out in two times. After the first immunization, the second immunization was carried out after an interval of two weeks.

[0081] Take the blood and kidney tissues of grass carp 28 days after the first immunization for the determination of immune-related physiological indexes. Six grass carps were sampled from the experimental group and the control group respectively, with 3 replicates, and 2 fish in each replicate. The determination indexes include: the respiratory burst activity of blood leukocytes, the activities of serum lysozyme, complement and superoxide dismutase. Among them, the blood leukocyte separation technique was the method established by Yeh et al. (2008), and the determination of the respiratory burst activity of blood leukocytes was the method established by Dan et al. (2013). The activities of serum lysozyme and complement were determined by the methods established by Yeh et al. (2008) and Sunyer et al. (1995), and measured by turbidimetry using a 96-well plate. The activities of superoxide dismutase (SOD) and alkaline phosphatase were determined using a superoxide dismutase assay kit and an alkaline phosphatase activity detection kit.

[0082] (1) Respiratory burst activity

[0083] The respiratory burst activity of the serum in the immunized group was 1.38 ± 0.25, and that in the control group was 0.56 ± 0.24. By calculation, P < 0.05, indicating that the respiratory burst activity of the serum of grass carp in the immunized group was significantly increased.

[0084] (2) Serum lysozyme activity

[0085] The serum lysozyme activity in the immunized group was 2.36 ± 0.15 g / L -1 , and that in the control group was 1.6 ± 0.25 g / L -1 . By calculation, P < 0.05, indicating that the serum lysozyme activity of grass carp in the immunized group was significantly increased.

[0086] (3) Complement activity

[0087] The complement activity in the immunized group was 1.28 ± 0.28 g / L -1 , and that in the control group was 0.76 ± 0.16 g / L -1 . By calculation, P < 0.05, indicating that the complement activity of grass carp in the immunized group was significantly increased.

[0088] (4) SOD activity

[0089] The SOD activity in the immunized group was 158 ± 5.2 U / mL -1 , and that in the control group was 129 ± 9.6 U / mL -1 . By calculation, P < 0.05, indicating that the SOD activity of grass carp in the immunized group was significantly increased.

[0090] (5) Alkaline phosphatase activity

[0091] The alkaline phosphatase activity in the immunized group was 2.56 ± 0.36 U / g -1, the alkaline phosphatase activity of the control group was 1.92 ± 0.19 U g -1 , by calculation, P < 0.05, and the alkaline phosphatase activity of grass carp in the immunization 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 immunization groups were injected intramuscularly. The grass carp were anesthetized with 100 mg / L of MS-222 (Sigma, United States), and then injected immunologically by intramuscular injection at the base of the dorsal fin. The immunization injection volume was 50 μL, and the immunization doses of the experimental groups were 10 μg / fish in sequence; the blank control group used Empty-LNP without encapsulating nucleic acid. The immunization was carried out in two times. After the first immunization, the second immunization was carried out again after an interval of two weeks.

[0094] The kidney tissues of grass carp 28 days after the first immunization were taken for the determination of immune-related gene expression. Six grass carp were sampled from each of the experimental group and the control group, with 3 parallels, and 2 fish in each parallel. The total RNA of the kidney samples was extracted by the Trizol method (Invitrogen, USA). The extracted total RNA samples were treated with DNase I (RNase Free) to remove genomic DNA contamination, and then the integrity of the RNA was detected by agarose gel electrophoresis, and the concentration and quality of the sample RNA were measured by a micro nucleic acid analyzer. The extracted RNA samples were reverse transcribed into cDNA using a reverse transcription kit. The primers for the determination of genes were designed with Prime 5 software according to the gene sequences in NCBI, and the primer synthesis was completed by Sangon Biotech (Shanghai). During the determination process, the 18S gene was used as an internal reference, and the specific primer sequences are shown in Table 3. The quantitative expression analysis of immune-related genes was carried out using a CFX96 real-time quantitative PCR instrument system (Bio-Rad, United States). The data obtained from the determination were analyzed by ΔΔCt (Livak and Schmittgen 2001).

[0095] Table 3 Primer sequences of genes detected by quantitative PCR

[0096]

[0097]

[0098] We selected genes related to immunity, including C-reactive protein (CRP) gene, type I interferon (IFN-I) gene, interferon-induced Mx-2 protein gene (Mx-2), tumor necrosis factor α (TNF-α) gene, interleukin 1β gene (IL-1β), interleukin 8 gene (IL-8), complement C3 gene (C3), immunoglobulin M (IgM) and D gene (IgD), CD8α receptor gene (CD8α), major histocompatibility complex I (MHC-I) and IIB gene (MHC-IIB). Our research showed that all the VP35-mRNA immunized groups were significantly up-regulated (P < 0.05), as Figure 3 shown.

[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 immunized groups were given intramuscular injection. The grass carp were anesthetized with 100 mg / L of MS-222 (Sigma, United States), and then immunized by intramuscular injection at the base of the dorsal fin. The immunization injection volume was 50 μL, and the immunization doses of the immunized groups were 10 μg / fish in sequence; the blank control group was given Empty-LNP without encapsulating nucleic acid. The immunization was carried out in two times. After the first immunization, the second immunization was carried out again after an interval of two weeks. At the 4th week after the first immunization, GCRV II virus was injected into the grass carp by intraperitoneal injection. The water temperature was controlled (25 ± 0.5 °C), and the morbidity was regularly checked and recorded. Finally, the mortality and immune protection rate of the grass carp were calculated, and the virus content in the blood and kidney of the grass carp was detected 1 day after the challenge. The virus infected in the blood of the control group grass carp could be detected after the challenge, and the average virus load was 103.8 copies / ml. The presence of GCRV II virus could not be detected in the blood of the immunized groups, indicating that the grass carp reovirus type II mRNA vaccine could completely clear the virus in the blood. In the kidney of the grass carp 1 day after the challenge, there was no residual virus in the immunized groups, while the average virus load in the control group was 106.5 copies / ml. One day after the challenge, the grass carp began to die. The mortality of the control group grass carp was 100%, while the mortality of the immunized group grass carp was 6.9%, and the relative protection rate was 93.1%.

Claims

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

7.

2. The grass carp reovirus type II mRNA vaccine according to claim 1, wherein 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.

3. The grass carp reovirus type II mRNA vaccine according to claim 1, characterized in that, The modified mRNA strand is encapsulated by nanoliposomes to form VP35-mRNA-LNP, and the encapsulation rate of the modified mRNA strand in VP35-mRNA-LNP > 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, wherein The raw materials for preparing the nanoliposomes include: ionizable cationic lipid, cholesterol, neutral lipid and polyethylene glycol lipid.

6. The grass carp reovirus type II mRNA vaccine according to claim 5, wherein 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-tetraene-19-yl ester; the neutral lipid is distearoyl phosphatidylcholine; the polyethylene glycol lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol.

7. The preparation method of the grass carp reovirus type II mRNA vaccine according to any one of claims 1-6, characterized in that, Comprising: Constructed plasmid; The constructed plasmid includes: Select GCRV II outer capsid protein VP35 as the target antigen; Codon-optimize the GCRV II outer capsid protein VP35 coding sequence with fish as the host to obtain the mRNA strand sequence; Insert the mRNA strand sequence into the Escherichia coli plasmid vector pOK12 containing the T7 promoter to obtain the complete plasmid; The codon optimization with fish as the host includes: adding the carp glyceraldehyde-3-phosphate dehydrogenase 5'UTR sequence, Kozak sequence, grass carp immunoglobulin μ heavy chain signal peptide coding sequence, 3 stop codons, Sparus aurata Ywhaz-like protein b 3'UTR sequence and polyA nucleic acid sequence to the GCRV II outer capsid protein VP35 coding sequence; the polyA nucleic acid sequence serves as the tail of the mRNA strand sequence.

8. The preparation method of the grass carp reovirus type II mRNA vaccine according to claim 7, characterized in that, Including the preparation of the modified mRNA strand; The preparation of the modified mRNA strand includes: The complete plasmid was linearized using the 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 preparation method of the grass carp reovirus type II mRNA vaccine according to claim 7, characterized in that, It includes the preparation of VP35-mRNA-LNP; The preparation of VP35-mRNA-LNP includes: Ionizable cationic lipids, cholesterol, neutral lipids and polyethylene glycol lipids were dissolved in absolute ethanol, and an organic phase was prepared according to a molar ratio of nitrogen atoms to phosphorus atoms of 7:1; The modified mRNA strand was diluted with a sodium citrate solution and used as the aqueous phase; The organic phase and the aqueous phase were mixed by microfluidics, dialyzed, concentrated and buffer exchanged to obtain VP35-mRNA-LNP.

10. The application method of the grass carp reovirus type II mRNA vaccine according to claim 1, characterized in that, Grass carp were immunized by intramuscular injection at the base of the dorsal fin, with an immunization injection volume of 50 μL and an immunization dose of 10 μg per fish.

Images