Encephalomyocarditis multi-epitope recombinant protein and its application in the preparation of encephalomyocarditis vaccine

Abstract

The present invention belongs to the technical field of vaccines and relates to an encephalomyocarditis multi-epitope recombinant protein, and the encephalomyocarditis multi-epitope recombinant protein comprises 8 B-cell epitopes and 1 signal peptide. The present invention also provides the application of the recombinant protein in the preparation of an encephalomyocarditis vaccine. With the goal of developing a safe and effective vaccine, the B-cell epitopes of VP1, VP2, and VP3 of the EMCV structural protein are screened, and a recombinant epitope protein with good hydrophilicity, strong antigenicity, and high surface accessibility is constructed. By immunizing BALB / c mice, it is found that it can induce the production of neutralizing antibodies and a Th1-dominated cellular immune response, and can protect the mice from EMCV attack. The results show that the MIgH-EMCV-II<supgt;2D2< / supgt; protein may be a potential candidate for developing a safe and effective multi-epitope vaccine to control EMCV infection.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccines, and in particular relates to a multi-antigen epitope recombinant protein of encephalomyocarditis and an application thereof in preparing an encephalomyocarditis vaccine. Background Art

[0002] Encephalomyocarditis virus (EMCV) is a small, non-enveloped, positive-sense single-stranded virus belonging to the genus Cardiovirus in the family Picornaviridae. It is a zoonotic pathogen. The virus has a wide host range and is endemic. Under natural conditions, EMCV can infect livestock, rodents, wild animals, and primates. Rodents are its natural reservoir and main transmitter. Mice are highly susceptible and are the most commonly used experimental animal model. The most widely infected and most seriously endangered livestock are pigs, which can cause miscarriage, stillbirth, and weak piglets in pregnant sows, and fatal myocarditis and encephalitis in piglets. Many countries and regions have reported outbreaks of the disease in pig herds.

[0003] The viral genome is about 7.8 kb in length and encodes a large open reading frame (ORF). During the formation of viral particles, the precursor protein of EMCV is eventually cleaved into structural proteins VP1, VP2, VP3, VP4 and non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C, and 3D. The structural proteins VP1, VP2, and VP3 are located on the surface of the viral particles, while VP4 is located on the inside of the capsid and close to the VP1-VP2-VP3 complex. These four proteins are all involved in the formation of viral antigenic sites. Among them, VP1 is the main structural protein of EMCV, located on the surface of the viral capsid, and contains the main antigenic epitopes of EMCV. Viral non-structural proteins mainly encode viral genome replication and processing. 2A and 3C encode proteases that are involved in polyprotein processing and cleavage, 2B and 2C are mainly responsible for cell membrane permeability and rearrangement of intracellular membranes, 3A is a membrane-bound protein that inhibits cellular protein secretion and mediates membrane protein transport, and 3B is covalently bound to the 5' end of the viral genome and, as the smallest protein encoded by the virus, cooperates with 3D in viral replication.

[0004] One of the main measures to prevent infectious diseases is vaccination. However, there is currently no effective vaccine for EMCV, so the development of a new vaccine is urgent. The advancement of genetic engineering technology and bioinformatics tools has provided a new direction for the research and development of new vaccines, among which epitope-based vaccines are a hot research topic. Compared with traditional inactivated vaccines, it is safer and more stable. It is constructed by integrating multiple B cell or T cell epitopes on viral proteins into expression vectors. Multi-epitope vaccines can effectively stimulate the body to produce an immune response, which is beneficial for controlling viral infection. It has been widely used in the prevention and control of viruses such as FMDV, HIV, HBV, HCV, SARS-CoV-2 and MPX. However, there are few studies on the identification of EMCV epitopes. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a multi-epitope recombinant protein of encephalomyocarditis and its application in the preparation of encephalomyocarditis vaccine. The present invention intends to screen the B cell epitopes of the structural proteins VP1, VP2, and VP3 of the EMCV PV21 strain (GenBank: X74312), construct a multi-epitope gene of encephalomyocarditis virus and optimize the codon of the gene, synthesize the recombinant plasmid and transfect it into CHO cells for expression. The immunogenicity of the EMCV multi-epitope recombinant protein is explored through animal immunization tests and virus challenge tests, providing new ideas for the prevention and treatment of EMCV.

[0006] The first object of the present invention is to provide a multi-epitope recombinant protein for encephalomyocarditis, wherein the multi-epitope recombinant protein for encephalomyocarditis comprises the following 8 B cell antigen epitopes and 1 signal peptide;

[0007] The 8 B cell antigen epitopes are: VP2 70-76 aa 、VP3 135-142 aa 、VP1 26-33 aa 、VP1 61-68 aa 、VP1 94-101 aa 、VP1 150-156 aa 、VP1 206-214aa 、VP1 259-272 aa ;

[0008] The VP2 70-76 aa The amino acid sequence is: WTSTQKP;

[0009] The VP3 135-142 aa The amino acid sequence is: KPTSRDQA;

[0010] The VP1 26-33 aa The amino acid sequence is: LPENQTKV;

[0011] The VP1 61-68 aaThe amino acid sequence is: SNKTCPNS;

[0012] The VP1 94-101 aa The amino acid sequence is: GNGNEETS;

[0013] The VP1 150-156 aa The amino acid sequence is: TPTKPTT;

[0014] The VP1 206-214aa The amino acid sequence is: KRFDNTGSL;

[0015] The VP1 259-272 aa The amino acid sequence is: PTSGDKIDMTPRAG;

[0016] The amino acid sequence of the signal peptide is: MNFGLSLIFLVLILKGVQC;

[0017] Preferably, the VP2 70-76 aa 、VP3 135-142 aa 、VP1 26-33 aa 、VP1 61-68 aa 、VP1 94-101 aa 、VP1 150-156 aa 、VP1 206-214aa 、VP1 259-272 aa Connect in series.

[0018] Preferably, the encephalomyocarditis multi-antigen epitope recombinant protein comprises a T cell epitope and a connecting peptide, and the signal peptide is connected to the B cell antigen epitope via the T cell epitope and the connecting peptide;

[0019] Preferably, the amino acid sequence of the T cell epitope is: ISISEIKGVIVHKIEGILF;

[0020] Preferably, the amino acid sequence of the connecting peptide between the T cell epitope and the B cell antigen epitope is: KK;

[0021] Preferably, the amino acid sequence of the connecting peptide between different B cell antigen epitopes is: GGGGS.

[0022] Preferably, the amino acid sequence of the encephalomyocarditis multi-antigen epitope recombinant protein is SEQ ID NO.3 in the sequence listing.

[0023] The second object of the present invention is to provide a gene encoding the above-mentioned encephalomyocarditis multi-antigen epitope recombinant protein;

[0024] Preferably, the gene sequence encoding the encephalomyocarditis multi-antigen epitope recombinant protein is SEQ ID NO.4 in the sequence listing.

[0025] The third object of the present invention is to provide an expression cassette, a recombinant vector or a cell containing the above gene;

[0026] Preferably, the expression cassette is pcDNA3.1;

[0027] Preferably, the cells are CHO cells.

[0028] The fourth object of the present invention is to provide a method for culturing the above-mentioned cells, wherein the above-mentioned cells are cultured in SFM4CHO medium, and during the culture period, feed medium Cell Boost 5 and glucose are supplemented;

[0029] Preferably, the amount of feed medium Cell Boost 5 added is 2.5-7.5% of the volume of SFM4CHO basal medium;

[0030] Preferably, when the glucose concentration in the culture medium is lower than 3.5 g / L, glucose is added to 7.0 g / L.

[0031] The fifth object of the present invention is to provide the use of the above-mentioned recombinant protein, gene, expression cassette, recombinant vector or cell in the preparation of encephalomyocarditis vaccine.

[0032] Preferably, the single dose of the vaccine is: based on a mouse weighing (18.0±2.0) g, the single immunization dose is 50-250 μg / mouse; preferably, the single immunization dose is 250 μg / mouse; the vaccine is used for immunization one to two times.

[0033] The sixth object of the present invention is to provide a vaccine, which comprises the above-mentioned recombinant protein, gene, expression cassette, recombinant vector or cell.

[0034] Preferably, the vaccine comprises an adjuvant; preferably, the adjuvant is Freund's complete adjuvant and Freund's incomplete adjuvant; and / or

[0035] The single dosage of the vaccine is: based on a mouse weighing (18.0±2.0) g, the single immunization dose is 50-250 μg / mouse; preferably, the single immunization dose is 250 μg / mouse; the vaccine is used for immunization one to two times.

[0036] The present invention aims to develop a safe and effective vaccine, screens the B cell epitopes of VP1, VP2, and VP3 of the EMCV structural protein, and constructs a recombinant epitope protein with good hydrophilicity, strong antigenicity, and high surface accessibility. By immunizing BALB / c mice, it was found that it can induce the production of neutralizing antibodies and Th1-dominated cellular immune responses, and can protect mice from EMCV attacks. The results showed that MIgH-EMCV-Ⅱ 2D2The protein may be a potential candidate for developing a safe and effective multi-epitope vaccine to control EMCV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0038] Figure 1 Schematic diagram for the construction of EMCV multi-epitope vector.

[0039] Figure 2 Prediction of secondary structure and antigenic epitopes of VP1, VP2 and VP3 proteins.

[0040] Figure 3 For identification and expression of recombinant plasmids.

[0041] Figure 4 For the screening and preparation of monoclonal cells.

[0042] Figure 5 To study the effects of sugar-supplemented and fed-culture on the growth of engineered cell lines and the expression of recombinant proteins.

[0043] Figure 6 This is the ultrafiltration result of the recombinant protein.

[0044] Figure 7 For the determination of neutralizing antibodies and cytokine IFN-γ.

[0045] Figure 8 The protective effect of EMCV multi-epitope candidate vaccine on mice.

[0046] Fig. 9 The figures are the results of the determination of virus titers in mouse serum and different tissues.

[0047] Fig.10 The effect of immune dose on immune effect.

[0048] Fig.11 The impact of immunization procedures on immune effects. DETAILED DESCRIPTION

[0049] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0050] Example 1

[0051] 1 Materials and methods

[0052] 1.1 Materials

[0053] 1.1.1 Cells, viruses, animals, and ethical statement

[0054] The CHO-K1-S2 cells, BHK-21, EMCV PV21 strain (GenBank accession number: X74312), and Escherichia coli competent cells BL21 required by the present invention are all purchased from ATCC. CHO-K1-S2 cells are cultured at a constant temperature in a 37°C, 5% CO2 shaker, and the required culture medium is SFM4CHO culture medium. BHK21 cells are cultured at a constant temperature in a 37°C, 5% CO2 incubator, and the required culture medium is a high glucose (DMEM) culture medium containing 10% newborn calf serum. The processes of cell recovery, culture, and passage must strictly follow the principle of sterility and be operated in a clean bench. 6-8 week old healthy BALB / c female mice were purchased from Lanzhou Veterinary Research Institute. The present invention has been approved by the Animal Welfare and Ethical Review Committee of Northwest Minzu University (CAU approval number: xbmu-sm-202367).

[0055] 1.1.2 Reagents

[0056] SFM4CHO basal medium and feed medium Cell Boost 5 (CB5) were purchased from Cytiva, USA. New bovine serum (New Bovine Serum) was purchased from Lanzhou Minhai Bioengineering Co., Ltd., high-glucose DMEM medium was purchased from Lanzhou Bailing Biotechnology Co., Ltd., HRP-labeled His tag antibody was purchased from Jackson, USA. G418 selective antibiotics were purchased from Gibco, USA. Mouse IFN-γ ELISA Kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd.

[0057] 1.1.3 Instruments and equipment

[0058] PowerPac Basic electrophoresis instrument was purchased from Bio-Rad, USA; Amersham Imager 600 electrophoresis gel imaging system was purchased from GE Healthcare Bio-Sciences AB, USA; MK3 microplate reader was purchased from Thermo, USA; and Countstar automatic cell counter was purchased from Shanghai Ruiyu Biotechnology Co., Ltd.

[0059] 1.2 Methods

[0060] 1.2.1 Screening of epitope genes

[0061] The amino acid sequences of VP1, VP2, and VP3 proteins of EMCV PV21 strain (GenBank accession number: X74312) were screened from NCBI, and the amino acid sequences of the above proteins were selected for analysis. The potential dominant epitopes of B cells were screened by using the online prediction website http: / / tools.iedb.org / bcell / and the protean module in DNAstar.

[0062] 1.2.2 Screening of signal peptides

[0063] Signal peptide (SP) is a short peptide chain located at the N-terminus of secretory proteins that can guide the transfer of newly synthesized proteins to the secretory pathway. SPs serve as signals to guide protein secretion and protein target location. In addition to protein targeting, it is also used in various fields such as recombinant protein production, disease diagnosis, and vaccination. Different exogenous proteins need to be expressed in mammalian cells through different signal peptides. Even if the same protein can be secreted and expressed under the action of different signal peptides, its secretion efficiency will vary greatly. Therefore, selecting a suitable signal peptide will help improve the secretory expression ability of CHO cells. The present invention selected 6 signal peptides as shown in Table 1 for subsequent experiments.

[0064] Table 1 Amino acid sequence and source of signal peptide

[0065]

[0066] 1.2.3 Construction of recombinant plasmid

[0067] The screened dominant antigen epitopes were concatenated through Linker (GGGGS), and the order of concatenation was: VP2-1 / VP2-2 / VP2-3 / VP2-4, VP3, VP1-1, VP1-2, VP1-3, VP1-4, VP1-5, and VP1-6, obtaining four concatenated antigen epitope sequences.

[0068] In order to enhance the immunogenicity of the recombinant protein and induce an effective immune response, a universal helper T cell epitope (amino acid sequence: ISISEIKGVIVHKIEGILF) was added to the N-terminus, and a signal peptide was introduced to the N-terminus of the T cell epitope to increase the secretion expression of the recombinant protein. At the same time, the kozak sequence (GCCACC) was inserted before the signal peptide sequence to improve the translation efficiency of the eukaryotic gene. In order to facilitate subsequent purification, a His tag and a stop codon were added to the C-terminus; then the recombinant protein gene sequence was optimized according to the codon preference of CHO cells, and the optimized sequence was inserted into the eukaryotic expression vector pcDNA3.1 Afl II and EcoR Ⅴ restriction site to prepare the recombinant plasmid.

[0069] The sequence design is shown below: Figure 1 shown.

[0070] Figure 1 Schematic diagram for the construction of EMCV multi-epitope vector.

[0071] Depend on Figure 1 It can be seen that the binding mode is: upstream restriction site Afl Ⅱ+Kozak sequence+signal peptide+universal T cell epitope+Linker1+VP2 B cell epitope+Linker2+VP3 B cell epitope+Linker2+VP1 B cell epitope+Linker2+6×His tag+downstream restriction site EcoR Ⅴ.

[0072] Among them, Linker1 represents the Linker connecting the T cell epitope and the B cell epitope, and the amino acid sequence is: KK; Linker2 represents the Linker connecting different B cell epitopes, and the amino acid sequence is: GGGGS.

[0073] In this experiment, the tandem antigen epitope sequence containing VP2-1 (named EMCV-Ⅰ) was first used to combine with the 6 different signal peptides in Table 1, and then codons were optimized and recombinant plasmids were synthesized by Nanjing GenScript. These 6 plasmids were named MIgH-EMCV-Ⅰ, Azu-EMCV-Ⅰ, tPA-EMCV-Ⅰ, VSV-G-EMCV-Ⅰ, MIgκ-EMCV-Ⅰ, and Alb-EMCV-Ⅰ.

[0074] The best signal peptide (MIgH) was then combined with the remaining three epitope sequences, namely, the tandem epitope sequence containing VP2-2 (named EMCV-Ⅱ), the tandem epitope sequence containing VP2-3 (named EMCV-Ⅲ), and the tandem epitope sequence containing VP2-4 (named EMCV-Ⅳ), respectively. Nanjing GenScript performed codon optimization and the optimized sequence was inserted into the eukaryotic expression vector pcDNA3.1. Afl II and EcoR V restriction site, and the recombinant plasmids MIgH-EMCV-Ⅱ, MIgH-EMCV-Ⅲ and MIgH-EMCV-Ⅳ were prepared in sequence.

[0075] 1.2.4 Electroporation of recombinant plasmid into CHO cells

[0076] First, place the electrofection kit at room temperature for 30 min. Then, use electrofection to transfect the recombinant plasmid into serum-free fully suspended CHO-K1-S2 cells. At the same time, set up a control group, and the control group transfection plasmid was pcDNA3.1. Take 1 mL of cell viability higher than 95%, and the live cell density is 2.0×10 6 cells / mL, centrifuged at 850 rpm for 10 min, discarded the supernatant to collect the cells, added 82 μL Nuclefector Solution and 18 μL Supplement, respectively, gently pipetted to mix, then added 10 μg of plasmid, immediately transferred the mixture into the electroporation cup, selected the appropriate electroporation program for electroporation. The electroporated cell suspension was transferred to a 6-well plate, supplemented with SFM4CHO medium to a total volume of 2 mL, placed in a 37 ℃, 5% CO2 incubator for static culture, and after 48 h, an appropriate amount of supernatant was taken for Western blot detection.

[0077] 1.2.5 Stable culture and monoclonal cell screening in shake flasks

[0078] 96 h after transfection, the cells in the 6-well plate were subcultured, and G418 at a concentration of 400 μg / mL was added for pressure screening for 2 to 3 generations, and then gradually amplified to shake flask culture. G418 is an aminosugar antibiotic that can prevent protein synthesis in mammalian cells by interfering with ribosome function. G418 is often used instead of neomycin on pcDNA3.1 vectors to selectively screen cell lines that can stably express the target protein or stably express specific genes. After the cells in the 6-well plate were amplified to shake flask culture, 1.0×10 6 The cells were passaged at a density of 10 cells / mL, and the culture volume was 50 mL. The cells were cultured in a shaker at 37 °C and 5% CO2 at a speed of 120 r / min. Western blot was used to detect whether the expression was stable. At the same time, the cells in the cell pool were screened for monoclonal clones, and 1 cell per well was inoculated into a 96-well culture plate, and G418 was added for preliminary screening, and the cells were continued to be cultured in a 37 °C incubator. After 15 days, the cells were observed to find the wells that formed a single cell colony, and the images were collected using an inverted fluorescence microscope. An appropriate amount of culture cell supernatant was taken for Western blot detection, and the monoclonal cells obtained from the initial screening were named one by one. From the four engineering cells MIgH-EMCV-Ⅰ, MIgH-EMCV-Ⅱ, MIgH-EMCV-Ⅲ, and MIgH-EMCV-Ⅳ, one monoclonal cell with high expression was selected, MIgH-EMCV-Ⅰ 1C4 MIgH-EMCV-Ⅱ 2D2 MIgH-EMCV-Ⅲ 3C8 and MIgH-EMCV-Ⅳ2G4 The cells were scaled up and cryopreserved step by step.

[0079] 1.2.6 Western blot detection

[0080] The collected cell supernatant was treated with 5× protein loading buffer and separated by 10% SDS-PAGE. The membrane was transferred to PVDF membrane by semi-dry transfer at 10V, 1A, 30min. The membrane was blocked with 2.5% skim milk powder at room temperature for 2h, and HRP-labeled anti-His tag antibody diluted 1:5000 was added. The membrane was incubated at 4℃ overnight, washed 5 times with TBST, and then developed with ECL high-sensitive colorimetric solution. The membrane was analyzed with Amersham Imager 600 gel imaging system.

[0081] 1.2.7 Effects of sugar-supplemented culture on the growth of engineered cell lines and recombinant protein expression

[0082] The commercially available feed medium Cell Boost 5 (CB5) was selected for use in combination with SFM4CHO basal medium. The feed scheme is shown in Table 2, and the sugar supplement scheme is shown in Table 3. MIgH-EMCV-Ⅰ, which is in good growth state and in the logarithmic growth phase, was added to 1C4 , MIgH-EMCV-Ⅱ 2D2 , MIgH-EMCV-Ⅲ 3C8 , MIgH-EMCV-Ⅳ 2G4 These four engineered cells were expressed at 1.0×10 6 The cells were passaged at a density of 1.5 cells / mL, with three groups in parallel, and the culture volume was 50 mL. The cells were cultured in a shaker at 37 °C and 5% CO2 at a speed of 120 r / min. The experimental group was fed according to Table 2, and the sugar was supplemented according to Table 3. The untreated group, i.e., the group without feed medium and the group without sugar supplement, was used as the control. From the second day, the countstar automatic cell counter was used to measure the live cell density and cell viability every day. The glucose concentration was monitored using a glucose detection kit, and glucose was added to 7.0 g / L when the concentration was lower than 3.5 g / L. The cell suspension was harvested when the viability dropped below 60%, and the supernatant was collected after centrifugation at 850 rpm for 10 minutes. The content of recombinant protein in the supernatant was detected by Western Blot.

[0083] Table 2 Feeding schedule

[0084]

[0085] Table 3 Sugar supplementation plan

[0086]

[0087] 1.2.8 Purification and BCA quantification of recombinant proteins

[0088] Tangential flow ultrafiltration is one of the key separation and purification technologies in biopharmaceutical processes. Through the tangential flow of liquid on the membrane surface, the accumulation of macromolecules / large particles on the membrane surface is reduced, thereby achieving a higher processing flux than direct current filtration. It is widely used in biopharmaceutical fields such as antibodies, vaccines, gene and cell therapy, nucleic acid drugs, etc., and is the main means of drug molecule concentration and buffer replacement. The present invention uses tangential flow technology to concentrate and purify four recombinant proteins. The specific method is: install a 10 kD Hydrosart membrane package to an ultrafiltration device, and pass pure water for 20 minutes of circulation cleaning. Then change to PBS, wash until the pH is neutral, and then pass the above-prepared recombinant protein, circulate and concentrate to the required volume, and collect the filtered part and the unfiltered part. Finally, pass pure water, wash for 10 minutes, and then change to 0.1 mol / L NaOH or 10% ethanol for 20 minutes. Hydrosart is a stabilized regenerated cellulose membrane with super hydrophilicity, extremely low adsorption, wide pH tolerance and excellent chemical compatibility, which is suitable for the concentration of special proteins. The protein concentrations before and after purification were measured using the BCA protein quantification method. According to the protein concentrations measured by the BCA protein quantification method, the concentrations of the four proteins were adjusted to 1 mg / mL. They were then fully emulsified with an equal volume of adjuvant (Freund's complete adjuvant was used for the first time, and Freund's incomplete adjuvant was used for the second time) to prepare a vaccine with a final concentration of 0.5 mg / mL for immunizing mice.

[0089] 1.2.9 Animal experiments and grouping

[0090] 85 healthy BALB / c female mice aged 6-8 weeks, weighing approximately (18.0±2.0) g, were selected and randomly divided into 6 groups according to Table 4. After one week of adaptive culture, they were used for immunization. All experimental mice were immunized twice, with an interval of two weeks. During the first immunization of each group, 0.5 mL of the vaccine (i.e., 250 μg / mouse) was inoculated in the muscles of both hind limbs; the second immunization was performed 14 days later, and the reagents, doses, and immunization sites of the second immunization were the same as those of the first immunization. The PBS group was inoculated with equal amounts of sterile PBS twice, and the adjuvant group (adjuvant) was inoculated with equal amounts of Freund's complete adjuvant for the first time and equal amounts of Freund's incomplete adjuvant for the second time. Eyeball blood was collected from mice in all experimental groups 14 days after the second immunization, and serum was prepared according to conventional methods. The challenge experiment was performed 14 days after the second immunization, and the mice were observed for 10 consecutive days after the challenge, and the clinical symptoms of the mice were recorded. The clinical symptom grade judgment is shown in Table 5. Ten days after infection, the mice were killed by cervical dislocation, and the brain, heart, and spleen tissues were isolated aseptically and analyzed by TCID 50 Determination of viral titers in serum and tissues.

[0091] Table 4 Immunization schedule

[0092]

[0093] Table 5 Clinical grade score

[0094]

[0095] 1.2.10 Effects of different immunization doses on immune effects

[0096] According to the above experimental screening, a vaccine with the best protective effect was finally obtained in order to verify the effect of different immunization doses of this vaccine on the immune effect. The present invention selected 28 healthy BALB / c female mice aged 6-8 weeks and randomly divided them into 4 groups, with a body weight of about (18.0±2.0) g. Each group was immunized with one injection, and the vaccine content of 50 μg / mouse, 150 μg / mouse, and 250 μg / mouse was inoculated in the muscles of both hind limbs, and the control group was inoculated with an equal volume of sterile PBS. The challenge experiment was carried out 14 days after immunization. After the challenge, the mice were observed for 7 consecutive days to observe the weight changes and clinical symptoms of the mice, and the vaccine protection rate was calculated. The specific steps are as described above.

[0097] 1.2.11 Effects of different immunization procedures on immune efficacy

[0098] In order to verify the effect of different immunization procedures on the immune effect, the optimal immune dose screened in step 1.2.10 was used for subsequent experiments. Similarly, 28 healthy BALB / c female mice aged 6-8 weeks were randomly divided into 4 groups, with a body weight of approximately (18.0±2.0) g. The vaccine was prepared as described above. The immunization group was vaccinated with one and two injections, respectively, and the control group was vaccinated with the same number of times with the same amount of sterile PBS. The specific immunization steps are:

[0099] One injection: the first immunization was on the first day, and the challenge experiment was conducted 14 days later. The immunization dose was 250 μg / mouse.

[0100] Two injections were given: the first immunization on the first day, the second immunization on the 14th day, and the challenge experiment was performed 7 days after the second immunization. The inoculation volume for both immunizations was the same, 250 μg / mouse.

[0101] The mice were observed for 7 consecutive days after the challenge test, and the weight changes, clinical symptoms and vaccine protection rate of the mice were determined as described above.

[0102] 1.2.12 Statistical analysis

[0103] GraphPad Prism 8.0 software was used for drawing, and the significant differences between the data of each group were calculated by single factor analysis ANOVAs or t test. Data are expressed as mean ± standard deviation (±s), with * p<0.05,** p <0.01,*** p <0.001 indicated that the difference was statistically significant.

[0104] 2 Results

[0105] 2.1 Screening of potential dominant B cell epitopes

[0106] The present invention uses the protean module in DNAstar to predict the secondary structures of the structural proteins VP1, VP2, and VP3 of the EMCV PV21 strain (GenBank: X74312) and screens its B cell potential dominant epitopes ( Figure 2 ). The secondary structure of proteins is predicted from different aspects by three methods: Garnier-Robson, ChouFasman and Eisenberg. The Garnier-Robson method predicts the secondary structure of proteins by calculating the possibility of specific amino acid residues in a specific structure; the Chou-Fasman method predicts the secondary structure of proteins by the crystal structure of sequence amino acid residues; the Eisenberg method predicts the secondary structure of proteins by analyzing the hydrophobicity and hydrophilicity. When predicting antigenic epitopes, the following basic principles are usually used for judgment: in the amino acid segments selected by flexibility, hydrophilicity, antigenic index and protein surface possibility (antigenic index ≥ 0, hydrophilicity index ≥ 0, and amino acid surface possibility index ≥ 1), if there is a flexible structure inside or near it, the area with fewer α helices and β folds and more turns and irregular curls is more likely to be a B cell epitope, and the antigenic epitope generally only occupies 5 to 7 amino acid residues, and does not exceed 20 at most.

[0107] The prediction results showed that 26-33 aa, 61-68 aa, 94-101 aa, 150-156 aa, 206-214 aa and 259-272 aa of the structural protein VP1 (referred to as VP1-1, VP1-2, VP1-3, VP1-4, VP1-5, VP1-6, respectively), 10-16 aa, 70-76 aa, 144-150 aa, 157-164 aa of the structural protein VP2 (referred to as VP2-1, VP2-2, VP2-3, VP2-4, respectively) and 135-142 aa of the structural protein VP3 (referred to as VP3- 135-142 aa ) is most likely to contain the antigenic epitope.

[0108] The amino acid sequence of the structural protein VP1-1 is: LPENQTKV.

[0109] The amino acid sequence of the structural protein VP1-2 is: SNKTCPNS.

[0110] The amino acid sequence of the structural protein VP1-3 is: GNGNEETS.

[0111] The amino acid sequence of the structural protein VP1-4 is: TPTKPTT.

[0112] The amino acid sequence of the structural protein VP1-5 is: KRFDNTGSL.

[0113] The amino acid sequence of the structural protein VP1-6 is: PTSGDKIDMTPRAG.

[0114] The amino acid sequence of the structural protein VP2-1 is: LSDRVSQ.

[0115] The amino acid sequence of the structural protein VP2-2 is: WTSTQKP.

[0116] The amino acid sequence of the structural protein VP2-3 is: NRWSKDN.

[0117] The amino acid sequence of the structural protein VP2-4 is: TQTNKKGP.

[0118] Structural protein VP3 135-142 aa The amino acid sequence is: KPTSRDQA.

[0119] Figure 2 Prediction of secondary structure and B cell antigen epitopes of VP1, VP2 and VP3 proteins. A: VP1 protein secondary structure and antigen epitope prediction, B: VP2 protein secondary structure and antigen epitope prediction, C: VP3 protein secondary structure and antigen epitope prediction.

[0120] 2.2 Construction of recombinant plasmid

[0121] According to step 1.2.3, 9 recombinant plasmids were constructed.

[0122] (1) The amino acid sequence of the recombinant plasmid MIgH-EMCV-Ⅰ is (SEQ ID NO.1 in the sequence listing):

[0123] MNFGLSLIFLVLILKGVQCISISEIKGVIVHKIEGILFKKLSDRVSQGGGGSKPTSRDQAGGGGSLPENQTKVGGGGSSNKTCPNSGGGGSGNGNEETSGGGGSTPTKPTTGGGGSKRFDNTGSLGGGGSPTSGDKIDMTPRAGHHHHHH.

[0124] Among them, positions 1-19 are signal peptides, positions 20-38 are T cell epitopes, positions 39-40 are Linker1, positions 41-47 are VP2-1, positions 48-52 are Linker2, and positions 53-60 are VP3 135-142 aa , positions 61-65 are Linker2, positions 66-73 are VP1-1, positions 74-78 are Linker2, positions 79-86 are VP1-2, positions 87-91 are Linker2, positions 92-99 are VP1-3, positions 100-104 are Linker2, positions 105-111 are VP1-4, positions 112-116 are Linker2, positions 117-125 are VP1-5, positions 126-130 are Linker2, positions 131-144 are VP1-6, and positions 145-150 are 6×His.

[0125] The nucleotide sequence of the recombinant plasmid MIgH-EMCV-Ⅰ after codon optimization is (SEQ ID NO.2 in the sequence list):

[0126] ATGAATTTTGGTCTAAGTCTAATCTTTCTAGTGCTGATCCTGAAAGGCGTGCAGTGCATCTCCATCAGCGAGATCAAGGGCGTCATCGTGCACAAGATCGAGGGCATCCTGTTCAAGAAGCTGTCCGACAGAGTGTCCCAAGGCGGCGGAGGCTCCAAGCCTACCTCCAGAGATCAGGCCGGCGGCGGCGGATCTCTGCCTGAGAACCAGACCAAAGTGGGCGGCGG AGGCTCCTCCAACAAGACCTGTCCTAACTCCGGCGGAGGTGGCAGCGGCAACGGCAATGAAGAAACCTCTGGCGGCGGAGGCTTCTACCCCAACCAAGCCCACCACAGGCGGCGGAGGCTCTAAGCGGTTCGACAACACCGGCAGCCTGGGCGGCGGCGGCTCTCCTACATCTGGCGACAAGATCGACATGACCCCTCGGGCTGGCCACCATCACCACCACCACTGA.

[0127] Among them, positions 1-57 are signal peptides, positions 58-114 are T cell epitopes, positions 115-120 are Linker1, positions 121-141 are VP2-1, positions 142-156 are Linker2, and positions 157-180 are VP3 135-142 aa , positions 181-195 are Linker2, positions 196-219 are VP1-1, positions 220-234 are Linker2, positions 235-258 are VP1-2, positions 259-273 are Linker2, positions 274-297 are VP1-3, positions 298-312 are Linker2, positions 313-333 are VP1-4, positions 334-348 are Linker2, positions 349-375 are VP1-5, positions 376-390 are Linker2, positions 391-432 are VP1-6, and positions 433-450 are 6×His.

[0128] (2) The amino acid sequence of the recombinant plasmid Azu-EMCV-Ⅰ is obtained by replacing the 1st to 19th amino acid sequence of MIgH-EMCV-Ⅰ with: MTRLTVLALLAGLLASSRA.

[0129] The nucleotide sequence of the recombinant plasmid Azu-EMCV-Ⅰ is obtained by replacing the nucleotide sequence of positions 1-57 of MIgH-EMCV-Ⅰ with: ATGACTCGACTAACTGTGCTAGCACTACTAGCTGGCCTGCTGGCCTCCTCTCGG

[0130] GCC.

[0131] (3) The amino acid sequence of the recombinant plasmid tPA-EMCV-Ⅰ is obtained by replacing the 1st to 19th amino acid sequence of MIgH-EMCV-Ⅰ with: MDAMKRGLCCVLLLCGAVFVSP.

[0132] The nucleotide sequence of the recombinant plasmid tPA-EMCV-Ⅰ is obtained by replacing the nucleotide sequence of positions 1-57 of MIgH-EMCV-Ⅰ with: ATGGATGCAATGAAGCGAGGTCTATGCTGCGTCCTGCTGCTGTGCGGCGCCGT

[0133] GTTCGTGTCCCCT.

[0134] (4) The amino acid sequence of the recombinant plasmid VSV-G-EMCV-Ⅰ is obtained by replacing the 1st to 19th amino acid sequence of MIgH-EMCV-Ⅰ with: MKCLLYLAFLFIGVNC.

[0135] The nucleotide sequence of the recombinant plasmid VSV-G-EMCV-Ⅰ is obtained by replacing the nucleotide sequence at positions 1 to 57 of MIgH-EMCV-Ⅰ with: ATGAAGTGCCTACTATATCTAGCATTTCTATTCATCGGCGTCAACTGC.

[0136] (5) The amino acid sequence of the recombinant plasmid MIgκ-EMCV-Ⅰ is obtained by replacing the 1st to 19th amino acid sequence of MIgH-EMCV-Ⅰ with: METDTLLLWVLLLWVPGSTGD.

[0137] The nucleotide sequence of the recombinant plasmid MIgκ-EMCV-Ⅰ is obtained by replacing the nucleotide sequence of positions 1-57 of MIgH-EMCV-Ⅰ with: ATGGAAACTGATACTCTACTACTATGGGTGCTGCTGCTGTGGGTCCCTGGCTCC

[0138] ACCGGCGAT.

[0139] (6) The amino acid sequence of the recombinant plasmid Alb-EMCV-Ⅰ is obtained by replacing the 1st to 19th amino acid sequence of MIgH-EMCV-Ⅰ with: MKWVTFISLLFLFSSAYS.

[0140] The nucleotide sequence of the recombinant plasmid Alb-EMCV-Ⅰ is obtained by replacing the nucleotide sequence at positions 1 to 57 of MIgH-EMCV-Ⅰ with: ATGAAGTGGGTGACTTTTATCAGTCTACTATTTCTGTTCTCCTCTGCCTACTCC.

[0141] (7) The amino acid sequence of the recombinant plasmid MIgH-EMCV-Ⅱ is (SEQ ID NO.3 in the sequence listing):

[0142] MNFGLSLIFLVLILKGVQCISISEIKGVIVHKIEGILFKKWTSTQKPGGGGSKPTSRDQAGGGGSLPENQTKVGGGGSSNKTCPNSGGGGSGNGNEETSGGGGSTPTKPTTGGGGSKRFDNTGSLGGGGSPTSGDKIDMTPRAGHHHHHH.

[0143] The amino acid sequence at positions 41-47 is VP2-2, and the rest is identical to the amino acid sequence of MIgH-EMCV-Ⅰ.

[0144] The nucleotide sequence of the recombinant plasmid MIgH-EMCV-Ⅱ after codon optimization is (SEQ ID NO.4 in the sequence list):

[0145] ATGAATTTTGGTCTAAGTCTAATCTTTCTAGTGCTGATCCTGAAGGGCGTGCAGTGCATCTCCATCTCCGAGATCAAGGGCGTCATCGTGCACAAGATCGAGGGCATCCTGTTCAAGAAGTGGACCTCCACCCAGAAGCCTGGCGGCGGCGGCAGCAAACCCACCTCTAGAGATCAAGCTGGCGGCGGCGGCTCTCTGCCTGAAAACCAGACCAAGGTGGGCGGAGG TGGCTCCTCCAACAAGACCTGTCCTAACTCCGGCGGCGGAGGCTCTGGCAATGGCAACGAGGAAACATCTGGCGGAGGAGGCTCTACCCCCACAAAACCTACCACCGGCGGAGGCGGCTCCAAGCGGTTCGACAACACCGGCTCCCTGGGCGGCGGCGGATCTCCAACAAGCGGCGACAAGATCGACATGACCCCTAGAGCCGGCCACCACCATCACCACCACTGA.

[0146] Among them, positions 1-57 are signal peptides, positions 58-114 are T cell epitopes, positions 115-120 are Linker1, positions 121-141 are VP2-2, positions 142-156 are Linker2, and positions 157-180 are VP3 135-142 aa , positions 181-195 are Linker2, positions 196-219 are VP1-1, positions 220-234 are Linker2, positions 235-258 are VP1-2, positions 259-273 are Linker2, positions 274-297 are VP1-3, positions 298-312 are Linker2, positions 313-333 are VP1-4, positions 334-348 are Linker2, positions 349-375 are VP1-5, positions 376-390 are Linker2, positions 391-432 are VP1-6, and positions 433-450 are 6×His.

[0147] (8) The amino acid sequence of the recombinant plasmid MIgH-EMCV-Ⅲ is: the amino acid sequence of positions 41-47 of MIgH-EMCV-Ⅱ is replaced by the amino acid sequence of VP2-3: NRWSKDN.

[0148] The nucleotide sequence of the recombinant plasmid MIgH-EMCV-Ⅲ after codon optimization is (SEQ ID NO.5 in the sequence list):

[0149] ATGAATTTTGGTCTAAGTCTAATCTTTCTAGTGCTGATCCTGAAAGGCGTGCAGTGCATCTCCATCAGCGAAATCAAGGGCGTCATCGTGCACAAGATCGAGGGCATCCTGTTCAAGAAGAACAGATGGTCCAAGGACAACGGCGGCGGCGGCTCCAAACCCACCTCTCGGGATCAGGCTGGCGGCGGAGGCAGCCTGCCTGAGAACCAGACCAAGGTGGGCGGAGG CGGATCCTCCAACAAGACCTGTCCTAACTCCGGCGGCGGCGGCTCTGGCAATGGCAACGAGGAAACATCTGGCGGCGGCGGCTCTACCCCAACAAAGCCTACCACCGGCGGAGGAGGCTCCAAGCGGTTCGACAACACCGGATCTCTGGGCGGTGGCGGCTCTCCTACCTCCGGCGACAAGATCGACATGACCCCTAGAGCCGGCCACCATCACCACCACCACTGA.

[0150] (9) The amino acid sequence of the recombinant plasmid MIgH-EMCV-Ⅳ is as follows: the amino acid sequence of VP2-2 of MIgH-EMCV-Ⅱ is replaced by the amino acid sequence of VP2-4: TQTNKKGP.

[0151] The nucleotide sequence of the recombinant plasmid MIgH-EMCV-Ⅳ after codon optimization is (SEQ ID NO.6 in the sequence list):

[0152] ATGAATTTTGGTCTAAGTCTAATCTTTCTAGTGCTGATCCTGAAGGGCGTGCAGTGCATCTCCATCTCCGAGATCAAGGGCGTCATCGTGCACAAGATCGAGGGCATCCTGTTCAAGAAGACCCAAACCAACAAGAAAGGCCCTGGCGGCGGCGGCTCCAAGCCTACATCCAGAGATCAGGCCGGCGGAGGCGGCAGCCTGCCTGAGAACCAGACCAAGGTGGGCGGC GGAGGCTCCTCCAACAAAACCTGTCCTAACTCCGGCGGAGGCGGCTCTGGCAACGGCAATGAAGAAACCTCTGGAGGTGGCGGATCTACCCAACCAAGCCCACAACAGGCGGCGGCGGAAGCAAGCGGTTCGACAACAACCGGCTCTCTGGGCGGCGGCGGCTCTCCCACCAGCGGCGACAAGATCGACATGACCCCTAGAGCTGGCCACCATCACCACCACCACTGA.

[0153] 2.3 Identification and expression of recombinant plasmids

[0154] Restriction endonuclease AflⅡ and EcoRⅤ After enzyme digestion, a specific DNA band size of about 485 bp appeared, which was consistent with the expected band ( Figure 3 A). After performing its function, the signal peptide will be automatically cleaved and separated from the recombinant protein, and the recombinant protein can be transported to the extracellular space. The results of Western blot showed that compared with the other five signal peptides, the plasmid synthesized with the signal peptide MIgH had the highest level of secretion and expression of recombinant protein in CHO cells, and its protein size was about 27KD. Therefore, the signal peptide MIgH was selected in subsequent experiments to synthesize the remaining three sequences ( Figure 3 B).

[0155] Four eukaryotic expression plasmids synthesized with signal peptide MIgH were purified by restriction endonuclease AflⅡ and EcoRⅤ After enzyme digestion, agarose electrophoresis results showed that all recombinant plasmids had a specific DNA band size of about 490 bp, which was consistent with the expected band ( Figure 3 C). Western blot analysis revealed that the four plasmids synthesized with the signal peptide mouse heavy chain (MIgH) could be secreted and expressed in CHO suspension cells, and the protein size was about 27 KD, which was consistent with the expected protein size ( Figure 3 D).

[0156] In order to verify whether it can be stably expressed, the culture supernatants of the 3rd, 6th, 9th and 12th generations were taken for Western blot detection. It was found that these four gene-engineered CHO cells that secrete and express EMCV multi-epitopes can stably secrete and express from the 1st to the 12th generation, and the secretion expression level of MIgH-EMCV-Ⅱ is the highest ( Figure 3 E). In order to explore the growth rules of these four engineered cells, cells that were in good growth state and in the logarithmic growth phase and secreted and expressed recombinant proteins were cultured at 1.0×10 6 cells / mL, the cell density and viability were monitored every day. The results showed that the growth patterns of these four cell lines were basically the same. The cell viability decreased to 60% on the 7th day of culture, and the highest viable cell density was 13.4×10 6 cells / mL、13.2×10 6 cells / mL、13.1×10 6 cells / mL、13.0×10 6 cells / mL( Figure 3 FG).

[0157] Figure 3 For identification and expression of recombinant plasmids. A: 6 recombinant plasmids were double-digested with Afl Ⅱ and EcoR Ⅴ restriction enzymes for verification; B: Western blot was used to detect the expression level of EMCV multi-epitope recombinant proteins mediated by different signal peptides; C: 4 recombinant plasmids were double-digested with Afl Ⅱ and EcoR Ⅴ restriction enzymes for verification; D: Western blot was used to detect the expression of 4 EMCV multi-epitope recombinant proteins; E: Western blot was used to detect the stable expression of 4 EMCV multi-epitope recombinant proteins; F: The viable cell density of 4 genetically engineered CHO cells; G: The viability of 4 genetically engineered CHO cells.

[0158] 2.4 Screening and preparation of monoclonal cells

[0159] After 15 days of pressure screening, the results of Western blot detection are as follows:

[0160] A total of 42 monoclonal cell lines were screened from MIgH-EMCV-Ⅰ cells, of which 6 positive clones were named as: MIgH-EMCV-Ⅰ 1C4 MIgH-EMCV-Ⅰ 1C11 MIgH-EMCV-Ⅰ 1H4 MIgH-EMCV-Ⅰ 2B10 MIgH-EMCV-Ⅰ3B4 MIgH-EMCV-Ⅰ 4F11 ( Figure 4 A). Select a monoclonal cell line with high expression level and stable expression, namely MIgH-EMCV-Ⅰ 1C4 Expand the culture ( Figure 4 E).

[0161] A total of 31 monoclonal cell lines were screened from MIgH-EMCV-Ⅱ cells, of which 6 were positive clones, which were named as: MIgH-EMCV-Ⅱ 1B1 MIgH-EMCV-Ⅱ 2D2 MIgH-EMCV-Ⅱ 3A9 MIgH-EMCV-Ⅱ 4A8 MIgH-EMCV-Ⅱ 2C5 MIgH-EMCV-Ⅱ 4D10 ( Figure 4 B) Select a monoclonal cell line with high expression level and stable expression, namely MIgH-EMCV-Ⅱ 2D2 Expand the culture ( Figure 4 F).

[0162] A total of 24 monoclonal cell lines were screened from MIgH-EMCV-Ⅲ cells, of which 6 were positive clones, which were named as: MIgH-EMCV-Ⅲ 1D7 MIgH-EMCV-Ⅲ 2B5 MIgH-EMCV-Ⅲ 3H9 MIgH-EMCV-Ⅲ 4A11 MIgH-EMCV-Ⅲ 3C8 MIgH-EMCV-Ⅲ 4B11 ( Figure 4 C). Select a monoclonal cell line with high expression level and stable expression, namely MIgH-EMCV-Ⅲ 3C8 Expand the culture ( Figure 4 G).

[0163] A total of 40 monoclonal cell lines were screened from MIgH-EMCV-Ⅳ cells, of which 6 were positive clones, which were named as follows: MIgH-EMCV-Ⅳ 1C11 MIgH-EMCV-Ⅳ 2G4 MIgH-EMCV-Ⅳ 2C10 MIgH-EMCV-Ⅳ 3C2 MIgH-EMCV-Ⅳ 3F2 MIgH-EMCV-Ⅳ 3E6 ( Figure 4D) Select a monoclonal cell line with high expression level and stable expression, namely MIgH-EMCV-Ⅳ 2G4 Expand the culture ( Figure 4 H).

[0164] Figure 4 Screening and preparation of monoclonal cells. A, B, C, D: respectively represent the images of MIgH-EMCV-Ⅰ, MIgH-EMCV-Ⅱ, MIgH-EMCV-Ⅲ, and MIgH-EMCV-Ⅳ monoclonal cells collected by inverted fluorescence microscope; E, F, G, H: respectively represent the levels of recombinant protein expressed by MIgH-EMCV-Ⅰ, MIgH-EMCV-Ⅱ, MIgH-EMCV-Ⅲ, and MIgH-EMCV-Ⅳ monoclonal cells detected by Western blot.

[0165] 2.5 Sugar and feed supplementation can increase the expression of recombinant proteins

[0166] The experimental results show that compared with the control group, the sugar-supplemented culture can extend the culture time of the four engineered cell lines by 2-3 days ( Figure 5 B. Figure 5 F. Figure 5 J. Figure 5 N). Western Blot was used to detect the content of recombinant proteins in the supernatant on the 6th day, and it was found that the addition of CB5 and glucose could significantly increase the expression levels of the four recombinant proteins ( Figure 5 D. Figure 5 H. Figure 5 L. Figure 5 P). This indicates that sugar and feed supplementation can prolong the culture period of CHO engineered cells and improve the expression of recombinant proteins.

[0167] Figure 5 The effects of glucose supplementation and feeding culture on the growth of engineered cell lines and the expression of recombinant proteins. A, E, I, M: viable cell density; B, F, J, N: cell viability; C, G, K, O: glucose concentration; D, H, L, P: Western blot detection of recombinant protein expression.

[0168] 2.6 Purification of recombinant protein

[0169] Western Blot results showed that ultrafiltration technology increased the concentration of recombinant protein ( Figure 6 The concentration of the recombinant protein was quantified by the BCA method. The protein concentration after concentration was: MIgH-EMCV-I 1C4 is 1.289 mg / mL, MIgH-EMCV-Ⅱ 2D2 is 1.315 mg / mL, MIgH-EMCV-Ⅲ 3C8is 1.504 mg / mL, MIgH-EMCV-Ⅳ 2G4 It is 1.288 mg / mL.

[0170] Figure 6 The ultrafiltration results of recombinant proteins. 1C4 Protein ultrafiltration results, B:MIgH-EMCV-Ⅱ 2D2 Protein ultrafiltration results, C:MIgH-EMCV-Ⅲ 3C8 Protein ultrafiltration results, D: MIgH-EMCV-Ⅳ 2G4 Protein ultrafiltration results. a: negative control, b: sample before ultrafiltration, c: ultrafiltration waste liquid, d: sample after ultrafiltration.

[0171] 2.7 Determination of neutralizing antibodies and cytokine IFN-γ

[0172] The TCID of EMCV was measured according to the Reed-Muench method. 50 For 10 -7.289 The serum collected 14 days after the second immunization was tested for neutralizing antibody titer. The results showed that the neutralizing antibody titer of the PBS group and the adjuvant group was less than 2 1 MIgH-EMCV-Ⅱ 2D2 The group produced the highest level of neutralizing antibodies, with a titer of about 1:16, and MIgH-EMCV-Ⅰ 1C4 The neutralizing antibody titer of the group was about 1:8, MIgH-EMCV-Ⅲ 3C8 MIgH-EMCV-Ⅳ 2G4 The neutralizing antibody titer of the group was 1:4 ( Figure 7 A). Cytokines play an important role in the pathogenesis of EMCV.

[0173] Mice infected with EMCV can produce IL-2, IL-12 and IFN-γ, and the body's resistance to EMCV infection mainly depends on the production of IFN-γ mediated by IL-12, thereby increasing the activity of natural killer cells (NK cells) to inhibit viral replication, play a protective role in the early stage of infection, and can effectively reduce the occurrence of myocardial cell necrosis and inflammation. The ELISA was used to measure the IFN-γ production level of each group of mice. The results showed that the IFN-γ levels of each immune group were significantly higher than those of the PBS group and the adjuvant group ( Figure 7 B).

[0174] Figure 7 The method is to measure the neutralizing antibody and cytokine IFN-γ, wherein A: the titer of the neutralizing antibody after immunization of mice, and B: the measurement of the cytokine IFN-γ in the serum.

[0175] 2.8 Protective effect of EMCV multi-epitope vaccine candidate on mice

[0176] The clinical symptoms of mice were observed every day after the challenge. The results showed that all mice in the PBS group showed mental depression on the first day after the challenge, and then showed symptoms such as decreased appetite, messy fur, and head edema in the following days. Mice No. 1, No. 4, No. 5, and No. 6 died on the 5th day, and No. 2 and No. 3 died on the 7th and 10th days, respectively ( Figure 8 A). In the adjuvant group, except for mice No. 7 and No. 9, which showed decreased appetite on the first day after the virus attack, the other seven mice showed mental depression. Subsequently, they gradually developed symptoms such as head edema, hind limb paralysis, and circling. No. 1 died on the 7th day, and No. 6, No. 7, No. 8, and No. 9 died on the 5th day. The remaining mice showed symptoms such as head edema ( Figure 8 B). MIgH-EMCV-Ⅰ 1C4 The mice in the group showed poor spirits on the first day after the virus attack. No. 1 died on the 5th day, No. 5 died on the 7th day, and No. 8 died on the 10th day. The remaining mice showed symptoms such as mental depression and loss of appetite, and then returned to normal ( Figure 8 C). MIgH-EMCV-Ⅱ 2D2 Except for the symptoms of mental depression and poor appetite 1-2 days after infection, the mice in the group quickly recovered to normal ( Figure 8 D). MIgH-EMCV-Ⅲ 3C8 The mice in the same group also showed depression on the first day after infection. Mice 7 and 8 died on the 4th and 5th day, respectively. Mice 1 and 2 died on the 7th day. Mice 3, 4, and 6 died on the 9th day. The remaining mice gradually developed symptoms such as hind limb paralysis ( Figure 8 E). MIgH-EMCV-Ⅳ 2G4 The mice in the group showed low spirits on the first day after infection. Mouse No. 6 died on the fifth day, and mice No. 5 and 7 died on the sixth day. Mouse No. 3 and 9 died on the seventh day. The rest of the mice gradually recovered to normal ( Figure 8 F).

[0177] The weight change was determined according to the formula. The results showed that the PBS group had the largest weight change and continued to lose weight. The weight loss was 9.2% compared to before the challenge. The weight change in the adjuvant group was relatively large, probably due to edema in the mice, but the weight loss was 5.5% compared to before the challenge. MIgH-EMCV-Ⅰ 1C4 The body weight of the group decreased by 2.4% compared with that before the challenge. 2D2 The body weight of the group was basically stable, and the body weight decreased by only 1.4% compared with that before the challenge. 3C8 Group and MIgH-EMCV-Ⅳ 2G4The body weight of the two groups also changed significantly, decreasing by 5.6% and 4.1% respectively compared with before the challenge. Figure 8 G). In summary, all mice in the PBS group died within 10 days after challenge. The protection rate in the adjuvant group was 44.4%. 1C4 The protection rate of the group was 66.66%. 2D2 The protection rate of the group was 100%, MIgH-EMCV-Ⅲ 3C8 The protection rate of the group was 22.22%. 2G4 The protection rate of the group was 33.33%. Therefore, compared with other groups, MIgH-EMCV-Ⅱ 2D2 The group was able to completely protect mice from EMCV challenge and could serve as an effective candidate EMCV vaccine ( Figure 8 H).

[0178] Figure 8 The protective effect of EMCV multi-epitope candidate vaccine on mice. Among them, A, B, C, D, E, F: PBS group, adjuvant group, MIgH-EMCV-I 1C4 Group, MIgH-EMCV-Ⅱ 2D2 Group, MIgH-EMCV-Ⅲ 3C8 Group, MIgH-EMCV-Ⅳ 2G4 The clinical symptom scores of mice in each group after challenge with the virus. G: The weight changes of mice in each group after challenge with the virus. H: The determination of vaccine protection rate.

[0179] 2.9 Determination of virus titers in mouse serum and different tissues

[0180] The TCID of EMCV in the serum of mice in the PBS group was obtained according to the above formula. 50 For 10 -5.655 , and the best protective effect is MIgH-EMCV-Ⅱ 2D2 The TCID of EMCV in the serum of mice in the group 50 Only 10 -1.825 ( Fig. 9 A), compared with the PBS group, MIgH-EMCV-Ⅱ 2D2 The viral load in the serum of the group was reduced by 6760 times.

[0181] TCID of EMCV in brain tissue of mice in PBS group 50 For 10 -6.62 , while MIgH-EMCV-Ⅱ 2D2 TCID of EMCV in the brain tissue of mice in the same group 50 Only 10 -0.62 ( Fig. 9B) Compared with the PBS group, MIgH-EMCV-Ⅱ 2D2 The viral load in the brain tissue of the group was reduced by 10 6 times.

[0182] TCID of EMCV in heart tissue of mice in PBS group 50 For 10 -2.525 , while MIgH-EMCV-Ⅱ 2D2 TCID of EMCV in heart tissue of group A mice 50 For 10 -0.79 ( Fig. 9 C) Compared with the PBS group, MIgH-EMCV-Ⅱ 2D2 The viral load in the heart tissue of the group was reduced by 54 times.

[0183] TCID of EMCV in spleen tissue of mice in PBS group 50 For 10 -2.405 , while MIgH-EMCV-Ⅱ 2D2 TCID of EMCV in spleen tissue of group A mice 50 For 10 -0.79 ( Fig. 9 D) Compared with the PBS group, MIgH-EMCV-Ⅱ 2D2 The viral load in the spleen tissue of the group was reduced by 41 times. According to the above results, EMCV infection mainly infects the brain tissue of mice. 2D2 Afterwards, it can significantly reduce the replication of the virus in mouse brain, heart, spleen and other tissues.

[0184] Fig. 9 The results of the determination of virus titers in mouse serum and different tissues are shown in Figure 1. A, B, C, and D are the determination of virus titers in mouse serum, brain tissue, heart, and spleen, respectively.

[0185] 2.10 Effects of different immunization doses on immune effects

[0186] Different immunization doses have different protective effects on mice. The experimental results show that the immunization effect is related to the immunization dose. All mice in the PBS group died within 7 days after the virus was attacked. There was no significant change in the weight of mice in the high-dose group, i.e., the 250 μg immunization group. Only one mouse developed hind limb paralysis on the 4th day and died on the 5th day. The protection rate for mice was 85.7%. The weight of mice in the medium-dose group, i.e., the 150 μg immunization group, decreased, and one mouse also developed hind limb paralysis and died on the 6th day after the virus was attacked. The protection rate for mice was 85.7%. The weight of mice in the low-dose group, i.e., the 50 μg immunization group, decreased significantly. Three mice had messy fur, systemic edema, hind limb paralysis and then died after the virus was attacked. The protection rate for mice was 57.1% ( Fig.10). In summary, the high-dose group had a better protective effect on mice, and the weight loss was lighter than that of the medium-dose group, so the high-dose group was selected for subsequent immunization program optimization experiments.

[0187] Fig.10 The figure shows the effect of the immunization dose on the immunization effect. A: change in mouse weight, B: vaccine protection rate.

[0188] 2.11 Effects of different immunization procedures on immune efficacy

[0189] Different immunization procedures have different effects on the immune effect. The results showed that all mice in the PBS group died within 7 days after the virus challenge, regardless of whether they were immunized once or twice. There was no significant change in the weight of mice in the 250 μg recombinant protein group, and no obvious clinical symptoms. The protection rate of mice can reach 100%. The weight of mice in the single immunization group decreased on the 6th and 7th days after the virus challenge, and one mouse died on the 5th day. The protection rate of mice was 85.7% ( Fig.11 ). Therefore, the vaccine can improve the immune efficacy after booster immunization and effectively prevent EMCV infection in mice, with a protection rate of 100% for mice.

[0190] Fig.11 The effect of immunization program on immune effect. A: change in mouse weight, B: vaccine protection rate.

[0191] 3 Discussion

[0192] The four structural proteins of EMCV are all involved in the formation of viral antigenic epitopes, among which VP1 is related to the topological structure, antigenicity, receptor adsorption and uncoating of the surface of the virus particle, and is the most important neutralizing antigenic epitope. The VP1 / VP3 binding region is the region with relatively large EMCV variation and is the main domain for studying EMCV genetic variation. The two B cell linear epitopes on VP2 have certain immunogenicity. VP3 is highly correlated with the cell tropism of the virus and also has a small number of antigenic epitopes. The VP4 gene is located at the innermost side of the viral structural protein and has the weakest antigenicity. Therefore, the present invention first uses the protean module in the online prediction website DNAstar and http: / / tools.iedb.org / bcell / to analyze the physicochemical properties and secondary structures of these three structural proteins, and finally screens out 6, 4 and 1 B cell antigenic epitopes on VP1, VP2 and VP3 proteins, respectively. It is cloned into the pcDNA3.1 plasmid, electrotransferred to CHO-K1-S2 cells, and its expression is detected by Western blot, and it is found that it can stably secrete and express. Four highly expressed monoclonal cell lines were obtained by adding G418 to a 96-well plate and screening by limiting dilution method, and were named MIgH-EMCV-Ⅰ. 1C4, MIgH-EMCV-Ⅱ 2D2 , MIgH-EMCV-Ⅲ 3C8 , MIgH-EMCV-Ⅳ 2G4 . Secondly, the recombinant proteins secreted by the monoclonal cell lines were harvested, concentrated by ultrafiltration, and then emulsified with adjuvants to prepare vaccines. The vaccine was immunized with BALB / c mice to preliminarily evaluate the immunogenicity of the vaccine. The results of the microcell neutralization experiment showed that each group could stimulate the body to produce neutralizing antibodies. The ELISA results showed that immunization with the four antigens could induce mice to produce high levels of IFN-γ. The results of the viral load test in various tissues after the virus attack showed that the viral load in the brain tissue of the dead mice in the same immunized group was higher than that in the heart and spleen, indicating that encephalitis caused by encephalomyocarditis infection may be the main cause of death in mice. In addition, MIgH-EMCV-Ⅱ 2D2 The viral load of the MIgH-EMCV-Ⅱ group was significantly lower than that of the other groups. 2D2 After immunization, the body can be stimulated to produce a stronger immune response, thereby more effectively inhibiting the proliferation of the virus in the body and protecting mice from the lethal attack of EMCV.

[0193] In summary, the present invention successfully expressed the EMCV multi-epitope recombinant protein using the CHO eukaryotic expression system, and evaluated the immune effect in mice. The results of the challenge experiment showed that the immune recombinant protein MIgH-EMCV-Ⅱ 2D2 The protection rate of mice was 100%. This provides a theoretical basis and technical support for the development of EMCV multi-epitope vaccines.

[0194] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant protein of multiple antigenic epitopes of encephalomyocarditis virus, characterized in that: The amino acid sequence of the encephalomyocarditis virus multi-antigen epitope recombinant protein is shown in SEQ ID NO.

3.

2. A nucleic acid molecule encoding the encephalomyocarditis virus multi-antigenic epitope recombinant protein according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

4. An expression cassette, recombinant vector or cell containing the nucleic acid molecule of claim 3.

5. The expression cassette, recombinant vector or cell according to claim 4, characterized in that: The recombinant vector includes pcDNA3.

1.

6. The expression cassette, recombinant vector or cell according to claim 4, characterized in that: The cells are CHO cells.

7. The cell culture method according to claim 6, characterized in that: The cells of claim 6 are cultured in SFM4CHO medium, and supplemented with feed medium Cell Boost 5 and glucose during the culture period.

8. The culture method according to claim 7, characterized in that: The amount of feed medium Cell Boost 5 added is 2.5-7.5% of the volume of SFM4CHO basal medium.

9. The culture method according to claim 8, characterized in that: When the glucose concentration in the culture medium was lower than 3.5 g / L, glucose was added to 7.0 g / L.

10. Use of the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the expression cassette, recombinant vector or cell according to any one of claims 4 to 6 in the preparation of an encephalomyocarditis vaccine.

11. A vaccine, characterized in that: The vaccine comprises the recombinant protein of claim 1, the nucleic acid molecule of claim 2 or 3, or the expression cassette, recombinant vector or cell of any one of claims 4 to 6.

12. The vaccine according to claim 11, characterized in that: The vaccine includes an adjuvant.

13. The vaccine according to claim 12, characterized in that: The adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant.

Images