MRNA vaccine and protein vaccine based on toxoplasma gondii multi-epitope gene and application of mRNA vaccine and protein vaccine in preparation of drugs for preventing toxoplasmosis

Abstract

The invention belongs to the technical field of biological medicines, and relates to an mRNA vaccine and a protein vaccine based on a toxoplasma gondii multi-epitope gene and application of the mRNA vaccine and the protein vaccine in preparation of medicines for preventing toxoplasmosis. And the nucleotide sequence of the mRNA vaccine is as shown in SEQ ID NO.3. The protein vaccine has an amino acid sequence as shown in SEQ ID NO. 1. The vaccine provided by the invention can effectively enhance humoral immunity and cellular immunity responses, and effectively prolong the survival time of mice attacked by the toxoplasma ME49 strain (type II).

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to an mRNA vaccine and a protein vaccine based on a multi-epitope gene of Toxoplasma gondii and their application in preparing a drug for preventing toxoplasmosis. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Toxoplasmosis is a parasitic disease caused by the intracellular protozoan Toxoplasma gondii, which can be transmitted by drinking water contaminated by oocysts in the host's feces or eating food containing tissue cysts. About one-third of the world's population is positive for Toxoplasma infection. The infection rate of cats is relatively high, about 24.5%; in terms of livestock and poultry, the average Toxoplasma antibody positivity rates of pigs, sheep, goats, cattle, and chickens are 29.5%, 13.9%, 17.1%, 7.5%, and 19.0%, respectively. In addition to causing economic losses to the breeding industry, the infection of these livestock and poultry has also become a potential risk factor for human infection. The clinical symptoms caused by Toxoplasma infection mainly depend on the species of Toxoplasma and the host's immune system. Its pathological changes are mainly manifested by tissue inflammation and necrosis caused by parasite proliferation, and secondly by cell apoptosis induced by some effector molecules. Infection in people with normal immune function is often latent infection, usually an asymptomatic infection, or only manifests mild clinical symptoms such as fever, malaise or lymphadenitis. However, when immunocompromised patients (such as those with AIDS) are infected, bradyzoites can reactivate and transform into cytotoxic tachyzoites and cause space-occupying lesions in the brain. Retinal choroiditis is also a common manifestation of T. gondii infection in healthy and immunocompromised people and may lead to retinal tissue loss and subsequent irreversible visual impairment. In addition, in pregnant women infected with the parasite for the first time, T. gondii tachyzoites can cross the placental blood barrier to reach the fetus, causing spontaneous abortion or congenital disabilities. Therefore, the prevention and treatment of toxoplasmosis is urgent. Summary of the invention

[0004] In order to address the deficiencies in the prior art, the purpose of the present invention is to provide an mRNA vaccine and a protein vaccine based on Toxoplasma multi-epitope genes and their use in the preparation of drugs for preventing toxoplasmosis. The present invention constructs mRNA vaccines and protein vaccines by screening the dominant epitope genes of Toxoplasma antigens. The mRNA vaccine and protein vaccine based on Toxoplasma multi-epitope genes show good humoral immunity and cellular immunity levels in immunized mice, can significantly prolong the survival time and survival rate of mice, and can be used for the prevention of toxoplasmosis in humans or animals.

[0005] The present invention uses the immune epitope database (IEDB) to screen the Toxoplasma gondii antigens SAG1, GRA7, and ROP16 for CD4 8 + T cell dominant epitopes and CD 4 + The T cell dominant epitope concentration area was predicted by comprehensive analysis of DNAStar-Protean software, and the B cell dominant epitope concentration area of ​​SAG1, GRA7, and ROP16 were connected to artificially synthesize the Toxoplasma gondii composite epitope peptide (T-SGR), thereby obtaining mRNA and protein vaccines based on Toxoplasma multi-epitope genes. Experiments on immunized mice showed that the vaccine provided by the present invention can effectively enhance humoral immunity and cellular immune responses, and effectively prolong the survival time of mice after being attacked by Toxoplasma ME49 strain (type II).

[0006] Based on the above research results, the present invention provides the following technical solutions:

[0007] In the first aspect, an mRNA vaccine based on a multi-epitope gene of Toxoplasma gondii, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0008] In the second aspect, a protein vaccine based on a Toxoplasma multi-epitope gene has an amino acid sequence as shown in SEQ ID NO.1.

[0009] In the third aspect, a nucleic acid molecule, the nucleotide sequence of which is complementary to the mRNA vaccine described in the first aspect of the present invention, or is a nucleotide capable of encoding the protein vaccine described in the second aspect of the present invention.

[0010] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.2.

[0011] In a fourth aspect, a recombinant vector comprises a backbone vector and the nucleic acid molecule described in the third aspect of the present invention.

[0012] The backbone vector of the present invention can be any one or more of a plasmid, a viral vector, a phage, a phagemid, a cosmid, a F cosmid or an artificial chromosome. In some embodiments, the backbone vector is a plasmid for preservation or amplification. Specifically, the backbone vector is a pET-28a plasmid.

[0013] A fifth aspect provides a host cell comprising the recombinant vector described in the fourth aspect of the present invention.

[0014] The host cell of the present invention may be a bacterial cell, a fungal cell, an animal cell, etc. Among them, the bacterial cell may be Escherichia coli (BL21), Gram-negative bacillus, Bacillus subtilis, Pseudomonas aeruginosa, etc.; the fungal cell may be yeast, etc. In some embodiments, the host cell is Escherichia coli, preferably Escherichia coli BL21.

[0015] The present invention connects the coding gene to the pET-28a vector, transforms the competent cell BL21, and then induces IPTG to express the fusion protein, and uses a His-tag nickel ion protein purification column to purify the expression product to obtain a protein vaccine based on Toxoplasma multi-epitopes. The target protein vaccine obtained by this method has higher purity.

[0016] In the sixth aspect, an mRNA vaccine based on the multi-epitope gene of Toxoplasma gondii as described in the first aspect of the present invention, a protein vaccine based on the multi-epitope gene of Toxoplasma gondii as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, a recombinant vector as described in the fourth aspect of the present invention, or a host cell as described in the fifth aspect of the present invention is used in the preparation of a drug for preventing toxoplasmosis.

[0017] In some embodiments, the drug is administered to a human or an animal. The animal described in the present invention may be a mouse, a rat, a pig, a dog, a cow, a sheep, a chicken, etc.

[0018] In some embodiments, the drug further comprises a pharmaceutical excipient, which may be an emulsifier, a stabilizer, a preservative, a filler, an antioxidant, a buffer, a pH adjuster, a diluent, and the like.

[0019] The drug of the present invention can be administered into the body in a known manner. The administration method can be intravenous injection, inguinal injection, subcutaneous injection, rectal administration, intramuscular injection, etc. When administering the drug, a single dose or multiple doses can be used.

[0020] In some embodiments, the drug has any of the following effects:

[0021] (1) Increase antibody titer levels;

[0022] (2) increase in the levels of IL-2, IFN-γ, or IL-10 in splenocytes;

[0023] (3) Increase the content of CD4 positive cells or CD8 positive cells.

[0024] The beneficial effects of the present invention are:

[0025] The mRNA and protein vaccines based on the Toxoplasma multi-epitope gene provided by the present invention can induce mice to produce effective humoral and cellular immune responses, which can not only increase positive cells, cytokines, CD4 positive cells and CD8 positive cells, but also improve the survival rate of mice. The vaccine can be used as an effective candidate vaccine for controlling acute or chronic infection of Toxoplasma gondii and for the prevention of toxoplasmosis in humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 The figures are the construction diagram and in vitro expression result diagram of the mRNA vaccine in the embodiment of the present invention; A, schematic diagram of the construction and coating of T-SGR mRNA vaccine; B, in vitro transcription of T-SGR mRNA, M: DNA marker, 1: DNA fragment, 2: in vitro transcription product; C, in vitro expression of T-SGR mRNA in 293T cells determined by Western blotting; D, in vitro expression of T-SGR mRNA in 293T cells.

[0028] Figure 2 The figure is an in vitro expression and detection diagram of the Toxoplasma multi-epitope protein in the embodiment of the present invention; A, schematic diagram of the construction of the T-SGR multi-epitope plasmid; B, SDS-PAGE analysis of the expression product of pET-28a(+)-TSGR-T-His in Escherichia coli, lane M: protein marker, 1: negative control (non-induced), 2: IPTG induction, 3: supernatant after ultrasonic treatment, 4: precipitate after ultrasonic treatment, 5-6: precipitate denatured with 8M urea; C, protein blotting analysis of purified TSGR-His using anti-His monoclonal antibody, M: protein marker.

[0029] Figure 3 The results of the determination of anti-Toxoplasma immunoglobulin IgG and IgG1, IgG2a antibody subtypes in the serum of immunized mice in the embodiment of the present invention are shown in Figure 1. A. The serum was collected before immunization and two weeks and four weeks after the last immunization (n=3), and the IgG antibody level in the serum of mice was detected by ELISA, and the data were analyzed using one-way ANOVA. B. The IgG1 and IgG2a antibody levels in the serum of mice were detected by ELISA before immunization and two weeks after the last immunization, and the data were analyzed using one-way ANOVA. The figure shows that after two immunizations, the antibody titer level of the protein vaccine immunization group increased significantly compared with the control group.

[0030] Figure 4The results of the determination of IL-2, IL-4, IFN-γ, and IL-10 in the spleen cells of immunized mice in the embodiment of the present invention are shown in Figure 1. A, IFN-γ cytokine level; B, IL-4 cytokine level; C, IL-2 cytokine level; D, IL-10 cytokine level. The figure shows that the cytokine content of the vaccine immunization group increased significantly compared with the control group PBS.

[0031] Figure 5 The results of flow cytometry for T cell clustering in spleen cells and quantitative analysis of CD8+ or CD4+ T cells producing IFN-γ in the sample in the embodiment of the present invention are shown in the figure. Two weeks after the last immunization, the spleen of the immunized mouse was aseptically removed, and a spleen single cell suspension was prepared by grinding with a 200-mesh copper mesh. After adding multi-epitope peptides for stimulation and culture, flow cytometry was used to detect T cell clustering. The figure shows that compared with the control group PBS, the content of CD4 and CD8 positive cells in the protein vaccine immunization group increased significantly (P<0.01).

[0032] Figure 6 The figures are the results of immune protection of Toxoplasma multi-epitope mRNA vaccine and protein vaccine in the examples of the present invention; A. Weight change of C57 mice (n=5 / group) after being challenged with 200 ME49 strains 4 weeks after the last immunization; B. Survival curve of immunized animals (n=5 / group) after being challenged with 200 ME49 strains 4 weeks after the last immunization, and the survival curve was statistically analyzed using the Mantel-Cox test; 200 ME49 strains were injected into the mice via the inguinal cavity, and their survival curves and weight changes were detected. Compared with the PBS control group, the survival rate of mice in the vaccine immunization group was significantly improved. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] The reagents and methods not described in detail in the following examples are all conventional reagents and methods in the relevant field.

[0035] Example 1. Synthesis of Toxoplasma gondii multi-epitopes

[0036] Toxoplasma gondii antigens SAG1, GRA7, and ROP16 were screened for CD using IEDB + 8T cell dominant epitopes and CD + 4T cell dominant epitope concentration areas. DNAStar-Protean software was used to comprehensively analyze and predict the B cell dominant epitope concentration areas of SAG1, GRA7, and ROP16, as shown in Table 1.

[0037] Table 1 Predominant epitopes of Toxoplasma gondii antigenic epitopes screened

[0038]

[0039]

[0040] The screened dominant epitopes of Toxoplasma gondii are connected together to artificially synthesize a Toxoplasma gondii composite epitope peptide, which can be used as an artificially synthesized Toxoplasma gondii composite epitope peptide and also as a target protein vaccine. Its amino acid sequence is as follows: QARASSVVNNVARCSYGADSTLGPVKLSAEGPTTMTLVCGKDGVKVPQDNNQYCSGTTLTGCNEKSFKDILPKLTENPWQGNASSDKGATLTIKKEAFPAESAPGTPLKKLALPAVGMGASYFAADRLVPELTEEQQRGDEPLTTGQNVGTVLGFAALAAAAAFLGMGLTRTYRHFS PRKNRSRQPALEQEVPESGEDGEDARQSAPGTPQSATLDSGIQSPAQERRGSPQRQIAMSTENPADSGASQLASSVSSYVAVQTPHVKRSERIRRVRLSEEEGLEEVQQLKAAAAQLLVAVPDYEAMRAVLQEAVLSEQRVATRKRKRKQPPGAVESAVDEVFPPNERVMMINANGVPIALYNRG, as in SEQ Shown as ID NO.1.

[0041] The coding gene corresponding to the amino acid sequence is as follows:

[0042] CAAGCCAGAGCCTCATCGGTCGTCAATAATGTCGCAAGGTGCTCCTACGG

[0043] TGCAGACAGCACTCTTGGTCCTGTCAAGTTGTCTGCGGAAGGACCCACT

[0044] ACAATGACCCTCGTGTGCGGGAAAGATGGAGTCAAAGTTCCTCAAGACA

[0045] ACAATCAGTACTGTTCCGGGACGACGCTGACTGGTTGCAACGAGAAATC

[0046] GTTCAAAGATATTTTGCCAAAATTAACTGAGAACCCGTGGCAGGGTAACG

[0047] CTTCGAGTGATAAGGGTGCCACGCTAACGATCAAGAAGGAAGCATTTCC

[0048] AGCCGAGAGCGCCCCCGGCACCCCCCTGAAGAAGCTCGCGCTGCCGGCT

[0049] GTTGGTATGGGTGCATCGTATTTTGCCGCTGATAGAATTCTGCCGGAACTA

[0050] ACAGAGCAGCAACAGACAGGCGAAGAACCCCTAACCACCGGCCAGAAT

[0051] GTGAGCACTGTGTTAGGCTTCGCAGCGCTTGCTGCTGCCGCAGCGTTCCT

[0052] TGGCATGGGTCTCACGAGGACGTACCGACATTTTTCCCCACGCAAAAAC

[0053] AGATCACGGCAGCCTGCACTCGAGCAAGAGGTGCCTGAATCAGGCAAA

[0054] GATGGGGAGGATGCCCGCCAGAGCGCCCCCGGCACCCCCCAGAGCGCTA

[0055] CATTGGATTCAGGAATACAGTCTCCGGCACAAGAGCGTCGGGGATCCCCT

[0056] CAAAGACAGATTGCGATGTCGACCGAAAATCCAGCGGATAGCGGCGCCT

[0057] CGCAGCTTGCCTCCAGTGTTTCTAGTTATGTAGCAGTACAAACTCCTCATG

[0058] TGAAACGTTCAGAACGCATCCGGCGCGTTCGACTTTCAGAAGAGGGTCT

[0059] GGAAGAAGTTCAGCAGCTGAAAGCAGCTGCCGCACAGCTTCTCGTAGCG

[0060] GTTCCGGACTATGAGGCAATGCGGGCTGTTCTGCAAGAGGCGGTCCTCTC

[0061] AGAACAGAGGGTTGCTACCCGTAAGCGGAAGAGAAAGCAACCTCCAGG

[0062] AGCGGTGGAGTCAGCTGTTGACGAAGTGTTCCCTCCAAATGAGCGTGTCATGATGATAAATGCCAACGGAGTGCCGATCGCTCTATACAATCGTGG, as shown in SEQ ID NO.2.

[0063] Example 2. Toxoplasma gondii multi-epitope mRNA vaccine

[0064] The coding gene of the target peptide was connected to the pcDNA3.1 vector, and the DNA fragment of the target peptide was amplified. After comprehensive consideration of multiple factors such as CAI, MFE, AUP, GC content, and restriction sites, T7 promoter, 5'UTR, Kozak, 3'UTR, and PolyA tail were added. The linear double-stranded DNA containing the T7 promoter sequence was used as a template, and ATP, GTP, CTP, and N1-Me-pUTP were used as substrates. The DNA sequence downstream of the promoter was transcribed by T7 RNA polymerase to efficiently synthesize mRNA, and a chemical one-step capping method (Cap3011) was used to finally obtain the mRNA transcription product. Using the SM102 lipid formula and microfluidic equipment, the mRNA was mixed with the buffer, and the two were incubated for self-assembly. After purification and concentration, the mRNA coating was completed ( Figure 2 ).

[0065] The sequence of the mRNA is as follows:

[0066] TAATACGACTCACTATA AGGTCCAACACAACATATACAAAACAAACGAATC

[0067] TCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTT

[0068] AAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAgccacc

[0069] ATGTACCGGATGCAGCTGCTGAGCTGCATCGCCTTGTCCCTGGCGCTCGT

[0070] GACCAACTCCCAGGCGCGCGCTTCCTCTGTGGTGAACAACGTGGCACGG

[0071] TGCTCTTACGGGGCCGACAGCACCCTCGGCCCCGTGAAGTTGTCGGCCG

[0072] AGGGGCCGACAACTATGACTCTCGTGTGCGGCAAGGACGGGGTGAAGGT

[0073] GCCCCAGGACAATAACCAGTATTGTTCGGGCACCACCCTGACCGGCTGC

[0074] AACGAGAAGTCATTCAAGGACATTCTGCCCAAGTTGACAGAGAACCCTT

[0075] GGCAGGGGAACGCCTCCTCTGATAAGGGGGCCACCCTGACTATCAAGAA

[0076] GGAGGCGTTCCCCGCCGAGAGTGCCCCCGGGACGCCGCTGAAGAAGCT

[0077] GGCCCTGCCCGCCGTGGGCATGGGGGCCAGCTACTTCGCGGCGGACCGG

[0078] ATCCTCCCCGAGCTGACTGAGCAGCAGCAGACCGGGGAGGAGCCTCTGA

[0079] CAACTGGGCAGAATGTGAGCACCGTGCTGGGATTCGCCGCGCTGGCTGC

[0080] CGCGGCCGCCTTCCTGGGGATGGGGCTGACGAGGACCTATCGTCACTTC

[0081] AGCCCCAGGAAGAACCGGTCGCGGCAGCCAGCTCTCGAGCAGGAGGTG

[0082] CCTGAGTCCGGCAAGGATGGCGAGGATGCTCGGCAGTCTGCCCCGGGGA

[0083] CTCCCCAGTCCGCCACGCTGGACTCTGGCATCCAGAGTCCAGCGCAGGA

[0084] GCGGAGGGGGAGTCCCCAGCGGCAGATTGCCATGAGCACCGAGAATCCT

[0085] GCCGACTCCGGCGCCTCCCAGCTCGCCTCTTCCGTGTCGTCATACGTTGC

[0086] CGTTCAGACACCGCACGTGAAGCGCAGCGAGCGCATCCGTCGAGTCCGG

[0087] CTCAGCGAGGAGGGGCTGGAGGAGGTGCAGCAGCTGAAGGCTGCTGCA

[0088] GCCCAGCTCCTCGTCGCTGTGCCGGACTACGAGGCGATGCGCGCTGTGC

[0089] TTCAGGAGGCGGTGCTGAGCGAGCAGCGTGTGGCGACACGGAAGAGGA

[0090] AGAGGAAGCAGCCGCCTGGGGCGGTCGAGAGCGCCGTGGACGAGGTGT

[0091] TCCCCCCGAATGAGAGGGTGATGATGATCAACGCCAATGGCGTGCCCATC

[0092] GCCCTCTACAATCGGGGGTGA GCTGCAGAATTCGTCGACGGATCCGATCT

[0093] GGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCC

[0094] CGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCC

[0095] CCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAAT

[0096] GCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTG

[0097] ATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAG

[0098] GGTTGGTCAATTTCGTGCCAGCCACACCCTCGAGCTAGC aaaaaaaaaaaaaaaa

[0099] aaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa, as shown in SEQ ID NO. 3.

[0100] Among them, the italic ones are promoters, the underlined ones are non-coding sequences (UTR), and the gray ones are start codons / stop codons.

[0101] Example 3. Purification of Toxoplasma gondii multi-epitope protein

[0102] The target peptide encoding gene (as shown in SEQ ID NO.2) was connected to the pET-28a vector, and the competent cell BL21 was transformed and induced by IPTG to express the fusion protein. The expression product was purified using a His-tag nickel ion protein purification column, and the expression of the recombinant protein was identified by SDS-PAGE and Western blot ( Figure 3 ).

[0103] Example 4. Immunization of C57 mice with Toxoplasma gondii multi-epitope gene vaccine

[0104] SPF female C57 mice (6 weeks old) were purchased from Jinan Weitong Lihua Co., Ltd. 30 mice were randomly divided into 3 groups. The experimental group was injected with 10 μg protein vaccine and mRNA vaccine through the hind leg groin, and the control group was injected with the same volume of PBS. The mice were immunized twice, with an interval of 2 weeks.

[0105] Example 5. Determination of humoral immunity level in immunized mice

[0106] Blood was collected from the tail of mice before and after the last immunization. The blood samples were allowed to stand at room temperature for 3 hours and then centrifuged at 3000 rpm for 30 minutes to collect serum. The total IgG content in the serum was determined using an enzyme-linked immunosorbent assay. The results are shown in Table 1. Figure 3 After two immunizations, both mRNA and protein vaccines induced mice to produce antibodies compared with the PBS control group ( Figure 3 ), by detecting IgG1 and IgG2a antibody subtypes, the level of IgG2a subantibody in the serum of mice in the mRNA vaccine immunization group and the protein immunization group was significantly higher than that of IgG1 subantibody, proving that mRNA vaccines and protein vaccines based on Toxoplasma multi-epitope genes can induce Th1 immune response in mice.

[0107] Example 6. Determination of cellular immunity level in immunized mice

[0108] Two weeks after the last immunization, the spleen of the immunized mouse was aseptically removed and ground through a 200-mesh copper mesh to prepare a spleen single cell suspension. After removing the red blood cells from the spleen cells using red blood cell lysis buffer, the cells were resuspended in 1640 medium containing 10% fetal bovine serum and the concentration of the cell suspension was adjusted to 1×10 6 cells / ml, and after adding composite epitope peptides for stimulation and culture, the content of cytokines was detected by ELISA. The results showed that the levels of IFN-γ and IL-10 in the spleen cells of mice in the mRNA vaccine immunization group and the protein vaccine immunization group were significantly higher than those in the control group (P<0.01)( Figure 4 ), which proved that mRNA vaccine and protein vaccine based on Toxoplasma multi-epitope genes can enhance the cellular immunity level of mice.

[0109] Example 7. Determination of T cell populations in spleen cells of immunized mice

[0110] Two weeks after the last immunization, the spleen of the immunized mouse was aseptically removed and ground through a 200-mesh copper mesh to prepare a spleen single cell suspension. After adding multi-epitope peptides for stimulation and culture, CD4 was labeled with fluorescent antibodies. + 、CD8 + T cells were detected by flow cytometry. Figure 5 The results showed that compared with the control group PBS, the CD4 + 、CD8 + The content of immune T cells increased significantly (P<0.01).

[0111] Example 8. Study on the immune protection of Toxoplasma gondii multi-epitope vaccine

[0112] Four weeks after the last immunization, the immunized mice were subjected to an attack experiment. The mice were intraperitoneally injected with 200 Toxoplasma gondii ME49 strains. After infection, their weight changes and survival curves were detected. The attack experiment of immunized mice showed that compared with the control group PBS, the weight of mice in the mRNA vaccine immunization group and the protein vaccine immunization group had no significant effect, and the survival rate could reach 80% ( Figure 6 ).

[0113] In summary, mRNA vaccines and protein vaccines based on multi-epitope genes of Toxoplasma gondii can induce effective humoral and cellular immune responses in mice and improve the survival rate of mice. This vaccine can be used as an effective candidate vaccine to control acute or chronic infection of Toxoplasma gondii and prevent toxoplasmosis in humans and animals.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. 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. An mRNA vaccine based on a multi-epitope gene of Toxoplasma gondii, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

3.

2. A protein vaccine based on a multi-epitope gene of Toxoplasma gondii, characterized in that: The protein vaccine has an amino acid sequence as shown in SEQ ID NO.

1.

3. A nucleic acid molecule, characterized in that: The nucleotide sequence of the nucleic acid molecule is complementary to the mRNA vaccine described in claim 1; or, the nucleic acid molecule is a nucleotide capable of encoding the protein vaccine described in claim 2.

4. A recombinant vector, characterized in that: It comprises a backbone vector and the nucleic acid molecule according to claim 3.

5. The recombinant vector according to claim 4, characterized in that: The backbone vector is a plasmid used for preservation or amplification; preferably, the backbone vector is a pET-28a plasmid.

6. A host cell, characterized in that Containing the recombinant vector according to claim 4 or 5.

7. The host cell according to claim 6, characterized in that The host cell is Escherichia coli, preferably Escherichia coli BL21.

8. Use of the mRNA vaccine based on Toxoplasma multi-epitope gene according to claim 1, the protein vaccine based on Toxoplasma multi-epitope gene according to claim 2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4 or 5, or the host cell according to claim 6 or 7 in the preparation of a drug for preventing toxoplasmosis.

9. The use according to claim 8, characterized in that: The drug is administered to humans or animals; Or, the medicine further comprises pharmaceutical excipients.

10. The use according to claim 8, characterized in that: The drug has any of the following effects: (1) Increase antibody titer levels; (2) increase in the levels of IL-2, IFN-γ, or IL-10 in splenocytes; (3) Increase the content of CD4 positive cells or CD8 positive cells.

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