MEV-hBD3 mRNA vaccine for combined prevention of brucellosis and tuberculosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses an MEV-hBD3 mRNA vaccine for combined prevention of brucellosis and tuberculosis, the amino acid sequence of the vaccine comprises a plurality of CD8 + T cell epitopes, and the amino acid sequence of the CD8 + T cell epitopes at least comprises one of SEQ ID NO: 6 to SEQ ID NO: 18; the amino acid sequence of the vaccine further comprises a plurality of CD4 + T cell epitopes, and the amino acid sequence of the CD4 + T cell epitopes at least comprises one sequence from SEQ ID NO: 19 to SEQ ID NO: 32; the amino acid sequence of the vaccine further comprises a plurality of B cell epitopes, and the amino acid sequence of the B cell epitopes at least comprises one sequence of SEQ ID NO: 33 to SEQ ID NO: 41. The MEV-hBD3 mRNA vaccine provided by the invention has good immunogenicity, and is expected to provide an immunological strategy and a theoretical basis for emergency prevention and control of further prevalence of brucellosis and tuberculosis in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a MEV-hBD3mRNA vaccine for combined prevention of brucellosis and tuberculosis. Background Art

[0002] Brucellosis (also known as undulant fever) is a zoonotic infectious disease caused by Brucella infection. It is characterized by abortion in cows and sheep, which is very harmful to the breeding industry. The pathogen of this disease is Brucella abortus. Both male and female animals are susceptible to this pathogen. Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. Humans, cattle, and sheep can all get sick. Among livestock, cattle (especially dairy cows) are the most susceptible.

[0003] There is currently no effective vaccine that can jointly prevent brucellosis and tuberculosis. Summary of the invention

[0004] The purpose of the present invention is to provide a MEV-hBD3 mRNA vaccine for the combined prevention of brucellosis and tuberculosis to solve the above technical problems.

[0005] One aspect of the present invention relates to a MEV-hBD3 mRNA vaccine for the combined prevention of brucellosis and tuberculosis, wherein the amino acid sequence of the vaccine includes several CD8 + T cell epitope, CD8 + The amino acid sequence of the T cell epitope includes at least one of SEQ ID NO:6 to SEQ ID NO:18; the amino acid sequence of the vaccine also includes several CD4 + T cell epitope, CD4 + The amino acid sequence of the T cell epitope includes at least one sequence from SEQ ID NO:19 to SEQ ID NO:32; the amino acid sequence of the vaccine also includes several B cell epitopes, and the amino acid sequence of the B cell epitope includes at least one sequence from SEQ ID NO:33 to SEQ ID NO:41.

[0006] Preferably, the amino acid sequence of the vaccine also includes Linker-AAY, Linker-GPGPG, Linker-KK and Linker-GGGS.

[0007] Preferably, the nucleotide sequence of the vaccine further includes a tissue plasminogen activator (tPA) leader sequence, 5' and 3' untranslated regions (UTRs), a Kozak sequence and a stop codon. The nucleotide sequence of the 5'UTR is: ACATTTGCTTCTGACACA ACTGTGTTCACTAGCAACCTCAAACAGACACC; the nucleotide sequence of Kozak is: GCCACCATGG; the nucleotide sequence of the tPA leader sequence is: ATGGACGCCATGAAGAGGCTGTGCTGCGTGCTGCTGCTGTG CGGCGCCGTGTTCGTGAGCCCC, the nucleotide sequence of the stop codon is: TAG, the nucleotide sequence of the 3'UTR is: GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCTCCCAACGGGCCCTCCTCCCC TCCTTGCACCGGCCCTTCCTGGTCTTTGAATAAAGTCTGAGTGGGCAGCA.

[0008] Preferably, the amino acid sequence of the MEV-hBD3 mRNA vaccine is SEQ ID NO: 1.

[0009] The brucellosis and tuberculosis dual prevention vaccine provided by the present invention can reduce the number of vaccinations, alleviate the pain and inconvenience of the vaccinated subjects, improve the compliance rate of immunization, increase the vaccine coverage rate, improve the vaccination efficiency, help reduce the regulatory costs and storage and transportation costs, and improve efficiency.

[0010] The biggest feature and advantage of the mRNA vaccine provided by the present invention compared to other types of vaccines lies in its rapid research and development capabilities, efficient production, and the ability to simultaneously activate humoral and cellular immune responses. It has good immunogenicity and is expected to provide immunological strategies and theoretical basis for emergency prevention and control of further spread of brucellosis and tuberculosis in the future.

[0011] In the context that Brucella and Mycobacterium tuberculosis seriously endanger public biosafety and antibiotic resistance increases year by year during clinical treatment, making clinical treatment more difficult, the present invention designs and develops a MEV-hBD3 mRNA vaccine based on a multi-epitope vaccine that can simultaneously prevent brucellosis and tuberculosis.

[0012] The present invention uses immunoinformatics technology to select the proteins Omp25, Omp31, MPT70 and MPT83 of Brucella melitensis, which are the most harmful to humans, and Mycobacterium tuberculosis H37Rv (L4 strain) as target antigens, and predicts cytotoxic T cell (CTL) epitopes, helper T lymphocyte (HTL) epitopes, B cell linear epitopes and B cell conformational epitopes based on Xinjiang high-frequency alleles HLA-A*1101 (13.46%), HLA-A*0201 (12.50%), HLA-A*0301 (10.10%), HLA-DRB1*0701 (16.35%), HLA-DRB1*1501 (8.65%) and HLA-DRB1*0301 (7.69%). Using linker protein connection technology, CD8 + T cells, CD4 + T cell, B cell linear epitope and B cell conformational epitope sequences were connected, and a 6X histidine tag was added to the C-terminus of the sequence. The leader sequence of tissue plasminogen activator (TPA) was added to the N-terminus. In order to further optimize the vaccine sequence, the present invention added 5' and 3' untranslated region (UTRs) sequences. In addition, a Kozak sequence was added to the 5' end of the ORF and a polyadenylation (PolyA) sequence was added to the 3' end. Finally, an mRNA vaccine based on a multi-epitope vaccine was designed and named "MEV-hBD3 mRNA vaccine".

[0013] The mRNA vaccine provided by the present invention has significant advantages over other types of vaccines: (1) Safety: there is no potential risk of infection or insertion of mutant genes, and mRNA is degraded through normal cellular processes; (2) Effectiveness: various modifications can be used to make mRNA more stable and highly translatable; (3) Productivity: the high yield of in vitro transcription reactions enables mRNA vaccines to have the potential for rapid, inexpensive and scalable production.

[0014] The present invention utilizes molecular docking technology to dock the MEV-hBD3 mRNA vaccine with the TRL-4 molecule, ensuring the correct binding of the TRL-4 immune cell receptor to the MEV-hBD3 mRNA vaccine and its subsequent effective immune response, thereby proving that the body's immune cells can efficiently take up and present antigens to the MEV-hBD3 mRNA vaccine.

[0015] The present invention selects human beta-defensin 3 (hBD3), a cationic antimicrobial peptide, as the adjuvant of the vaccine, which can amplify inflammatory signal transduction and induce activation of the adaptive immune system through antigen presenting cells, such as dendritic cells.

[0016] The present invention uses an EAAAK linker to connect to the N-terminus of the vaccine structure to improve the immunity of the vaccine.

[0017] The present invention connects the high-frequency antigen epitopes screened using linker proteins, while focusing on the compatibility between linkers and different epitopes. Linker-AAY is used to connect CD8 + For T cell epitopes, Linker-GPGPG was used to connect CD4+T cell epitopes, Linker-KK was used to connect B cell epitopes, and Linker-GGGS was used to connect 6XHis tags. The use of these high-frequency antigen epitopes and the optimized design of linker proteins can theoretically enhance the immunogenicity of vaccine proteins; at the same time, in order to ensure that the mRNA vaccine can be successfully translated in cells, the 5' and 3' UTR elements on both sides were added, which can be derived from viruses or eukaryotic genes, which can significantly affect the stability and translation of mRNA, and the half-life of mRNA is extended and the expression level is significantly increased. The 5' cap structure and poly (A) tail play an important role in the translation and stability of mRNA. The Kozak sequence is recognized by the ribosome as a translation start site. TPA43 is a sequence located before the coding region of the structural gene, which can be transcribed but not translated, and can guide mRNA to the correct position to start translation.

[0018] The present invention expresses MEV-hBD3 multi-epitope protein through a prokaryotic expression system, verifies the protein after purification using a nickel column with the His tag at the N-terminus of the vaccine protein through Western Blotting experiments, and verifies the immunoreactivity of the vaccine using patient serum.

[0019] The mice were inoculated with MEV-hBD3 mRNA vaccine protein by nasal drops, and an in vivo animal experimental model was established. After three inoculations, the specific antibody secreting cells (ASCs) of the mice were detected using the enzyme-linked immunosorbent assay (ELISPOT) technique, thus proving that MEV-hBD3 mRNA can activate the specific humoral immunity of mice; at the same time, the enzyme-linked immunosorbent assay technique was used to measure the secretion capacity of specific T cells in the mice to IFN-γ and IL-4, thus proving that MEV-hBD3 mRNA can activate the specific cellular immunity of mice. Therefore, the present invention has proved through in vitro and in vivo experiments that BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 SignalP 5.0 software was used to analyze the protein sequence of potential signal peptides.

[0021] Figure 2These are the HTL epitopes, CTL epitopes, LB epitopes and CB epitopes finally selected for vaccine design. In the figure, "green" represents the T cell epitope of Omp25, "blue" represents the T cell epitope of Omp31, "purple" represents the T cell epitope of MPT70, and "red" represents the T cell epitope of MPT83.

[0022] Figure 3 To predict the secondary structure of mRNA using the RNAfold website. A: Optimal secondary structure: corresponding minimum free energy. B: Center of mass secondary structure.

[0023] Figure 4 shows the secondary structure predicted by NetSurfP-2.0, where A: The secondary structure was predicted by NetSurfP-2.0. When the threshold is 25%, the red color rises upward to indicate residue exposure, while the sky blue indicates buried residues. As shown in the figure, "orange" represents α-helix, "blue" represents β-folding, and "purple" represents random curling. Among them, the extended chain is represented as an expanded black line, the thickness of which is equal to the probability of disordered residues. B: The secondary structure of MEV was predicted by SOPMA. C: The tertiary structure was predicted by AlphaFold.

[0024] Figure 5 (A) Immunoglobulin levels after vaccination. In the figure, "yellow" represents IgM+IgG, "green" represents IgM, "blue" represents IgG1+IgG2, "purple" represents IgG1, and "red" represents IgG2. (B) Cytokine and interleukin concentrations after vaccination. As shown in the figure, "D" represents a danger signal. (C) B cell population levels after vaccination. As shown in the figure, "black" represents the total number of B cells and "green" represents the total number of memory B cells. (D) B lymphocyte counts for each entity state. (E) Plasma B lymphocyte levels after vaccination. (F) CD4T helper lymphocyte levels after vaccination. (G) CD4 + T regulatory lymphocyte levels. Total, memory, and counts of each entity state are plotted here. (H) CD8 + T cytotoxic lymphocyte levels. (I) Natural killer cell levels after vaccination. (J) Dendritic cell levels after vaccination.

[0025] Figure 6 Schematic diagram of the construction of vaccine protein expression vector.

[0026] Figure 7 This is an SDS-PAGE image; M in the figure: marker; 1: precipitation after induced expression; 2: protein after purification using a nickel column.

[0027] Figure 8WB images: M: marker; 1-2: the purified protein reacts with the serum of Brucella patients; 3-4: the purified protein reacts with the serum of tuberculosis patients; 5-6: the purified protein does not react with the serum of healthy people.

[0028] Fig. 9 These are the results of the ELISA experiment. In the figure, pbs is the control group; vaccine is the vaccine group. DETAILED DESCRIPTION

[0029] The following is further described in detail through specific implementation methods:

[0030] Development of MEV-hBD3 mRNA vaccine

[0031] The specific steps include:

[0032] S1. Design of candidate antigens

[0033] Access the Uniprot online website to obtain the FASTA sequences of Omp25 (accession: Q45321), Omp31 (accession: P0A3U4), MPT70 (accession: P9WNF5), and MPT83 (accession: P9WNF3).

[0034] The e-values ​​of Omp25, Omp31, MPT70 and MPT83 detected by BLASTp were 4.6e-156, 1.3e-173, 1.7e-130 and 8.8e-153, respectively, indicating that these four proteins are non-homologous to humans, so using the above four proteins to construct a vaccine can reduce the possibility of stimulating the body to produce an immune response.

[0035] After obtaining the sequences, the signal peptides of the four proteins were predicted and analyzed. The present invention used SignalP 5.0 software to analyze the protein sequences of potential signal peptides and predict the signal peptides (SPs) and their related cleavage sites in Gram-negative bacterial proteins. Figure 1 SignalP 5.0 software was used to analyze the protein sequence of potential signal peptides. The pink part represents the extracellular domain.

[0036] After analysis: the Omp25 signal peptide sequence is (SEQ ID NO: 2): MRTLKSLVIVSAALLPFSATAFAA.

[0037] The signal peptide sequence of Omp31 is (SEQ ID NO: 3): MKSVILASIAAMFATSAMAA. The signal peptide sequence of MPT70 is (SEQ ID NO: 4): MKVKNTIAATSFAAAGLAALAVAVSPPAAA. The signal peptide sequence of MPT83 is (SEQ ID NO: 5): MINVQAKPAAAASLAAIAIAFLAGC.

[0038] S2. Identification of epitopes

[0039] (1) Identification of CD8+ T cell epitopes of Omp25, Omp31, MPT70, and MPT83 proteins

[0040] The high-frequency alleles HLA-A*1101 (13.46%), HLA-A*0201 (12.50%), HLA-A*0301 (10.10%), HLA-DRB1*0701 (16.35%), HLA-DRB1*1501 (8.65%) and HLA-DRB1*0301 (7.69%) in Xinjiang were selected, and the online software IEDB and NetCTLpan1 server were used to predict CD8+ T cell epitopes.

[0041] Specifically, the antigenicity of the epitope was evaluated using VaxiJen online web server. An epitope with an E value greater than 0.5 was considered antigenic. The allergenicity of the epitope was evaluated using AllerTopV.2.0 (http: / / www.ddg-pharmfac.net / AllerTOP). The toxicity of the epitope was evaluated using ToxinPred server. Only epitopes that were antigenic, non-allergenic, and non-toxic were included in the selection of epitopes. Finally, 13 CD8 + T cell epitopes.

[0042] Table 1: 13 CD8 + T cell epitope (CTL epitope)

[0043]

[0044]

[0045] (2) Identification of CD4+ T cell epitopes

[0046] The high-frequency alleles HLA-A*1101 (13.46%), HLA-A*0201 (12.50%), HLA-A*0301 (10.10%), HLA-DRB1*0701 (16.35%), HLA-DRB1*1501 (8.65%) and HLA-DRB1*0301 (7.69%) in Xinjiang were selected.

[0047] CD4 prediction using IEDB and NetMHC-IIpan-4.0 online server + T cell epitopes were evaluated for antigenicity using VaxiJenonline web server. An epitope with an E value greater than 0.5 was considered antigenic. AllerTopV.2.0 was used to evaluate the allergenicity of the epitope. ToxinPred server was used to evaluate the toxicity of the epitope. Only epitopes with antigenicity, non-allergenicity, and non-toxicity were included in the selection of epitopes. Finally, 14 CD4+T cell epitopes were screened.

[0048] Table 2: 13 CD4 + T cell epitope (HTL epitope)

[0049] Serial number Serial Number <![CDATA[CD4 + Amino acid sequence of T cell epitope]]> Protein 1 SEQ ID NO:19 LYLGYGWNKAKTSTV Omp25 2 SEQ ID NO:20 YLGYGWNKAKTSTVG Omp25 3 SEQ ID NO:21 AGWNFQQDQIVYGVE Omp25 4 SEQ ID NO:22 RARLGYTATERLMVY Omp31 5 SEQ ID NO:23 VRARLGYTATERLMV Omp31 6 SEQ ID NO:24 GTVRARLGYTATERL Omp31 7 SEQ ID NO:25 LLTSILTYHVVAGQT MPT70 8 SEQ ID NO:26 VQGMSQDPVAVAASN MPT70 9 SEQ ID NO:27 LTSILTYHVVAGQTS MPT70 10 SEQ ID NO:28 PASVQGMSQDPVAVA MPT70 11 SEQ ID NO:29 ASVQGMSQDPVAVAA MPT70 12 SEQ ID NO:30 PTNAAFDKLPAATID MPT83 13 SEQ ID NO:31 DQLKTDAKLLSSILT MPT83 14 SEQ ID NO:32 QLKTDAKLLSSILTY MPT83

[0050] (3) Identification of conformational B cell epitopes

[0051] B cell epitopes were identified using online servers IEDB and Ellipro of ABCpred. The antigenicity of the epitopes was evaluated using the VaxiJen online server, and an E value of >0.5 was considered antigenic. The allergenicity of the epitopes was evaluated using the AllerTop online server, and the toxicity of the epitopes was evaluated using the ToxinPred online server. Only epitopes that were antigenic, non-allergic, and non-toxic were included in the selection of epitopes. Finally, four B cell conformational epitopes were selected.

[0052] Table 3: Conformational B cell epitopes (CB epitopes)

[0053]

[0054] (4) Identification of linear B cell epitopes

[0055] ABCpred (www.imtech.res.in / raghava / abcpred / ) was used to predict linear B cell (LB) epitopes. Therefore, the overall prediction accuracy was 65.93%, sensitivity was 67.14%, specificity was 64.71%, and positive predictive value was 65.61% when the software was tested using five-fold cross validation. Finally, epitopes with a score of 0.9 or above were selected as alternative LB epitopes. After screening, 2 LB epitopes were obtained in Omp25, 2 LB epitopes were obtained in Omp31, and 1 LB epitope was obtained in MPT70.

[0056] Table 4: Linear B cell epitopes (LB epitopes)

[0057]

[0058]

[0059] From Tables 1 to 4, we can see that:

[0060] Select 3 CD4 from Omp25 protein + T cell epitope (HTL epitope), 2 CD8 + T lymphocyte epitope (CTL epitope), 2 linear B cell epitopes (LB epitopes) and 2 conformational B cell epitopes (CB epitopes).

[0061] Three HTL epitopes, six CTL epitopes, two LB epitopes and one CB epitope were selected from the Omp31 protein.

[0062] Five HTL epitopes, three CTL epitopes, one LB epitope and one CB epitope were selected from MPT70.

[0063] Three HTL epitopes and two CTL epitopes were selected from MPT83 for subsequent vaccine construction.

[0064] Figure 2 These are the HTL epitopes, CTL epitopes, LB epitopes and CB epitopes finally selected for vaccine design, among which "green" represents the HTL cell epitope of Omp25, "blue" represents the T cell epitope of Omp31, "purple" represents the T cell epitope of MPT70, and "red" represents the T cell epitope of MPT83.

[0065] Table 5: List of selected CB epitopes and MEV-hBD3 mRNA vaccine design

[0066]

[0067]

[0068] Note: The selected B cell epitopes are antigenic, non-toxic, and non-allergenic

[0069] S3. Construction of recombinant multivalent epitope vaccine

[0070] The construction structure of the MEV-hBD3 mRNA vaccine includes the following components from the N-terminus to the C-terminus: 5'm7GCap-5'UTR-Kozak sequence-tPA (signal peptide) leader sequence-AAY (Linker)-CD8+T cell epitope-GPGPG (Linker)-CD4+T cell epitope-KK (Linker)-B cell linear epitope-KK (Linker)-B cell conformational epitope-EAKKK (Linker)-HBD3-stop codon-3'UTR-Poly (A) tail.

[0071] Human β-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent in vitro bactericidal activity. It has the ability to enhance the transmission of inflammatory signals and trigger the initiation of adaptive immune responses through antigen presenting cells, such as dendritic cells. HBD3 was selected as an adjuvant for the vaccine, and the specific sequence is:

[0072] MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRG RKCCRRKK.

[0073] The adjuvant sequence was linked to the N-terminus of the vaccine construct using the EAAAK linker.

[0074] In order to ensure the correct translation and expression of proteins after administration of the mRNA vaccine, the nucleotide sequence of the MEV-hBD3 mRNA vaccine provided by the present invention also adds other components, such as a tissue plasminogen activator (tPA) leader sequence, 5' and 3' untranslated regions (UTRs), a Kozak sequence and a stop codon.

[0075] The vaccine constructed in the present invention is named MEV-hBD3 mRNA vaccine, and the amino acid sequence is SEQ ID NO: 1.

[0076] An amino acid sequence of the final constructed vaccine (SEQ ID NO: 1) is as follows:

[0077] AAYTAGIAGSQIKAMYKLTDNILGRVAMYTIDQLKTDAKAMYKLPAATIDQLAMYYAINNNWTLKAMYSAGASGLEGKAMYASALHTWSDKAMYSGLEGKAETKAMYTLGAGAEYAIAMYSGLEGKAETKAMYTVTGQGNSLKAMYGQLNPQVNLVAMYGMSQDPVAVAGPGPGLYLGYGWNKAKTSTVGPGPGYLGYGWNKAKTSTVGGPGPGAGWNFQQDQIVYGVEGPGPGRARLGYTATERLMVYGPGPGVRARLGYTATERLMVGPGPGGTVRARLGYTATERLGPGPGLLTSILTYHVVAGQTGPGPGVQGMSQDPVAVAASNGPGPGLTSILTYHVVAGQTSGPGPGPASVQGMSQDPVAVAGPGPGASVQGMSQDPVAVAAGPGPGPTNAAFDKLPAATIDGPGPGDQLKTDAKLLSSILTGPGPGQLKTDAKLLSSILTYKKAGIAGSQIKLNGLDDKKQDQIVYGVEGDAGYSWKKQGMSQDPVAVAASNNPKKYIGINAGYAGGKFKHPKKLHTWSDKTKAGWTLCAKKKKTSTVGSKPSKDGLKLNNGLDDESKGNKNYDLAGTTVRNKLDKKQPPVPAPVEVAPQYLLNPVALTDNIGIGYKKKFIGIGGVRYINNNWGLNYKKKVGPGPASGQLNPQVNLVDTNSGQTASKLPASTIDELKTNSSAGQTSPANVVGTRQTGASTVTGQGKKNSLKVGNADTANDEAAAKMRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK

[0078] Experiment 1: Analysis of the Physicochemical Properties and Antigenicity of the MEV-hBD3 mRNA Vaccine

[0079] The present invention analyzes the antigenicity and allergenicity of the MEV-hBD3 mRNA vaccine constructed by the above amino acid sequence. SOLpro (http: / / scratch.proteomics.ics.uci.edu / ) is used to predict the solubility of the mRNA vaccine. Through multiple runs, 10-fold cross validation is performed with a threshold of 0.5, and the overall accuracy of the server is 74.15%. ProtParam (http: / / web.expasy.org / protparam / ) is used to predict the physicochemical properties of the mRNA vaccine, namely the average molecular weight, isoelectric point (PI) and hydrophilicity.

[0080] Table 4: Analysis results of the physical and chemical properties and antigenicity of the MEV-hBD3 mRNA vaccine

[0081]

[0082] Experiment 2: Prediction of secondary and tertiary structures of MEV-hBD3 mRNA vaccine

[0083] The RNAfold tool was used to predict the secondary structure of the MEV-hBD3 mRNA vaccine. The corresponding minimum free energy (MFE) was predicted to be -807.30 kcal / mop. The smaller the value, the greater the stability. This proves that the mRNA secondary structure of MEV-hBD3 mRNA is very stable. Figure 3 The secondary structure of the MEV-hBD3 mRNA vaccine was predicted using the RNAfold website. A: optimal secondary structure; B: centroid secondary structure.

[0084] In order to determine the secondary and tertiary structures of the polypeptide, the present invention used the SOPMA tool and NetSurfP-2.0 to conduct experiments respectively. The SOPMA results showed 23.67% of extended chains and 43.67% of irregular curls, proving that the secondary structure was stable. The high-precision supercomputer running software NetSurfP-2.0 was used to predict the tertiary structure of MEV-hBD3mRNA. The final results showed that the secondary and tertiary structures of the MEV-hBD3 mRNA vaccine could match each other, and the model rationality was high. It shows that the MEV-hBD3mRNA vaccine has a stable and reasonable structure and can be used as a vaccine for subsequent experiments.

[0085] Figure 4 shows the secondary structure of the MEV-hBD3 mRNA vaccine predicted by NetSurfP-2.0. When the threshold is 25%, the red color rises upward to indicate residue exposure, while the sky blue represents buried residues. As shown in the figure, "orange" represents α-helix, "blue" represents β-folding, and "purple" represents random curling. Among them, the extended chain is represented as an expanded black line, the thickness of which is equal to the probability of disordered residues.

[0086] Experiment 3: Molecular docking

[0087] The toll-like receptor (TLR) family, including toll-like receptor 4 (TLR-4) (PDB ID: 4G8A), is involved in the innate immune response and can recognize bacterial endotoxin- or lipopolysaccharide-induced inflammatory responses. In order to ensure the proper binding of the TRL4 immune cell receptor to the MEV-hBD3 mRNA vaccine and its subsequent effective immune response, the present invention simulates the docking of immune molecules. The MEV-hBD3 mRNA vaccine polypeptide was docked with TLR-4 (PDB ID: 4G8A) using the HDOCK server. The resulting three-dimensional structure was visualized using PyMOL. The results of the molecular docking analysis showed that the optimal docking complex was selected for analysis. The results showed that the docking score of the optimal complex of MEV1-TLR4 was -352.36 and the ligand RMSD was 109.48A. The present invention demonstrates the interaction between the MEV-hBD3 mRNA vaccine and TLR4 at the 2D and 3D structural levels. Among them, the force mainly includes hydrogen bonds and van der Waals forces.

[0088] Experiment 4: Immune simulation

[0089] The immunogenicity of the MEV-hBD3 mRNA vaccine was calculated by the C-ImmSim server. In all simulations, a specific position scoring matrix (PSSM) was used, with time intervals of 1, 84, and 164 (each time step was 8 hours), and three injections were simulated to study the functions and roles of central immune organs such as bone marrow, thymus, and lymph. The simulation parameters were set as random seed establishment of 12345, simulation volume specified as 50, and simulation step configuration of 1050. Through immune simulation, it was found that after the injection of the MEV-hBD3 mRNA vaccine, it may cause fluctuations in the relevant immune cells of the innate and adaptive immune response. These cells include HTL, CTL, T regulatory lymphocytes, natural killer cells, dendritic cells, and other immune cells.

[0090] After three doses of the vaccine, immunoglobulin levels (IgM, IgG1, IgG2, IgG1+IgG2, IgM+IgG) all showed an upward trend. After the last dose of the vaccine, immunoglobulin levels peaked ( Figure 5 A). After three vaccinations, cytokines (IFN-γ, TGF-β, IL-10 and IL-12) in the host also increased ( Figure 5 B). Among them, the increase of IFN-γ is the main factor. However, in this process, the risk factors did not fluctuate significantly. The presence of general B cells gradually increased, mainly based on the increase of memory B cells ( Figure 5 C, Figure 5 D) Plasma cells ( Figure 5 E) also showed a gradual upward trend, and the antibodies produced were mainly IgM and IgG1. Similarly, TH cells ( Figure 5 F) also gradually increased, mainly due to the increase of memory T cells. T regulatory lymphocytes ( Figure 5 G) gradually decreased after rising. CTL( Figure 5 H), natural killer cells ( Figure 5 I), dendritic cells ( Figure 5 J), macrophages ( Figure 5 K) and epithelial cells ( Figure 5 L) is in a relatively stable dynamic state. In particular, dendritic cells can present antigenic peptides on MHC class I and class II molecules, including presenting antigens to T cells.

[0091] This comparative study demonstrated that MEV-hBD3 mRNA is an effective vaccine that induces robust immune responses against B. melitensis and M. tuberculosis H37Rv (L4 strain).

[0092] Figure 5 (A) Immunoglobulin levels after vaccination. In the figure, "yellow" represents IgM+IgG, "green" represents IgM, "blue" represents IgG1+IgG2, "purple" represents IgG1, and "red" represents IgG2. (B) Cytokine and interleukin concentrations after vaccination. As shown in the figure, "D" represents a danger signal. (C) B cell population levels after vaccination. As shown in the figure, "black" represents the total number of B cells and "green" represents the total number of memory B cells. (D) B lymphocyte counts for each entity state. (E) Plasma B lymphocyte levels after vaccination. (F) CD4T helper lymphocyte levels after vaccination. (G) CD4 + T regulatory lymphocyte levels. Total, memory, and counts of each entity state are plotted here. (H) CD8 + T cytotoxic lymphocyte levels. (I) Natural killer cell levels after vaccination. (J) Dendritic cell levels after vaccination.

[0093] Experiment 5: In vivo experiments in mice

[0094] The present invention uses plasmid vector pVAX1 to complete the expression of vaccine protein (plasmid vector pVAX1 was purchased from Shanghai Shenggong), and after purification, it is used for in vivo vaccination experiments in mice. The present invention uses nasal drip to vaccinate mice, and 8-week-old SPF-level BALB / c mice provided by the Animal Experiment Center of Xinjiang Medical University are randomly divided into MEV-hBD3mRNA group (n=8) and health control (health control, HC) group (n=8). Mice in the MEV-hBD3 mRNA group are instilled with vaccine protein through the nose, and mice in the HC group are instilled with normal saline through the nose. The concentration of the vaccine protein solution is 500μg / ml, and 60μl (30μl in each nostril) is given to mice each time, once every 2 weeks, for a total of 3 times. The endotoxin of the vaccine solution is negative with horseshoe crab reagent (LAL) before use. Two weeks after the third inoculation, the serum of mice in the MEV-hBD3 mRNA and HC groups was collected under sterile conditions for subsequent experimental detection. The present invention was approved by the Animal Ethics Committee of Xinjiang Medical University, and the entire animal experiment process was carried out in strict accordance with the experimental requirements and operating guidelines of the Animal Ethics Committee of Xinjiang Medical University.

[0095] Figure 6 Schematic diagram of the construction of vaccine protein expression vector.

[0096] Experiment 6: WB experiment

[0097] MEV-hBD3 was induced to express with the help of a prokaryotic expression system, and the vaccine protein was subsequently purified with the help of a His tag, with a molecular weight of approximately 81 kDa. SDS-PAGE and WB experiments verified that MEV-hBD3 was successfully expressed and had good immunoreactivity, reacting with sera from patients with brucellosis and tuberculosis, but not with sera from healthy people.

[0098] Figure 7 This is an SDS-PAGE image; M in the figure: marker; 1: precipitation after induced expression; 2: protein after purification using a nickel column.

[0099] Figure 8 WB images: M: marker; 1-2: the purified protein reacts with the serum of Brucella patients; 3-4: the purified protein reacts with the serum of tuberculosis patients; 5-6: the purified protein does not react with the serum of healthy people.

[0100] Experiment 7: ELISA experiment

[0101] Mouse serum antibodies were detected by indirect ELISA. After the mouse immunization cycle, blood was drawn from the tail vein of the mouse and coagulated at room temperature for 30 minutes. The supernatant was collected after centrifugation at 2000 rpm for 15 minutes. If there was precipitation, it was centrifuged again. The purified MEV-hBD3 was coated on a 96-well plate, blocked with skim milk powder for 2 hours, incubated with primary and secondary antibodies, and detected by OD at 37°C using an enzyme reader after TMB color development and termination. 450nm value.

[0102] Fig. 9 The results of the ELISA experiment are shown in Figure 1. In the figure, PBS is the control group and Vaccine is the vaccine group. The experiment proves that MEV-hBD3 has good immunogenicity and can stimulate the body to produce IgG antibodies.

[0103] In summary, the present invention adopts immunoinformatics methods to identify and screen antigen epitopes. Conventional vaccines target single bacteria of brucellosis and tuberculosis, while the present vaccine can target both brucellosis and tuberculosis at the same time. In theory, it has a preventive effect on these three Brucella species, thereby improving the protective efficiency of the vaccine and the population coverage.

[0104] The present invention successfully verified the immunoreactivity and immunity of the MEV-hBD3 mRNA vaccine through WB and ELISA experiments, laying a new theoretical and experimental foundation for subsequent brucellosis and tuberculosis vaccines.

[0105] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A MEV-hBD3 mRNA vaccine for combined prevention of brucellosis and tuberculosis, characterized in that: The amino acid sequence of the vaccine includes several CD8 + T cell epitope, CD8 + The amino acid sequence of the T cell epitope includes at least one of SEQ ID NO:6 to SEQ ID NO:18; the amino acid sequence of the vaccine also includes several CD4 + T cell epitope, CD4 + The amino acid sequence of the T cell epitope includes at least one sequence from SEQ ID NO:19 to SEQ ID NO:32; the amino acid sequence of the vaccine also includes several B cell epitopes, and the amino acid sequence of the B cell epitope includes at least one sequence from SEQ ID NO:33 to SEQ ID NO:

41.

2. The vaccine according to claim 1, characterized in that: The amino acid sequence of the vaccine also includes Linker-AAY, Linker-GPGPG, Linker-KK and Linker-GGGS.

3. The vaccine according to claim 1, characterized in that: The nucleotide sequence of the vaccine also includes the tissue plasminogen activator (tPA) leader sequence, 5' and 3' untranslated regions (UTRs), Kozak sequence and stop codon.

4. The vaccine according to claim 1, characterized in that: The amino acid sequence of the MEV-hBD3 mRNA vaccine is SEQ ID NO: 1.

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