CN111939247B - A protein mixed vaccine for preventing human and animal toxoplasmosis - Google Patents

Abstract

The invention discloses a mixed protein vaccine for preventing toxoplasmosis of human and animals. One technical scheme to be protected by the invention is an immunogenic composition, and the active ingredients of the composition are a recombinant protein mixture consisting of Toxoplasma gondii recombinant protein 14-3-3, MIF and CDPK 3. In the embodiment of the invention, the BALB/c mouse is immunized by the recombinant protein mixture obtained by mixing the toxoplasma recombinant protein 14-3-3, MIF and CDPK3, and the result shows that the humoral immunity and the cellular immune response can be effectively enhanced by using the recombinant protein for immunization, the survival time of the mouse infected by the toxoplasma RH strain is obviously prolonged, the encysted number of the toxoplasma PRU chronic infection midbrain is obviously reduced, and a good immune effect is obtained. The vaccine prepared by the recombinant protein can be used as an effective candidate vaccine for preventing acute and chronic toxoplasma infection.

Description

A protein mixed vaccine for preventing human and animal toxoplasmosis

technical field

The invention relates to the field of medical biotechnology, in particular to a protein mixed vaccine for preventing human and animal toxoplasmosis.

Background technique

Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii is mostly a recessive infection in normal human body and will not have obvious symptoms, but it can cause serious complications in people with low immunity or immunodeficiency, such as cancer patients, AIDS patients and organ transplant recipients who have undergone long-term chemotherapy. Serious harm to human health. Infection of Toxoplasma gondii in early pregnancy can lead to fetal infection, which can lead to miscarriage, premature birth, fetal malformation and stillbirth, etc., affecting prenatal and postnatal care.

14-3-3 proteins are a group of highly conserved protein families widely found in yeast, plants, worms, protozoa, insects and humans. Studies have shown that 14-3-3 protein is involved in the regulation of a variety of important cellular life activities in vivo, such as cell signal transduction, cell differentiation and apoptosis. Toxoplasma gondii 14-3-3 (Tg14-3-3) is a cytoplasmic signal transduction protein, because Toxo14-3-3 can be screened from the cDNA library in the sexual and asexual stages of different virulent strains Coding genes, which are presumed to exist in different developmental stages of Toxoplasma gondii. In addition, Tg14-3-3 can also be secreted by the parasite and localized in the Toxoplasma gondii vesicle, which is a member of the Toxoplasma gondii secreted antigen.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory factor with tautomerase and oxidoreductase activities in mammals. Homologs of MIF are expressed in many parasites and are involved in host immune responses. Through sequence alignment, it was found that the Toxoplasma-derived MIF protein (TgMIF) has only 26% homology with mammalian MIF [1].

As an important second messenger, calcium ion can cause a variety of specific responses in cells. In apicomplexan, the change of Ca ²⁺ concentration regulates downstream effector molecules, affecting multiple processes such as parasite invasion, escape from host cells, and migration in the host. Calcium-dependent protein kinases (CDPKs) are a class of effector molecules that receive and transmit extracellular Ca ²⁺ signals, and are serine/threonine protein kinases. Studies have found that CDPKs are mainly involved in multiple processes such as parasite adhesion to host cells, invasion, escape, and protein secretion.

是来自弓形虫S48菌株的减毒速殖子，仅被用于预防绵羊流产的发生[2]。此外，基因修饰的弓形虫，如尿嘧啶营养缺陷型突变体，已被实验证明具有疫苗的作用[3-6]。然而，这些基因突变弓形虫疫苗有一定几率恢复为新型的致病虫株，不适合人类使用，因而研制有效的弓形虫疫苗刻不容缓。In the acute infection stage of Toxoplasma gondii, chemical drugs (pyrimethamine, sulfadiazine, etc.) can be used to control, but the chronic infection of Toxoplasma gondii cannot be eliminated. Vaccination is considered to be effective in preventing and controlling parasitic infection. At present, inactivated and attenuated vaccines, subunit vaccines, nucleic acid vaccines and recombinant protein vaccines have been developed to prevent Toxoplasma gondii infection. The only commercially available live attenuated vaccine

is an attenuated tachyzoite from the S48 strain of Toxoplasma gondii, which has only been used to prevent abortion in sheep [2]. In addition, genetically modified Toxoplasma gondii, such as uracil auxotrophic mutants, have been experimentally demonstrated to have vaccine effects [3-6]. However, these gene-mutated Toxoplasma gondii vaccines have a certain chance of reverting to new pathogenic strains, which are not suitable for human use. Therefore, it is urgent to develop effective Toxoplasma gondii vaccines.

references

1. Sommerville C, Richardson JM, Williams RA, Mottram JC, Roberts CW, Alexander J, Henriquez FL (2013) Biochemical and immunological characterization of Toxoplasma gondii macrophage migration inhibitory factor. The Journal ofbiological chemistry 288(18):12733-12741.doi :10.1074/jbc.M112.419911.

2. Buxton D, Innes EA (1995) A commercial vaccine for ovinetoxoplasmosis. Parasitology 110Suppl:S11-16.doi:10.1017/s003118200000144x.

3. Fox BA, Bzik DJ (2002) De novo pyrimidine biosynthesis is required forvirulence of Toxoplasma gondii. Nature 415(6874):926-929.doi:10.1038/415926a.

4. Fox BA, Bzik DJ (2010) Avirulent uracil auxotrophs based on disruption of orotidine-5'-monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii. Infection and immunity 78(9):3744-3752.doi:10.1128/IAI.00287-10 .

5. Fox BA, Bzik DJ (2015) Nonreplicating, cyst-defective type IIToxoplasma gondii vaccine strains stimulate protective immunity against acute and chronic infection. Infection and immunity 83(5):2148-2155.doi:10.1128/IAI.02756-14.

6. Xia N, Zhou T, Liang X, Ye S, Zhao P, Yang J, Zhou Y, Zhao J, Shen B (2018) ALactate Fermentation Mutant of Toxoplasma Stimulates Protective Immunity Against Acute and Chronic Toxoplasmosis. Frontiers in immunology 9:1814 .doi:10.3389/fimmu.2018.01814.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to prevent toxoplasmosis or how to prepare a stable and effective vaccine for preventing human and animals from being infected with toxoplasmosis.

In order to solve the above technical problems, the present invention first provides an immunogenic composition, which is immunogenic composition A or immunogenic composition B.

The active components of the immunogenic composition A provided by the present invention are composed of the recombinant protein 14-3-3 of Toxoplasma gondii, the recombinant protein MIF of Toxoplasma gondii and the recombinant protein CDPK3 of Toxoplasma gondii; the activity of the immunogenic composition B The components are composed of the recombinant protein 14-3-3 of the Toxoplasma gondii and the recombinant protein CDPK3 of the Toxoplasma gondii.

The recombinant protein 14-3-3 of Toxoplasma gondii is the following A1), A2), A3) or A4) protein:

A1) the amino acid sequence is the protein of sequence 1 in the sequence listing,

A2) the amino acid sequence is the protein at positions 37-302 of sequence 1 in the sequence listing,

A3) a fusion protein obtained by fusing a protein tag at the carboxy terminus or/and the amino terminus of the protein shown in A1) or A2),

A4) The amino acid sequence shown in Sequence 1 in the sequence listing is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function as derived from A1) or A2) or with A1) Or the protein shown in A2) has more than 80% identity;

The Toxoplasma gondii recombinant protein MIF protein is the protein of the following B1), B2), B3) or B4):

B1) the amino acid sequence is the protein of sequence 3 in the sequence listing,

B2) the amino acid sequence is the protein at positions 35-150 of sequence 3 in the sequence listing,

B3) a fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of B1) or B2),

B4) The amino acid sequence shown in Sequence 3 in the Sequence Listing is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function derived from B1) or B2) or with B1) Or the protein shown in B2) has more than 80% identity;

Said TgCDPK3 protein is the following C1), C2), C3) or C4) protein:

C1) the amino acid sequence is the protein of sequence 5 in the sequence listing,

C2) the amino acid sequence is the protein at positions 35-571 of sequence 5 in the sequence listing,

C3) a fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of C1) or C2),

C4) The amino acid sequence shown in Sequence 5 in the sequence listing is obtained by substitution and/or deletion and/or addition of one or several amino acid residues and has the same function, derived from C1) or C2) or with C1) or the protein shown in C2) having more than 80% identity.

The name of the protein of the above A2) is Tg14-3-3, which is a protein from Toxoplasma gondii, and the name of the protein of the above A1) is his-Tg14-3-3, which is fused to the amino terminus of the protein of A2) pET-28a (+) A fusion protein of 36 amino acid residues encoded by the sequence between the recognition sites of Nco I and EcoR I.

The name of the protein of the above-mentioned B2) is TgMIF, which is a protein from Toxoplasma gondii, and the name of the protein of the above-mentioned B1) is his-TgMIF, which is a fusion of Nco I and BamH on pET-28a(+) at the amino terminus of the protein of B2). I recognize the fusion protein of 34 amino acid residues encoded by the sequence between the recognition sites.

The name of the protein of the above-mentioned C2) is TgCDPK3, which is a protein from Toxoplasma gondii, and the name of the protein of the above-mentioned C1) is his-TgCDPK3, which is a fusion of Nco I and BamHI on pET-28a(+) at the amino terminus of the protein of C2). A fusion protein of 34 amino acid residues encoded by sequences between recognition sites.

The above Toxoplasma gondii Tg14-3-3 protein comes from the Toxoplasma gondii RH strain and consists of 266 amino acid residues. The predicted protein molecular weight is about 30kD and the isoelectric point is 4.61; the above Toxoplasma gondii TgMIF protein comes from the Toxoplasma gondii ME49 strain, which consists of 116 It is composed of amino acid residues, the predicted protein molecular weight is about 13kD, and the isoelectric point is 8.91. The above Toxoplasma gondii TgCDPK3 protein comes from Toxoplasma gondii ME49 strain and consists of 537 amino acid residues. The predicted protein molecular weight is about 59kD and the isoelectric point is 5.98.

The above proteins can be artificially synthesized or obtained by first synthesizing their coding genes and then carrying out biological expression.

Among the above proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag and the like.

In the above-mentioned proteins, the identity refers to the identity of the amino acid sequence. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

In the above proteins, the identity of more than 80% may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above immunogenic composition A, the mass ratio of the recombinant protein 14-3-3 of Toxoplasma gondii, the recombinant protein MIF of Toxoplasma gondii and the recombinant protein CDPK3 of Toxoplasma gondii is 1:1:1 (each mouse protein 4μg-8μg, total 12-24μg/per mouse); in the above immunogenic composition B, the mass ratio of the recombinant protein 14-3-3 of Toxoplasma gondii and the recombinant protein CDPK3 of Toxoplasma gondii is 1:1 (4 μg-8 μg of each protein per mouse, total 12-24 μg/mouse).

The above-mentioned immunogenic composition may be any of the following products:

F1. Products for the treatment and/or prevention and/or adjunctive treatment of Toxoplasma gondii infection;

F2. Products that improve the immune response of hosts after Toxoplasma infection;

F3. Products that reduce the host lethality caused by Toxoplasma gondii infection;

F4. Products that reduce the production of host brain cysts caused by Toxoplasma gondii infection.

The above product can be a drug or a vaccine.

The above-mentioned improvement of the host's immune response ability after Toxoplasma infection can be to improve the cytokine secretion ability of the host's spleen lymphocytes after Toxoplasma infection and/or improve the proliferation of the host's spleen lymphocytes after Toxoplasma infection and/or improve the body's ability to produce antibodies ; The cytokine may be INF-γ, IL-2, IL-4 and/or IL-10.

The above-mentioned product for the treatment and/or prevention and/or adjuvant treatment of Toxoplasma gondii infection may be a vaccine for the prevention of Toxoplasma gondii infection, and the vaccine for the prevention of Toxoplasma gondii infection contains an adjuvant.

The above adjuvants include Freund's adjuvant (FA) complete adjuvant and Freund's incomplete adjuvant.

The volume ratio of the recombinant protein mixture to the adjuvant in the vaccine can be 1:1.

The vaccine can be a liquid, and the protein concentration of the recombinant protein mixture in the liquid can be 0.12 mg/mL.

In practical applications, the immunogen composition or immune vaccine of the present invention can be directly administered to patients or animals as medicines, or mixed with suitable carriers or excipients and administered to patients or animals to prevent Toxoplasma gondii infection. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers, etc. Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Of these, water-soluble carrier materials are preferred. A variety of dosage forms can be prepared using these materials, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Buccal tablets, suppositories, freeze-dried powder injections, etc. It can be general formulation, sustained-release formulation, controlled-release formulation and various microparticle delivery systems. For tableting the unit administration dosage form, a wide variety of carriers well known in the art can be used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid Aluminum, etc.; wetting agents and binders, such as water, glycerin, polyethylene glycol, ethanol, propanol, starch syrup, dextrin, syrup, honey, glucose solution, acacia mucilage, gelatin pulp, sodium carboxymethylcellulose , shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch, alginate, agar powder, alginate, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid esters, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oils, etc.; absorption promotion agents, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets can also be further prepared as coated tablets, such as sugar-coated, film-coated, enteric-coated, or bilayer and multi-layer tablets. For formulating the unit administration form into a pill, a wide variety of carriers well known in the art can be used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin, etc. , ethanol, honey, liquid sugar, rice paste or batter, etc.; disintegrating agents, such as agar powder, dry starch, alginate, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc. For formulating the unit administration dosage form as a suppository, a wide variety of carriers well known in the art can be used. Examples of carriers are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like. In order to make unit dosage forms into injection preparations, such as solutions, emulsions, lyophilized powders and suspensions, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation, and in addition, conventional cosolvents, buffers, pH adjusters and the like can be added. In addition, colorants, preservatives, flavors, flavors, sweeteners, or other materials can also be added to the pharmaceutical preparations, if desired. The above-mentioned dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal; respiratory tract administration, such as nasal cavity; mucosal administration. The above-mentioned route of administration is preferably injection administration.

In order to solve the above technical problems, the present invention also provides a method for preparing the above immunogenic composition, comprising the following steps: preparing the recombinant protein 14-3-3 of the Toxoplasma gondii, the recombinant protein MIF of the Toxoplasma gondii and the The recombinant protein CDPK3 of Toxoplasma gondii, the recombinant protein 14-3-3 of Toxoplasma gondii, the recombinant protein MIF of Toxoplasma gondii and the recombinant protein CDPK3 of Toxoplasma gondii are mixed as the active ingredients of the immunogenic composition , to obtain the immunogenic composition;

The recombinant protein 14-3-3 of Toxoplasma gondii is prepared according to a method comprising the following steps: expressing the gene encoding the recombinant protein 14-3-3 of Toxoplasma gondii in a prokaryotic microorganism to obtain the recombinant protein of Toxoplasma gondii 14-3-3;

The recombinant protein MIF of Toxoplasma gondii is prepared according to a method comprising the following steps: expressing the encoding gene of the recombinant protein MIF of Toxoplasma in a prokaryotic microorganism to obtain the recombinant protein MIF of Toxoplasma gondii;

The recombinant protein CDPK3 of Toxoplasma gondii is prepared according to a method comprising the following steps: expressing the gene encoding the recombinant protein CDPK3 of Toxoplasma gondii in a prokaryotic microorganism to obtain the recombinant protein CDPK3 of Toxoplasma gondii.

The adjuvant described above may also contain other components, which can be determined by those skilled in the art according to the preventive effect of the vaccine on the disease.

In the above, the host may be human or animal.

In the above, the Toxoplasma gondii infection can be acute Toxoplasma gondii infection, such as Toxoplasma gondii RH strain infection; it can also be chronic Toxoplasma gondii infection, such as Toxoplasma gondii Pru strain infection.

The encoding gene of the recombinant protein 14-3-3 of the above-mentioned Toxoplasma gondii is the gene shown in the following a1), a2) or a3):

a1) The coding sequence of the coding strand is the DNA molecule shown in sequence 2;

a2) The coding sequence of the coding strand is the DNA molecule shown at positions 109-909 of sequence 2;

a3) has more than 80% identity with the DNA molecule defined in a1) or a2) and encodes the recombinant protein 14-3-3 of the Toxoplasma gondii.

The encoding gene of the recombinant protein MIF of the above-mentioned Toxoplasma gondii is the gene shown in the following b1), b2) or b3):

b1) The coding sequence of the coding strand is the DNA molecule shown in sequence 4;

b2) the coding sequence of the coding strand is the DNA molecule shown at positions 103-453 of sequence 4;

b3) has more than 80% identity with the DNA molecule defined by b1) or b2) and encodes the recombinant protein MIF of said Toxoplasma gondii.

The encoding gene of the recombinant protein 14-3-3 of the above-mentioned Toxoplasma gondii is the gene shown in the following c1), c2) or c3):

c1) The coding sequence of the coding strand is the DNA molecule shown in sequence 6;

c2) the coding sequence of the coding strand is the DNA molecule shown at positions 103-1716 of sequence 6;

c3) has more than 80% identity with the DNA molecule defined by c1) or c2) and encodes the recombinant protein CDPK3 of the Toxoplasma gondii.

The above-mentioned expression of the encoding gene of the recombinant protein 14-3-3 of Toxoplasma gondii in a prokaryotic microorganism includes introducing the encoding gene of the recombinant protein 14-3-3 of Toxoplasma gondii into the recipient Escherichia coli to obtain the expression The recombinant Escherichia coli of the Toxoplasma gondii recombinant protein 14-3-3 is cultured, and the recombinant Toxoplasma gondii protein 14-3-3 is obtained by expressing the recombinant Escherichia coli.

Expressing the gene encoding the recombinant protein MIF of Toxoplasma gondii in a prokaryotic microorganism comprises introducing the gene encoding the recombinant protein MIF of Toxoplasma into a recipient Escherichia coli to obtain a recombinant large intestine expressing the recombinant protein MIF of Toxoplasma gondii Bacillus, culture the recombinant Escherichia coli, and express the recombinant protein MIF of the Toxoplasma gondii.

Expressing the gene encoding the recombinant protein CDPK3 of the Toxoplasma gondii in a prokaryotic microorganism comprises introducing the gene encoding the recombinant protein CDPK3 of the Toxoplasma gondii into a recipient Escherichia coli to obtain a recombinant large intestine expressing the recombinant protein CDPK3 of the Toxoplasma gondii Bacillus, culturing the recombinant Escherichia coli, and expressing the recombinant protein CDPK3 of the Toxoplasma gondii.

The recombinant Escherichia coli expressing the recombinant protein 14-3-3 of the Toxoplasma gondii described above is the Toxoplasma gondii whose expressed amino acid sequence obtained by introducing pET-28a-Tg14-3-3 into Escherichia coli BL21 (DE3) is the sequence 1 The recombinant microorganism of the recombinant protein 14-3-3, the recombinant microorganism is named BL21(DE3)/pET-28a-Tg14-3-3, and the pET-28a-Tg14-3-3 is the vector pET-28a (+) A recombinant vector obtained by replacing the small fragment between the EcoR I and Xho I recognition sites with the DNA fragment shown at positions 109-909 of sequence 2;

The recombinant Escherichia coli expressing the recombinant protein MIF of Toxoplasma gondii is a recombinant microorganism obtained by introducing pET-28a-TgMIF into Escherichia coli BL21 (DE3) and expressing the recombinant protein MIF of the Toxoplasma gondii whose amino acid sequence is sequence 3, the The recombinant microorganism was named BL21(DE3)/pET-28a-TgMIF, and the pET-28a-TgMIF was a small fragment between the BamHI and Xho I recognition sites of the vector pET-28a(+) was replaced with sequence 4, the 103rd- The recombinant vector obtained from the DNA fragment shown at position 453;

The recombinant Escherichia coli expressing the recombinant protein CDPK3 of Toxoplasma gondii is a recombinant microorganism whose amino acid sequence obtained by introducing pET-28a-TgCDPK3 into Escherichia coli BL21 (DE3) is the recombinant protein CDPK3 of the Toxoplasma gondii in sequence 5, and the The recombinant microorganism was named BL21(DE3)/pET-28a-TgCDPK3, and the pET-28a-TgCDPK3 was a small fragment between the BamH I and Xho I recognition sites of the vector pET-28a(+) was replaced with sequence 6, the 103rd - A recombinant vector obtained from the DNA fragment shown at position 1716.

In order to solve the above technical problem, the present invention also provides the biological material related to Toxoplasma gondii protein in the above immunogenic composition. The biological material is any of the following:

H1) a nucleic acid molecule encoding the Toxoplasma gondii recombinant protein 14-3-3, MIF and/or CDPK3 described in claim 1;

H2) an expression cassette containing the nucleic acid molecule described in H1);

H3) a recombinant vector containing the nucleic acid molecule described in H1) or a recombinant vector containing the expression cassette described in H2);

H4) a recombinant microorganism containing the nucleic acid molecule described in H1), or a recombinant microorganism containing the expression cassette described in H2), or a recombinant microorganism containing the recombinant vector described in H3);

H5) a recombinant cell line containing the nucleic acid molecule of H1), or a recombinant cell line containing the expression cassette of H2);

H6) a transgenic animal tissue containing the nucleic acid molecule described in H1), or a transgenic animal tissue containing the expression cassette described in H2);

H7) a host cell containing the nucleic acid molecule of H1), or a host cell containing the expression cassette of H2).

In the above-mentioned biological material, the nucleic acid molecule of H1) may specifically be the encoding gene of the Toxoplasma recombinant protein 14-3-3, MIF and/or CDPK3 shown in the following b1) b2) and/or b3):

b1) The coding sequence (ORF) of the coding strand is the DNA molecule of nucleotides 1-909 of sequence 2 in the sequence listing; or the nucleotide sequence of the coding strand is the DNA molecule of sequence 2 in the sequence listing; or A DNA molecule that hybridizes with the DNA molecule defined in 2) under conditions and encodes a protein with the same function;

b2) The coding sequence (ORF) of the coding strand is the DNA molecule of nucleotides 1-453 of sequence 4 in the sequence listing; or the nucleotide sequence of the coding strand is the DNA molecule of sequence 4 in the sequence listing; or A DNA molecule that hybridizes with the DNA molecule defined in 2) under conditions and encodes a protein with the same function;

b3) The coding sequence (ORF) of the coding strand is the DNA molecule of nucleotides 1-1716 of sequence 6 in the sequence listing; or the nucleotide sequence of the coding strand is the DNA molecule of sequence 6 in the sequence listing; or A DNA molecule that hybridizes to the DNA molecule defined in 2) under the conditions and encodes a protein with the same function.

The above stringent conditions were hybridized in a solution of 2 × SSC, 0.1% SDS at 68 °C and washed twice for 5 min, and then in a solution of 0.5 × SSC, 0.1% SDS at 68 °C. Wash the membrane twice for 15 min each; or, hybridize and wash the membrane in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS at 65°C.

In the above-mentioned biological materials, the expression cassette containing nucleic acid molecules described in H2) refers to DNA capable of expressing the above-mentioned proteins in host cells. The expression cassette may also include a single- or double-stranded nucleic acid molecule of all regulatory sequences necessary for the nucleic acid molecule to express any of the above proteins. The regulatory sequences are capable of directing the coding sequence to express any of the proteins described above in a suitable host cell under compatible conditions. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include a promoter and transcriptional and translational termination signals. The regulatory sequences with linkers can be provided for the purpose of introducing specific restriction enzyme sites of the vector for ligation of the regulatory sequences with the coding region of the protein-encoding nucleic acid sequence. The regulatory sequence may be a suitable promoter sequence, ie, a nucleic acid sequence that is recognized by the host cell in which the nucleic acid sequence is expressed. Promoter sequences contain transcriptional regulatory sequences that mediate protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and hybrid promoters, and can be derived from extracellular or intracellular encoding homologous or heterologous to the host cell protein genes. The regulatory sequence may also be a suitable transcription termination sequence, ie, a sequence recognized by the host cell to terminate transcription. Termination sequences are operably linked to the 3' terminus of the protein-encoding nucleic acid sequence. Any terminator that is functional in the host cell of choice can be used in the present invention. The regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the protein-encoding nucleic acid sequence. Any leader sequence that is functional in the host cell of choice can be used in the present invention. The control sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein, which can guide the encoded protein into the cell secretion pathway. Any signal peptide coding region capable of directing the expressed protein into the secretory pathway of the host cell used can be used in the present invention. It may also be desirable to add regulatory sequences that regulate protein expression depending on the growth conditions of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of regulatory sequences are those that enable gene amplification. In these instances, the nucleic acid sequence encoding the protein is operably linked to the regulatory sequences.

The present invention also relates to a recombinant expression vector comprising a nucleic acid molecule of the present invention encoding any of the above proteins, a promoter and transcriptional and translational stop signals. When an expression vector is prepared, a nucleic acid molecule encoding any of the above proteins can be located in the vector so as to be operably linked to the appropriate expression control sequences. A recombinant expression vector can be any vector (eg, plasmid or virus) that facilitates recombinant DNA manipulation and expression of nucleic acid sequences. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it is to be introduced. Vectors can be linear or closed circular plasmids. The vector may be an autonomously replicating vector (ie, a complete structure that exists extrachromosomally and can replicate independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any mechanism to ensure self-replication. Alternatively, the vector is one that, when introduced into a host cell, will integrate into the genome and replicate with the chromosome into which it is integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids that collectively contain the entire DNA to be introduced into the genome of the host cell, or transposons, can be used. The vector contains one or more selectable markers that facilitate selection of transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline. The vector contains elements that enable stable integration of the vector into the host cell genome, or ensure that the vector replicates autonomously in the cell independently of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication that enables the vector to replicate autonomously in the target host cell. The origin of replication may carry mutations that render it temperature sensitive in the host cell (see eg, fEhrlich, 1978, Proceedings of the National Academy of Sciences 75:1433). One or more copies of a nucleic acid molecule of the invention encoding any of the above proteins can be inserted into a host cell to increase the yield of the gene product. The copy number of the nucleic acid molecule can be increased by inserting at least 1 additional copy of the nucleic acid molecule into the genome of the host cell, or by inserting an amplifiable selectable marker together with the nucleic acid molecule, by culturing the cell in the presence of a suitable selection reagent , cells are selected that contain amplified copies of the selectable marker gene and thus contain additional copies of the nucleic acid molecule. Procedures for ligating the above elements to construct recombinant expression vectors of the present invention are well known to those skilled in the art (see, e.g., Sambrook et al., Molecular Cloning Laboratory Handbook, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. , New York, 1989).

The term "operably linked" is defined herein as a conformation in which the regulatory sequence is positioned in place relative to the coding sequence of the DNA sequence such that the regulatory sequence directs the expression of the protein.

The present invention also relates to a recombinant cell containing a nucleic acid molecule encoding any of the above proteins. Recombinant cells can be prokaryotic or eukaryotic cells, such as bacteria (eg, E. coli cells) or yeast cells.

In the examples of the present invention, immunizing mice with Toxoplasma 14-3-3+MIF+CDPK3 mixed recombinant protein can induce mice to produce effective humoral and cellular immune responses, and effectively improve the survival of mice acutely infected with Toxoplasma RH strain rate, significantly prolong the survival time of mice, and significantly reduce the number of cysts in the brain tissue of mice chronically infected with Pru strains of Toxoplasma gondii. The vaccine prepared by the mixed recombinant protein can be used as an effective candidate vaccine for controlling acute or chronic infection of Toxoplasma gondii for the prevention of toxoplasmosis in humans and animals.

Description of drawings

Figure 1 is a graph showing the level of specific IgG in serum of immunized mice. Blood was collected one day before each immunization, namely 0d, 14d, 28d, and 42d, the serum was separated, and the content of IgG in mouse serum was determined by ELISA. "***" represents a very significant difference, p<0.001.

Figure 2 is a graph showing the proliferation of mouse spleen lymphocytes. The ordinate is the average stimulation index; different letters represent the multiple comparison results showing significant difference, p<0.01.

Figure 3 is a graph showing the detection of cytokines in mouse spleen lymphocytes. Different letters represent multiple comparison results showing significant difference, p<0.01.

Figure 4 is a graph showing the survival rate of mice infected with RH strain tachyzoites (A) and the number of brain cysts in each group of mice infected with Pru strain (B). "***" represents a very significant difference, p<0.001. Different letters represent multiple comparison results showing significant difference, p<0.01.

Figure 5 is the plasmid map of the prokaryotic expression vector pET-28a(+).

Figure 6 is the plasmid map of the recombinant vector pET-28a-Tg14-3-3, the restriction sites are Ecol I and Xho I.

Figure 7 is the plasmid map of the recombinant vector pET-28a-TgMIF, the restriction sites are BamH I and Xho I.

Figure 8 is the plasmid map of the recombinant vector pET-28a-TgCDPK3, the restriction sites are BamH I and Xho I.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product specification. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Toxoplasma gondii RH strain and ME49 strain in the following examples (preserved in our laboratory, refer to the papers published in our laboratory Encephalitis is mediated by ROP18 of Toxoplasma gondii, a severe pathogen in AIDS patients. and Vaccination with recombinant Toxoplasma gondii CDPK3 induces protective immunity against experimental toxoplasmosis.)

The data obtained from the test were processed by Excel software, and the data were analyzed by one-way ANOVA program in SPSS software for analysis of variance and multiple comparisons.

Example 1 Preparation of mixed protein vaccine and evaluation of its immune effect

(1) Construction of prokaryotic expression plasmids pET-28a-Tg14-3-3, pET28a-TgMIF and pET-28a-TgCDPK3

1. Extraction of total RNA of Toxoplasma gondii: Take out Toxoplasma gondii RH strain and ME49 strain from liquid nitrogen, quickly thaw at 37°C and add to HFF cells for culture. After three consecutive passages, collect Toxoplasma gondii RH strain and ME49 strain, and centrifuge at 4000 rpm at room temperature After 10 min, precipitation was used for total RNA extraction, Trizol was added, and it was carried out according to conventional RNA extraction methods.

2. Specific amplification of Tg14-3-3, TgMIF and TgCDPK3 genes: using the total RNA of Toxoplasma gondii RH strain as a template, and referring to the instruction manual of PrimeScript ^TM RT Master Mix (Perfect Real Time) kit (TaKaRa), specific amplification Increase the target gene of Tg14-3-3; take the total RNA of Toxoplasma gondii ME49 strain as the template, and use the same kit to specifically amplify the target gene of TgMIF and TgCDPK3. The reaction system was: 2 μl of 5×PrimeScript RT Master Mix (Perfect Real Time), 3 μl of total RNA template, supplemented with RNase Free dH ₂ O to 10 μl, and the reverse transcription reaction conditions were: 37 °C for 15 min, and 85 °C for inactivation for 5 s.

The specific amplification reaction conditions of Tg14-3-3 gene were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20s, annealing at 57°C for 20s, extension at 72°C for 30s, a total of 35 cycles, and a final extension at 72°C for 10 min. Upstream primer: GCA GAATTC ATGGCGGAGGAAATCA, downstream primer: GCA CTCGAG TTACTGATCAGCTTG. The single-line marks represent the restriction sites EcoR I and XhoI, respectively.

The specific amplification reaction conditions of TgMIF gene were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20s, annealing at 60°C for 20s, extension at 72°C for 30s, a total of 35 cycles, and a final extension at 72°C for 10 min. Upstream primer: GGATCC ATGCCCAAGTGCATGA, downstream primer: CTCGAG TCAGCCGAAAGTTCGGT. The single-line marks represent the enzyme cleavage sites BamH I and Xho I, respectively.

The specific amplification reaction conditions of TgCDPK3 gene were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 20s, annealing at 58°C for 20s, extension at 72°C for 30s, a total of 35 cycles, and a final extension at 72°C for 10 min. Upstream primer: GGATCC ATGGGGTGCGTCCAC, downstream primer: CTCGAG TCAGTGCTTCACTTTGACGTCG. The single-line marks represent the enzyme cleavage sites BamH I and Xho I, respectively.

3. The connection, transformation and sequencing of the target gene: The RT-PCR product of the Tg14-3-3 gene is recovered and purified with pET-28a(+) (referred to as pET-28a) after double digestion with EcoR I and Xho I, The ligation was performed overnight at 16°C, and the ligation product was transferred into TOP10 competent cells. Positive clones were sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

The RT-PCR product of TgMIF gene was recovered and purified, double digested with BamH I and Xho I with pET-28a(+), and then ligated overnight at 16°C, and the ligated product was transferred into TOP10 competent cells. Positive clones were sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

After recovery and purification, the RT-PCR product of TgCDPK3 gene was double digested with BamH I and Xho I with pET-28a(+), and then ligated overnight at 16°C, and the ligated product was transferred into TOP10 competent cells. Positive clones were sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing.

The sequencing results showed that the ligation product of the Tg14-3-3 gene and the vector pET-28a(+) was the replacement of pET-28a(+) with the Tg14-3-3 gene shown in positions 109-909 of sequence 2 in the sequence listing. The fragment (small fragment) between the recognition sites of EcoR I and Xho I, and the recombinant expression vector obtained by keeping other sequences of pET-28a(+) unchanged, was named pET-28a-Tg14-3-3. pET-28a-Tg14-3-3 is the his-Tg14-3-3 gene containing a His tag, the nucleotide sequence of the his-Tg14-3-3 gene is sequence 2, and encodes the protein his-Tg14 shown in sequence 1 -3-3. BL21(DE3)/pET-28a-Tg14-3-3 was introduced into Escherichia coli and named BL21(DE3) to express the protein his-Tg14-3-3 shown in SEQ ID NO: 1.

The ligation product of the TgMIF gene and the vector pET-28a(+) is to replace the fragment between the BamH I and Xho I recognition sites of pET-28a(+) with the TgMIF gene shown at positions 103-453 of SEQ ID NO: 4 in the sequence listing (Small fragment), the recombinant expression vector obtained by keeping other sequences of pET-28a(+) unchanged, named pET-28a-TgMIF. pET-28a-TgMIF is a his-TgMIF gene containing a His tag, and the nucleotide sequence of the his-TgMIF gene is SEQ ID NO: 4, which encodes the protein his-TgMIF shown in SEQ ID NO: 3. BL21(DE3)/pET-28a-TgMIF was introduced into Escherichia coli and named as BL21(DE3) to express the protein his-TgMIF shown in SEQ ID NO:3.

The ligation product of the TgCDPK3 gene and the vector pET-28a(+) is to replace the fragment between the BamH I and Xho I recognition sites of pET-28a(+) with the TgCDPK3 gene shown at positions 103-1716 of SEQ ID NO: 6 in the sequence listing (Small fragment), the recombinant expression vector obtained by keeping other sequences of pET-28a(+) unchanged, named pET-28a-TgCDPK3. pET-28a-TgCDPK3 is a his-TgCDPK3 gene containing a His tag, and the nucleotide sequence of the his-TgCDPK3 gene is sequence 6, which encodes the protein his-TgCDPK3 shown in sequence 5. BL21(DE3)/pET-28a-TgCDPK3 was introduced into Escherichia coli and named as BL21(DE3) to express the protein his-TgCDPK3 shown in SEQ ID NO:5.

4. Minimal extraction of plasmid: refer to the instructions for use of Hangzhou AXYGEN plasmid mini-extract.

(2) Purification and quantification of prokaryotic expression proteins of Tg14-3-3, TgMIF and TgCDPK3

1. Prokaryotic expression of Tg14-3-3, TgMIF and TgCDPK3 proteins: The prokaryotic expression plasmids pET-28a-Tg14-3-3, pET-28a-TgMIF and pET-28a-TgCDPK3 were individually transferred into E. coli BL21 (DE3) (Kang Wei Century), the recombinant E. coli containing pET-28a-Tg14-3-3 was named BL21(DE3)/pET-28a-Tg14-3-3, BL21(DE3)/pET- 28a-Tg14-3-3 can express the protein his-Tg14-3-3 shown in SEQ ID NO: 1. The recombinant E. coli containing pET-28a-TgMIF was named BL21(DE3)/pET-28a-TgMIF, and BL21(DE3)/pET-28a-TgMIF can express the protein his-TgMIF shown in sequence 3. The recombinant E. coli containing pET-28a-TgCDPK3 was named BL21(DE3)/pET-28a-TgCDPK3, and BL21(DE3)/pET-28a-TgCDPK3F could express the protein his-TgCDPK3 shown in sequence 5.

The three strains of BL21(DE3)/pET-28a-Tg14-3-3, BL21(DE3)/pET-28a-TgMIF and BL21(DE3)/pET-28a-TgCDPK3 were individually inoculated on cards containing 50 μg/ml. In the LB liquid medium of kanamycin (the medium obtained by adding kanamycin to the LB liquid medium to a concentration of 50 μg/ml of kanamycin), 37° C., 220 rpm shaking culture to OD ₆₀₀ value (with When the LB liquid medium containing 50 μg/ml kanamycin was a blank control), when it reached 0.6, isopropylthio-β-D-galactoside (IPTG) was added to induce expression, and BL21(DE3)/pET were obtained respectively. -28a-Tg14-3-3-induced expression of bacteria, BL21 (DE3)/pET-28a-TgMIF-induced expression of bacteria and BL21 (DE3)/pET-28a-TgCDPK3-induced expression of bacteria. The induced expression of the above three strains was induced with 1 mM IPTG at 30°C for 5 hours.

2. Ultrasound and purification of Tg14-3-3, TgMIF and TgCDPK3 proteins: Centrifuge the above-mentioned inducible bacterial solution at 4°C and 4000 rpm for 10 min, collect the bacterial cells, and add 10 ml of pre-cooled lysis buffer (containing 1% Triton X-100, 200 μl PMSF), resuspend the cells, and then sonicate (ultrasonic 5s, interval 5s, total sonication time 10min, power 300W). As a result, the supernatant containing the protein his-Tg14-3-3 was obtained from the bacterial solution induced by BL21(DE3)/pET-28a-Tg14-3-3, and the supernatant containing the protein his-Tg14-3-3 was obtained from BL21(DE3)/pET-28a-TgMIF. The supernatant containing the protein his-TgMIF was obtained from the inducible bacterial solution, and the supernatant containing the protein his-TgCDPK3 was obtained from the bacterial solution induced by BL21(DE3)/pET-28a-TgCDPK3. All liquids were supernatants containing the target protein. Take 1 ml of Ni-beads into the column, then wash three times with pre-cooled PBS, and then wash three times with lysis buffer, add the above-mentioned supernatant containing the target protein, and incubate at 4°C for 1 h. - Wash the beads twice with Wash buffer 1 (solute and its concentration are as follows: 50mM NaH ₂ PO ₄ , 300mM NaCl, 20mM imidazole, the solvent is water, pH 8.0 solution) 2 times, 5ml/time, collect Wash buffer 1 with a centrifuge tube Washing solution, Wash buffer 2 (the solute and its concentration are as follows: 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 40 mM imidazole, the solvent is water, pH 8.0 solution) washed twice, 10 ml/time, and the Wash buffer 2 washing solution was collected. Then use Elution buffer (solute and its concentration are as follows: 50mM NaH ₂ PO ₄ , 300mM NaCl, 300mM imidazole, the solvent is water, pH 8.0 solution) to elute the target protein 3 times, 0.5ml/time, collect the eluted protein three times The liquid was obtained to obtain the liquid containing the target protein (the liquid containing his-Tg14-3-3, the liquid containing his-TgMIF, and the liquid containing his-TgCDPK3). A small amount of the target protein liquid was added to the protein lysis buffer to cook the sample, and then subjected to SDS-PAGE gel electrophoresis, and then stained with Coomassie brilliant blue to detect the protein purification effect. The results showed that the target protein his-Tg14-3-3 with a size of 30kD, his-TgMIF with a size of 13kD and his-TgCDPK3 with a size of 59kD were obtained.

3. Quantification of Tg14-3-3, TgMIF and TgCDPK3 proteins: Detect the protein concentration according to the steps of Biyuntian BCA protein concentration assay kit (Item No.: P0012), in which his-Tg14 in the liquid containing his-Tg14-3-3 The concentration of -3-3 was 8 mg/ml, the concentration of his-TgMIF in the liquid containing his-TgMIF was 5 mg/ml, and the concentration of his-TgCDPK3 in the liquid containing his-TgCDPK3 was 7 mg/ml.

(3) Evaluation of the immune effect

1. Grouping of BALB/c mice: 240 6-8 week SPF female BALB/c mice, weighing 50±10g (purchased from Anhui Animal Experiment Center), were randomly divided into six groups (control group) , PBS group, MIF+CDPK3 group, 14-3-3+MIF group, 14-3-3+CDPK3 group, 14-3-3+MIF+CDPK3 group), 40 mice in each group.

2. Immunization program:

Blank group: without any treatment, it is blank control group;

PBS group: sterile PBS and adjuvant were mixed in equal volume, completely emulsified on ice (blowing with a syringe for 1 h, the same below), and BALB/c mice were immunized by intramuscular injection (the same below) in the legs, each injection of 0.1 per mouse per mouse ml;

MIF+CDPK3 group: mix the liquid containing his-TgMIF and the liquid containing his-TgCDPK3 obtained in step (2), and mix them with an equal volume of adjuvant. After complete emulsification, immunize BALB/c mice. The mice were injected with 0.1ml each time, and the immunization dose of each mouse was 6μg his-TgMIF and 6μg his-TgCDPK3;

14-3-3+MIF group: After mixing the liquid containing his-Tg14-3-3 and his-TgMI obtained in step (2), the adjuvant was mixed with equal volume, and after complete emulsification, BALB/c mice were immunized. , each injection of 0.1ml per mouse, the immunization dose of each mouse is 6μg his-Tg14-3-3 and 6μg his-TgMIF;

14-3-3+CDPK3 group: after mixing the liquid containing his-Tg14-3-3 and his-TgCDPK3 obtained in step (2), it was mixed with an equal volume of adjuvant, and after complete emulsification, immune BALB/c Mice, each injection of 0.1ml per mouse, the immunization dose of each mouse is 6μg his-Tg14-3-3 and 6μg his-TgCDPK3;

14-3-3+MIF+CDPK3 group: after mixing the liquid containing his-Tg14-3-3, the liquid containing his-TgMIF and the liquid containing his-TgCDPK3 obtained in step (2), with adjuvant etc. After the volume was mixed and completely emulsified, BALB/c mice were immunized with 0.1 ml per injection per mouse, and the immunization dose of each mouse was 4 μg his-Tg14-3-3, 4 μg his-TgMIF and 4 μg his-TgCDPK3;

The immunization time was the first day, the 15th day and the 29th day, a total of three times, and the operation method was the same. Freund's complete adjuvant was used for the first emulsified protein, followed by incomplete Freund's adjuvant twice. Each BALB/c mouse was immunized with a total volume of 100 μL per site.

3. Serum IgG was detected by ELISA: blood was collected one day before each immunization, namely 0d, 14d, 28d, and 42d. Three mice were randomly selected from each group each time, and blood was collected from the tail vein of mice. Add 100 μl of coating solution to the empty ELISA plate, and coat the corresponding proteins according to the grouping, and the concentration of each protein is 10 μg/ml. A conventional ELISA experiment is performed to detect the content of mouse serum IgG. The results showed that the 14-3-3+MIF+CDPK3 group (corresponding to MIF+1433+CDPK3 in Figure 1) in the immunization group had the best immunization effect after 2 weeks of immunization, and its IgG antibody level was significantly higher than that of the blank group (corresponding to Figure 1). control) and PBS groups (p<0.01); after 4 weeks of immunization, most of the IgG antibody levels in the immunized group were significantly higher than those in the blank group (corresponding to the control in Figure 1) and the PBS group (p<0.05). The specific results are shown in Figure 1.

4. Detection of the proliferation level of spleen lymphocytes in mice: Two weeks after the last immunization of all experimental groups, 3 mice were randomly selected from each group to obtain spleen cells for culture. The protein concentration was 10 μg/ml) for stimulation (see “(II) 2” for purification method). Subsequently, the proliferation of spleen lymphocytes was detected by CCK-8 assay. For the specific experimental method, refer to the steps of Cell Counting Kit-8 (CCK-8 kit) (Item No.: C0038) of Biyuntian Company. Stimulation index = _OD450 of stimulated cells/ _OD450 of unstimulated cells. The results showed that compared with the control group (corresponding to control and PBS in Figure 2), the 14-3-3+MIF+CDPK3 group (corresponding to MIF+1433+CDPK3 in Figure 2) had higher levels of his-Tg14-3-3, his-Tg14-3-3, his- The mean stimulation index of the three proteins, TgMIF and his-TgCDPK3, increased significantly, showing a more significant spleen lymphocyte proliferation response (p<0.001), indicating that his-Tg14-3-3, his-TgMIF and his-TgCDPK3 The three proteins mixed recombinant protein immunization of BALB/c mice can induce antigen-specific splenic lymphocyte proliferation. The specific results are shown in Figure 2.

5. Cytokine detection of mouse spleen lymphocytes: Two weeks after the last immunization of all experimental groups, three mice were randomly selected from each group, and spleen cells were cultured. The protein concentration was 10 μg/ml) for stimulation (see “(II) 2” for purification method). Cell supernatants were collected, and ELISA kits (Beijing Sizhengbai Biotechnology) were used to detect cytokines INF-γ (product number: CME0003), IL-2 (product number: CME0001), IL-4 (product number: CME0005) and IL- 10 (Product Number: CME0016). The results showed that the levels of INF-γ, IL-2, IL-4 and IL-10 in the immunized group after protein stimulation were significantly higher than those in the control group (corresponding to control and PBS in Figure 3), especially 14-3-3 +MIF+CDPK3 group (corresponding to MIF+1433+CDPK3 in Figure 3), INF-γ (p<0.001), IL-2 (p<0.001), IL-4 (p<0.001) and IL-10 (p<0.001) The level of 0.05) was significantly higher than that of the control group and other immunization groups, which more effectively caused cell-mediated immune responses, mainly Th1-type immune responses. In addition, the levels of cytokines INF-γ (p<0.01) and IL-2 (p<0.001) in the 14-3-3+CDPK3 group (corresponding to 1433+CDPK3 in Figure 3) were also significantly higher than those in the control group. The specific results are shown in Figure 3.

6. Attack infection of Toxoplasma gondii RH strain:

Intraperitoneal injection of Toxoplasma gondii RH strain:

Two weeks after the last immunization of all experimental groups, 15 mice from each group ^were randomly selected for intraperitoneal injection of Toxoplasma gondii RH strain. BALB/c mice of all experimental groups (15 mice in each group) were infected by intraperitoneal injection with an injection dose of 100 μl per mouse, and the survival time of the experimental mice was observed and recorded. The results showed that compared with the control group (shown in control and PBS in Figure 4), the survival time of the mice in the immunized group was prolonged, especially the Toxoplasma 14-3-3+MIF+CDPK3 mixed recombinant protein immunization group of the present invention. (MIF+1433+CDPK3 in A in Figure 4), 90% of the mice survived for more than 30 days, effectively prolonging the survival time of BALB/c mice acutely infected with Toxoplasma RH strain (p<0.001). In addition, more than 75% of the mice in the 14-3-3+CDPK3 immunized group (corresponding to 1433+CDPK3 in A in Figure 4) survived for more than 30 days, which also effectively prolonged the acute infection of BALB/c mice by the RH strain of Toxoplasma gondii survival time (p<0.001). The specific results are shown in A in Figure 4.

Infection of Toxoplasma gondii Pru strain by gavage: Two weeks after the last immunization of all experimental groups, 15 mice from each group were randomly selected for intraperitoneal injection of Toxoplasma gondii RH strain. BALB/c mice in all experimental groups (3 mice in each group) were intragastrically infected according to the oral dose of 100 μl per mouse, and the survival time of the experimental mice was observed and recorded. One month after infection, the surviving mice in each group were sacrificed, and the mouse brains were isolated and physically ground to prepare brain tissue homogenate. After mixing the brain tissue homogenate, take 10 μl and count it three times under the microscope to take the average value. The number of mouse brain cysts is 10 times the average value. The results showed that, compared with the control group (shown as control and PBS in B in Figure 4), the number of brain cysts in the immunized group mice decreased after chronic infection with the Toxoplasma gondii Pru strain, especially the Toxoplasma gondii 14-3-3+ of the present invention. In the MIF+CDPK3 mixed recombinant protein immunization group (shown by MIF+1433+cdpk3 in Figure 4 B), the number of midbrain cysts in the chronic infection of Toxoplasma gondii Pru strain was significantly reduced (p<0.001); 14-3-3+CDPK3 immunization The number of midbrain cysts in the group (indicated by 1433+CDPK3 in B in Figure 4) was also significantly decreased (p<0.01) in the chronic infection of Toxoplasma gondii Pru strain. The specific results are shown in B in 4.

To sum up, using Toxoplasma gondii 14-3-3+MIF+CDPK3 mixed recombinant protein immunization can induce mice to produce effective humoral and cellular immune responses, which can effectively improve the survival rate of mice with acute Toxoplasma gondii infection. Prolonged mouse survival and significantly reduced the number of cysts in the brain tissue of chronically infected mice. The vaccine prepared by the mixed recombinant protein can be used as an effective candidate vaccine for controlling acute or chronic infection of Toxoplasma gondii for the prevention of toxoplasmosis in humans and animals. In addition, using Toxoplasma 14-3-3+CDPK3 mixed recombinant protein immunization can also induce mice to produce a certain effective humoral and cellular immune response, effectively improve the survival rate of acute Toxoplasma infection in mice, significantly prolong the survival time of mice, significantly The number of cysts in the brain tissue of chronically infected mice is reduced, and the vaccine prepared using the mixed recombinant protein can also be used as an effective candidate vaccine for the control of acute or chronic infection of Toxoplasma gondii for the prevention of toxoplasmosis in humans and animals.

sequence listing

<110> Anhui Medical University

<120> A protein mixed vaccine for preventing human and animal toxoplasmosis

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<170> PatentIn version 3.5

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Val Tyr Lys Ala Lys Leu Ala Glu Gln Ala Glu Arg Tyr Asp Glu Met

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Ala Glu Ala Met Lys Asn Leu Val Glu Asn Cys Leu Asp Glu Gln Gln

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Pro Lys Asp Glu Leu Ser Val Glu Glu Arg Asn Leu Leu Ser Val Ala

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Tyr Lys Asn Ala Val Gly Ala Arg Arg Ala Ser Trp Arg Ile Ile Ser

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Ser Val Glu Gln Lys Glu Leu Ser Lys Gln His Met Gln Asn Lys Ala

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Leu Ala Ala Glu Tyr Arg Gln Lys Val Glu Glu Glu Leu Asn Lys Ile

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Cys His Asp Ile Leu Gln Leu Leu Thr Asp Lys Leu Ile Pro Lys Thr

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Ser Asp Ser Glu Ser Lys Val Phe Tyr Tyr Lys Met Lys Gly Asp Tyr

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Tyr Arg Tyr Ile Ser Glu Phe Ser Gly Glu Glu Gly Lys Lys Gln Ala

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Ala Asp Gln Ala Gln Glu Ser Tyr Gln Lys Ala Thr Glu Thr Ala Glu

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Ala Glu Leu Pro Ser Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn

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Tyr Ser Val Phe Phe Tyr Glu Ile Leu Asn Leu Pro Gln Gln Ala Cys

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Ala Gln Gln Asp Ala Leu Leu Lys Asp Ala Glu Lys Ala Val Ala Asp

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Ala Leu Gly Lys Pro Leu Ser Tyr Val Met Val Gly Tyr Ser Gln Thr

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Gly Gln Met Arg Phe Gly Gly Ser Ser Asp Pro Cys Ala Phe Ile Arg

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Val Ala Ser Ile Gly Gly Ile Thr Ser Ser Thr Asn Cys Lys Ile Ala

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Ala Ala Leu Ser Ala Ala Cys Glu Arg His Leu Gly Val Pro Lys Asn

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Arg Ile Tyr Thr Thr Phe Thr Asn Lys Ser Pro Ser Glu Trp Ala Met

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<212> PRT

<213> Artificial Sequence

<400> 5

Met Gly Ser Ser His His His His His His Ser Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Met Gly Cys Val His Ser Lys Asn Pro His Ser Lys His Ala

35 40 45

Gly Ala Ala Gly Glu Lys Pro Asp Ala Ser Leu Glu Lys Gly Gly Gln

50 55 60

Ser Lys Gly Ser Ala Pro Ser Ser Gly Thr Gly Asp Ser Gly Lys Gly

65 70 75 80

Thr Gly Ser Pro Asp Thr Lys Arg Asp Ser Met Pro Met Thr Pro Gly

85 90 95

Met Tyr Ile Thr Gln Gln Lys Ala His Leu Ser Asp Arg Tyr Gln Arg

100 105 110

Val Lys Lys Leu Gly Ser Gly Ala Tyr Gly Glu Val Leu Leu Cys Lys

115 120 125

Asp Lys Leu Thr Gly Ala Glu Arg Ala Ile Lys Ile Ile Lys Lys Ser

130 135 140

Ser Val Thr Thr Thr Ser Asn Ser Gly Ala Leu Leu Asp Glu Val Ala

145 150 155 160

Val Leu Lys Gln Leu Asp His Pro Asn Ile Met Lys Leu Tyr Glu Phe

165 170 175

Phe Glu Asp Lys Arg Asn Tyr Tyr Leu Val Met Glu Val Tyr Arg Gly

180 185 190

Gly Glu Leu Phe Asp Glu Ile Ile Leu Arg Gln Lys Phe Ser Glu Val

195 200 205

Asp Ala Ala Val Ile Met Lys Gln Val Leu Ser Gly Thr Thr Tyr Leu

210 215 220

His Lys His Asn Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu

225 230 235 240

Leu Glu Ser Lys Ser Arg Asp Ala Leu Ile Lys Ile Val Asp Phe Gly

245 250 255

Leu Ser Ala His Phe Glu Val Gly Gly Lys Met Lys Glu Arg Leu Gly

260 265 270

Thr Ala Tyr Tyr Ile Ala Pro Glu Val Leu Arg Lys Lys Tyr Asp Glu

275 280 285

Lys Cys Asp Val Trp Ser Cys Gly Val Ile Leu Tyr Ile Leu Leu Cys

290 295 300

Gly Tyr Pro Pro Phe Gly Gly Gln Thr Asp Gln Glu Ile Leu Lys Arg

305 310 315 320

Val Glu Lys Gly Lys Phe Ser Phe Asp Pro Pro Asp Trp Thr Gln Val

325 330 335

Ser Asp Glu Ala Lys Gln Leu Val Lys Leu Met Leu Thr Tyr Glu Pro

340 345 350

Ser Lys Arg Ile Ser Ala Glu Glu Ala Leu Asn His Pro Trp Ile Val

355 360 365

Lys Phe Cys Ser Gln Lys His Thr Asp Val Gly Lys His Ala Leu Thr

370 375 380

Gly Ala Leu Gly Asn Met Lys Lys Phe Gln Ser Ser Gln Lys Leu Ala

385 390 395 400

Gln Ala Ala Met Leu Phe Met Gly Ser Lys Leu Thr Thr Leu Glu Glu

405 410 415

Thr Lys Glu Leu Thr Gln Ile Phe Arg Gln Leu Asp Asn Asn Gly Asp

420 425 430

Gly Gln Leu Asp Arg Lys Glu Leu Ile Glu Gly Tyr Arg Lys Leu Met

435 440 445

Gln Trp Lys Gly Asp Thr Val Ser Asp Leu Asp Ser Ser Gln Ile Glu

450 455 460

Ala Glu Val Asp His Ile Leu Gln Ser Val Asp Phe Asp Arg Asn Gly

465 470 475 480

Tyr Ile Glu Tyr Ser Glu Phe Val Thr Val Cys Met Asp Lys Gln Leu

485 490 495

Leu Leu Ser Arg Glu Arg Leu Leu Ala Ala Phe Gln Gln Phe Asp Ser

500 505 510

Asp Gly Ser Gly Lys Ile Thr Asn Glu Glu Leu Gly Arg Leu Phe Gly

515 520 525

Val Thr Glu Val Asp Asp Glu Thr Trp His Gln Val Leu Gln Glu Cys

530 535 540

Asp Lys Asn Asn Asp Gly Glu Val Asp Phe Glu Glu Phe Val Glu Met

545 550 555 560

Met Gln Lys Ile Cys Asp Val Lys Val Lys His

565 570

<210> 6

<211> 1716

<212> DNA

<213> Artificial Sequence

<400> 6

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggggtg cgtccactcc 120

aagaatcccc actccaagca tgcaggcgca gctggagaaa aacccgacgc cagcctcgaa 180

aaggggggcc agagcaaggg gagcgcgccg tcgtcgggga ccggcgacag cggaaaagga 240

actgggtctc ccgacaccaa gagagactcc atgcccatga ctccaggcat gtacatcacg 300

cagcagaagg cccatttgtc tgaccgctac cagcgcgtga agaagctcgg aagcggtgct 360

tacggcgagg tgctgctgtg caaggacaaa ctgacaggcg cagagcgagc aatcaaaatc 420

atcaaaaagt cttctgtcac gaccacgagc aacagcgggg ctctcctcga cgaagtcgcc 480

gtgctgaaac agctcgacca cccgaacatc atgaagctct acgagttctt cgaggacaag 540

cgcaactact accttgtcat ggaggtgtac cgaggaggcg agttgtttga cgaaatcatt 600

cttcgtcaga agttcagcga agtggacgcc gctgtcatca tgaaacaggt gctctctggc 660

accacttacc tgcacaaaca caacattgtg catcgcgacc tgaagcccga aaaccttctt 720

ctcgagtcga agagccggga cgctcttatc aagatcgtcg actttggtct ctctgcgcac 780

tttgaagtcg gcggaaagat gaaggagcgc cttggcacag cctactacat tgccccagaa 840

gttctgagaa agaagtacga cgaaaaatgc gatgtctggt cttgcggcgt tatcctctac 900

attctccttt gcggctaccc gcccttcgga ggtcaaaccg accaggat cctcaagagg 960

gtcgagaaag gaaagttttc cttcgacccg cctgactgga ctcaagtgtc ggacgaggcg 1020

aagcagctgg tcaagctgat gctgacctac gagccttcga agagaatttc tgctgaggag 1080

gcgctgaatc atccgtggat cgtcaagttc tgctcccaga aacacaccga cgtcggcaaa 1140

cacgctctca cgggcgccct gggcaacatg aagaaattcc agtcctcaca gaagctggcg 1200

caagcggcca tgctgtttat ggggagcaag ctgacgactc tggaggagac gaaggagctg 1260

actcagatct ttcgtcaact tgacaacaac ggggacggcc aactggatcg caaggaacta 1320

attgaaggtt acagaaagct catgcagtgg aagggcgaca ccgtatctga cttggacagc 1380

agccagatag aggcagaggt ggatcacatt ctccaatctg tcgactttga cagaaatgga 1440

tacattgagt actctgaatt tgtgactgtg tgcatggaca agcagcttct gctgtcgcgc 1500

gagcgacttc ttgctgcctt ccaacagttc gacagcgacg gctcaggaaa aatcacgaat 1560

gaggaactcg gcagactctt tggtgtgacg gaagtcgacg acgaaacgtg gcaccaggtt 1620

ctgcaagagt gcgacaagaa caacgatgga gaggtcgact ttgaggagtt tgtggaaatg 1680

atgcagaaga tctgcgacgt caaagtgaag cactga 1716

Claims (7)

1. An immunogenic composition, characterized by: the immunogenic composition is immunogenic composition A or immunogenic composition B;

the active ingredients of the immunogenic composition A consist of recombinant protein 14-3-3 of toxoplasma, recombinant protein MIF of toxoplasma and recombinant protein CDPK3 of toxoplasma;

the active ingredients of the immunogenic composition B consist of the recombinant protein 14-3-3 of the toxoplasma gondii and the recombinant protein CDPK3 of the toxoplasma gondii;

the recombinant protein 14-3-3 of the toxoplasma gondii is a protein of A1) or A2) as follows:

A1) the amino acid sequence is protein of a sequence 1 in a sequence table,

A2) the amino acid sequence is protein at 37 th-302 th site of sequence 1 in the sequence table;

the Toxoplasma gondii recombinant protein MIF is the protein of the following B1) or B2):

B1) the amino acid sequence is the protein of the sequence 3in the sequence table,

B2) the amino acid sequence is protein at 35 th to 150 th site of the sequence 3in the sequence table;

the toxoplasma recombinant protein TgCDPK3 protein is the protein of the following C1) or C2):

C1) the amino acid sequence is protein of a sequence 5 in a sequence table,

C2) the amino acid sequence is protein at 35-571 th site of sequence 5 in the sequence table; in the immunogenic composition A, the mass ratio of the recombinant protein 14-3-3 of the toxoplasma gondii, the recombinant protein MIF of the toxoplasma gondii and the recombinant protein CDPK3 of the toxoplasma gondii is 1:1: 1;

in the immunogenic composition B, the mass ratio of the recombinant protein 14-3-3 of the toxoplasma gondii to the recombinant protein CDPK3 of the toxoplasma gondii is 1: 1.

2. The immunogenic composition of claim 1, wherein: the immunogenic composition is any one of the following products:

f1, products for the treatment and/or prevention and/or co-treatment of toxoplasma infection;

f2, a product for enhancing the immune response of a host after Toxoplasma gondii infection;

f3, a product for reducing host mortality caused by Toxoplasma gondii infection;

f4, reducing the production of host brain encapsulation caused by Toxoplasma gondii infection.

3. The immunogenic composition of claim 2, wherein: the product for treating and/or preventing and/or assisting in treating the Toxoplasma gondii infection is a vaccine for preventing the Toxoplasma gondii infection, and the vaccine for preventing the Toxoplasma gondii infection contains an adjuvant.

4. A process for the preparation of the immunogenic composition of any one of claims 1-3 comprising the steps of: preparing the recombinant protein 14-3-3 of the toxoplasma gondii, the recombinant protein MIF of the toxoplasma gondii and the recombinant protein CDPK3 of the toxoplasma gondii, and mixing the recombinant protein 14-3-3 of the toxoplasma gondii, the recombinant protein MIF of the toxoplasma gondii and the recombinant protein CDPK3 of the toxoplasma gondii as active ingredients of the immunogenic composition to obtain the immunogenic composition;

the recombinant protein 14-3-3 of the Toxoplasma gondii is prepared according to the method comprising the following steps: expressing the coding gene of the recombinant protein 14-3-3 of the toxoplasma gondii in prokaryotic microorganisms to obtain the recombinant protein 14-3-3 of the toxoplasma gondii;

the recombinant protein MIF of the toxoplasma gondii is prepared according to the method comprising the following steps: expressing the encoding gene of the recombinant protein MIF of the toxoplasma gondii in prokaryotic microorganisms to obtain the recombinant protein MIF of the toxoplasma gondii;

the recombinant protein CDPK3 of the Toxoplasma gondii is prepared according to the method comprising the following steps: expressing the encoding gene of the recombinant protein CDPK3 of the toxoplasma in prokaryotic microorganisms to obtain the recombinant protein CDPK3 of the toxoplasma.

5. The method of claim 4, wherein: the coding gene of the recombinant protein 14-3-3 of the toxoplasma is the gene shown in a1) or a 2):

a1) the coding sequence of the coding strand is a DNA molecule shown in a sequence 2;

a2) the coding sequence of the coding strand is the DNA molecule shown in the 109-909 position of the sequence 2;

the coding gene of the recombinant protein MIF of the toxoplasma is the gene shown in the following b1) or b 2):

b1) the coding sequence of the coding strand is a DNA molecule shown in a sequence 4;

b2) the coding sequence of the coding strand is a DNA molecule shown in the 103-453 th position of the sequence 4;

the encoding gene of the recombinant protein CDPK3 of the Toxoplasma gondii is the gene shown in the following c1) or c 2):

c1) the coding sequence of the coding strand is a DNA molecule shown in sequence 6;

c2) the coding sequence of the coding strand is the DNA molecule shown in position 103-1716 of the sequence 6.

6. The method of claim 4, wherein: the coding gene of the recombinant protein 14-3-3 of the toxoplasma gondii is expressed in prokaryotic microorganisms, and the coding gene of the recombinant protein 14-3-3 of the toxoplasma gondii is introduced into receptor escherichia coli to obtain recombinant escherichia coli for expressing the recombinant protein 14-3-3 of the toxoplasma gondii, and the recombinant escherichia coli is cultured and expressed to obtain the recombinant protein 14-3-3 of the toxoplasma gondii;

the method comprises the following steps of expressing the coding gene of the recombinant protein MIF of the toxoplasma gondii in prokaryotic microorganisms, wherein the coding gene of the recombinant protein MIF of the toxoplasma gondii is introduced into receptor escherichia coli to obtain recombinant escherichia coli expressing the recombinant protein MIF of the toxoplasma gondii, and the recombinant escherichia coli is cultured and expressed to obtain the recombinant protein MIF of the toxoplasma gondii;

the method comprises the steps of enabling the coding gene of the recombinant protein CDPK3 of the toxoplasma to be expressed in prokaryotic microorganisms, introducing the coding gene of the recombinant protein CDPK3 of the toxoplasma into receptor escherichia coli to obtain the recombinant escherichia coli expressing the recombinant protein CDPK3 of the toxoplasma, culturing the recombinant escherichia coli, and expressing to obtain the recombinant protein CDPK3 of the toxoplasma.

7. The method of claim 6, wherein: the recombinant Escherichia coli for expressing the recombinant protein 14-3-3 of the toxoplasma is a recombinant microorganism which is obtained by introducing pET-28a-Tg14-3-3 into Escherichia coli BL21(DE3) and expresses the recombinant protein 14-3-3 of the toxoplasma with an amino acid sequence of sequence 1, the recombinant microorganism is named as BL21(DE3)/pET-28a-Tg14-3-3, and the pET-28a-Tg14-3-3 is a vector pET-28a (+) (see)EcoR I andXho a small segment between the I recognition sites is replaced by a DNA segment shown by the 109-909 site of the sequence 2 to obtain a recombinant vector;

the recombinant Escherichia coli for expressing the recombinant protein MIF of the toxoplasma is a recombinant microorganism of the recombinant protein MIF of the toxoplasma, the expression amino acid sequence of which is sequence 3 and is obtained by introducing pET-28a-TgMIF into Escherichia coli BL21(DE3), the recombinant microorganism is named as BL21(DE3)/pET-28a-TgMIF, and the pET-28a-TgMIF is a carrier pET-28a (+) (Bacillus subtilis)BamHI andXho a small segment between the I recognition sites is replaced by a DNA segment shown in the 103 th-453 th site of the sequence 4 to obtain a recombinant vector;

the recombinant escherichia coli expressing the recombinant protein CDPK3 of the toxoplasma is a recombinant microorganism which is obtained by introducing pET-28a-TgCDPK3 into escherichia coli BL21(DE3) and expresses the recombinant protein CDPK3 of the toxoplasma, the amino acid sequence of the recombinant microorganism is sequence 5, the recombinant microorganism is named as BL21(DE3)/pET-28a-TgCDPK3, and the pET-28a-TgCDPK3 is a recombinant microorganism which is obtained by introducing a vector pET-28a-TgCDPK3 into escherichia coli BL21(DE3), and the recombinant microorganism is named as BL21(DE3)/pET-28a-TgCDPK3BamHI andXho a small segment between I recognition sites is replaced by a DNA segment shown in the 103 th 1716 th site of the sequence 6 to obtain a recombinant vector.

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