CN111138553B

CN111138553B - Fusion protein, toxoplasma subunit vaccine and vaccine composition thereof

Info

Publication number: CN111138553B
Application number: CN202010051245.2A
Authority: CN
Inventors: 杨阳; 涂正坤; 安倍莹; 高修竹; 李海军; 郭魏莹; 李天阳; 李天祺
Original assignee: First Hospital Jinlin University
Current assignee: First Hospital Jinlin University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-09-25
Anticipated expiration: 2040-01-17
Also published as: CN111138553A

Abstract

The invention provides a fusion protein and a nucleotide sequence thereof, which comprise a toxoplasma gondii P30 protein subunit and a T cell epitope. And a vaccine composition thereof for preventing abortion of a pregnant sow due to toxoplasmosis using the fusion protein. The invention has the advantages that the fusion protein has the characteristics of high expression quantity and high immunogenicity. The vaccine and the vaccine composition can effectively prevent abortion of pregnant sows caused by toxoplasmosis, and have high vaccine stability.

Description

Fusion protein, toxoplasma subunit vaccine and vaccine composition thereof

Technical Field

The invention relates to the fields of immunology and biology, in particular to a swine toxoplasma gondii genetic engineering subunit vaccine, a preparation method and application thereof, and mainly relates to a nucleotide sequence, a fusion protein, a vaccine for the swine toxoplasma gondii vaccine, a preparation method and application thereof.

Background

Toxoplasmosis, also known as toxoplasmosis or toxoplasmosis, is a worldwide distributed parasitic disease of humans and animals caused by the Toxoplasma gondii (Toxoplasma gondii), widely spread in humans, animals and wild animals, and seriously threatens the public health of humans and animals. Toxoplasma gondii can infect more than 200 vertebrates. According to foreign reports, the average infection rate of people is 25-50%, and at least 5 hundred million people in the world are predicted to be infected with toxoplasma gondii by people. According to statistics, the infection rate of toxoplasma of residents in China is 0.1-47.3%. The toxoplasma infection rate of pigs in China is 4.78-85.7%, the infection rate of cattle is 0.2-43.4%, and the infection rate of sheep is 0.35-37%. Seriously affecting the human health and the development of animal husbandry.

The infection of the toxoplasma gondii in the pregnant women can affect the development of fetuses, and even cause teratogenesis and even death of serious people, and meanwhile, the pregnant women suffer abortion or premature delivery; toxoplasma gondii is a major opportunistic causative agent in immunosuppressed or immunodeficient patients. It is reported that 30-46% of pregnant women have a chance to transmit toxoplasma to the fetus after infecting it, causing congenital infection of the fetus, resulting in abortion, stillbirth or congenital toxoplasmosis; 6-10% of AIDS patients are complicated by toxoplasmosis, and 50% of encephalitis of AIDS patients are caused by toxoplasmosis infection. The positive rate of toxoplasma antibody of pregnant and lying-in women and tumor patients is as high as 10-60%. The pig infected with toxoplasma also can cause 'innominate hyperpyrexia', and the fatality rate reaches 60%. The dog and cat as companion animals lift a crowd of 'pet fever of dog and cat', the dog as an important intermediate host of toxoplasma, and the cat as a unique terminal host is closely contacted with human and becomes a main infection source of human toxoplasmosis.

So far, no ideal commercial vaccine and medicine for preventing and treating toxoplasmosis exists at home and abroad, and host protective immune response can be caused after the toxoplasmosis is infected, so that the development of safe and effective vaccine is a good prevention measure for the toxoplasmosis. The development of toxoplasma gondii vaccines has gone through several stages, including whole worm vaccines, subunit vaccines, genetically engineered vaccines and nucleic acid (or DNA) vaccines, since the 60 s. Whole worm vaccines include inactivated vaccines and attenuated live vaccines, but the inactivated vaccines lack immunoprotection for mice, so that the whole worm vaccines have no practical application value; toxoplasma tachyzoite is weakened in toxicity after being treated by ultraviolet rays, radioactive rays, chemical reagents and the like, and can induce stronger immune response such as Ts-4, T-263 and S48, but attenuated live vaccines have the risks of insufficient attenuation, reversion of toxicity and the like, so that attenuated live vaccines cannot be widely used; the subunit vaccine is prepared by extracting specific components from a lysate of an insect body or an excretion-secretion antigen, has good immunogenicity, but is time-consuming and labor-consuming in purification and expensive in price; the gene engineering subunit vaccine is to express toxoplasma antigen gene in high efficiency expression vector to obtain great amount of purified single antigen. The above discussion can find that the genetic engineering vaccine is hope for the development of the toxoplasma vaccine and has potential development and application values.

Disclosure of Invention

The invention aims to provide a fusion protein capable of preventing a pig from being infected with a toxoplasma epidemic disease and a vaccine composition thereof. The invention also provides pharmaceutical compositions, such as vaccine compositions, containing the fusion proteins. In addition, the invention also provides a nucleotide for encoding the fusion protein, a method for preparing the fusion protein and the like.

Aiming at the current problems, the invention provides a nucleotide sequence, a vector, a protein, a vaccine, a preparation method and application thereof for preventing pig toxoplasma infection, wherein a toxoplasma P30 mutant gene is artificially synthesized (the artificially synthesized toxoplasma P30 mutant gene provided by the invention is the gene of the toxoplasma P30 epitope mutant in the invention), the P30 mutant gene is transferred into engineering bacteria for expression, the recombinant antigen is efficiently expressed after induction, and the genetic engineering vaccine with ideal immunogenicity is obtained through processes of fermentation, purification, emulsification and the like, so that the epidemic of the pig toxoplasma epidemic disease can be prevented.

One of the objectives of the present invention is to provide a novel vaccine polypeptide and a vaccine composition thereof, which can be used for preventing swine toxoplasma gondii.

The second purpose of the invention is to provide a construction and obtaining method of the protein genetic engineering vaccine.

The invention also aims to provide a genetic engineering strain capable of expressing the swine toxoplasma gondii genetic engineering subunit vaccine.

The fourth purpose of the invention is to provide a preparation method of the swine toxoplasma gondii genetic engineering subunit vaccine.

The fifth purpose of the invention is to provide the pig genetic engineering subunit vaccine which can induce humoral immune response in the body of an immune target animal.

In the first aspect, the invention provides a novel vaccine polypeptide capable of preventing swine toxoplasma and a vaccine composition thereof, wherein the vaccine polypeptide contains a mutant of a toxoplasma conserved structural gene P30 (the toxoplasma P30 mutant gene provided by the invention, namely the toxoplasma P30 epitope mutant gene), the 1 st to 480 th nucleotides of the toxoplasma P30 gene are analyzed by using a molecular biology software DNAstar, and the sequence of the toxoplasma P30 gene is subjected to 17-nucleotide point mutation and modification. The toxoplasma P30 mutant gene was followed by a T cell epitope. The protein engineering vaccine for preventing the swine toxoplasma provided by the invention contains non-immune active substances besides main immunogenic proteins. The nonimmunogenic substances include mainly a purification tag and C-terminal polyadenylic acid. Pharmaceutically acceptable salts are those which are non-toxic, irritating and allergenic and are suitable for use in human or animal tissues. Inactive substances and pharmaceutically acceptable salts are well known to those skilled in the art. Preferably, the adjuvant is 206 adjuvant; the antigen protein buffer solution is PBS; the purification tag and the C-terminal polynucleotide are carried on a vector, and the purification tag and the C-terminal polynucleotide carried on pET-28a are preferred.

In a second aspect, the present invention provides a nucleotide molecule encoding a swine toxoplasma genetically engineered subunit vaccine protein according to the first aspect of the present invention. The nucleotide can be in a DNA form, is synthesized by an artificial synthesis mode, is subjected to gene engineering operation, is cloned into a vector, is transformed into escherichia coli, and is screened, fermented and purified to obtain the swine toxoplasma vaccine polypeptide. The nucleic acid may be subjected to conventional molecular biological procedures in the present invention, such as: PCR, restriction enzyme digestion, ligation, etc. Nucleic acid design 5 'end and 3' end both add enzyme cutting sites, the gene manipulation technology is known to the technicians in this field.

In a third aspect, the present invention provides a vector comprising, in addition to the nucleotide molecule encoding the amino acids of the genetically engineered subunit vaccine of swine toxoplasma according to the second aspect of the present invention, expression control elements operably linked to the nucleotide sequence required for expression (transcription and translation) in prokaryotic cells. The most basic expression control elements include promoters, transcription terminators, enhancers, selectable markers, and the like, and these control elements are well known in the art. In a preferred embodiment, the expression vector is an E.coli expression vector.

In a fourth aspect, the invention provides a host cell, preferably E.coli. Comprising a vector according to the third aspect of the invention. The host cell is transformed or transfected with a gene sequence containing the coding protein, and then the gene sequence is detected to have good heredity and expression stability, so that the gene sequence can be used for producing the required swine toxoplasma protein engineering vaccine through fermentation expression.

In a fifth aspect, the invention provides a preparation method of a swine toxoplasma protein engineering vaccine, which comprises the following steps: the engineering bacteria ferment and express protein engineering vaccine protein, and the required protein is obtained through a purification process and a subsequent emulsification process. The methods involved include, but are not limited to, cell disruption, inclusion body washing, centrifugation, affinity chromatography, hydrophobic chromatography, emulsification, etc. The preparation processes involved in the present invention are well known to those skilled in the art.

In a sixth aspect, the present invention provides a vaccine for preventing toxoplasmosis in pigs, comprising the protein of the first aspect of the invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is an immunopotentiator or an immunoadjuvant, and preferably the immunoadjuvant is 206 adjuvant.

In a seventh aspect, the invention provides the use of the protein engineered vaccine of the sixth aspect. The vaccine can be injected into an immune pig through muscle injection, intradermal injection or subcutaneous injection with a certain effective dose, and can stimulate the humoral immune response and the cellular immunity of an organism.

The invention provides a fusion protein, which comprises a toxoplasma gondii P30 protein subunit and also comprises a T cell epitope. T cell epitope obviously improves the immunogenicity of the fusion protein.

Preferably, the toxoplasma gondii P30 protein subunit is a toxoplasma gondii P30 epitope mutant, and the amino acid sequence of the toxoplasma gondii P30 epitope mutant is the amino acid sequence shown in Seq ID NO. 2. The invention intercepts 160 amino acids (shown in Seq ID NO: 2) of the toxoplasma gondii P30 protein subunit and uses the amino acids as the toxoplasma gondii P30 epitope mutant for constructing the fusion protein, compared with the prior art, the obtained fusion protein is soluble expressed and is not expressed in an inclusion body, the expression quantity of the fusion protein is effectively improved, and meanwhile, the immunogenicity of the toxoplasma gondii P30 epitope mutant is kept.

Preferably, in any of the above cases, the amino acid sequence of the T cell epitope is the amino acid sequence shown in Seq ID No. 4. The T cell epitope sequence selected by the invention is firstly used for preparing fusion protein vaccines, particularly for preparing swine toxoplasmosis vaccines, and has the functions of improving the immunogenicity of toxoplasmosis P30 epitope mutants, simultaneously enabling the toxoplasmosis P30 epitope mutants-T cell epitope fusion protein to be efficiently expressed, and the obtained fusion protein is soluble expression and is not expressed in inclusion bodies.

Preferably, in any of the above, the coding sequence of the Toxoplasma gondii P30 epitope mutant comprises the nucleotide sequence shown in Seq ID No. 1. The invention intercepts 1-480 bases of a Toxoplasma gondii P30 gene, codon optimization is carried out on the gene in order to improve the protein expression quantity, 17 bases are mutated in total, and the gene is called a P30 mutant gene, namely the Toxoplasma gondii P30 epitope mutant gene.

Preferably, in any of the above, the coding sequence for the T cell epitope comprises the nucleotide sequence of the T cell epitope represented by Seq ID No. 3.

Preferably, in any of the above, the fusion protein comprises 1 Toxoplasma gondii P30 epitope mutant and 1T cell epitope. It is further preferred that the last amino acid of the mutant toxoplasma P30 epitope is directly linked to the first amino acid of the T cell epitope and that the last amino acid of the mutant toxoplasma P30 epitope does not comprise any linking amino acids to the first amino acid of the T cell epitope.

The fusion protein of any one of the above mentioned items, vaccine polypeptide for preventing swine toxoplasma and vaccine composition thereof, swine toxoplasma gene engineering subunit vaccine protein, swine toxoplasma gene engineering subunit vaccine, swine toxoplasma protein engineering vaccine, vaccine for preventing swine toxoplasmosis.

The invention also provides a nucleic acid molecule encoding the fusion protein of any one of the above.

Preferably, the coding sequence of the nucleic acid molecule comprises the nucleotide sequence set forth in Seq ID No. 5.

Preferably in any of the above, the amino acid sequence of the nucleic acid molecule is the amino acid sequence of Seq ID No. 6.

The invention also provides a toxoplasma subunit vaccine and a vaccine composition thereof, which comprise the amino acid sequence of the fusion protein and/or the nucleic acid molecule.

Preferably, the toxoplasma subunit vaccine and the compositions thereof are used as vaccines for preventing abortion of pregnant sows due to toxoplasma infection.

The fusion gene of the swine toxoplasma gene engineering subunit vaccine is divided into two parts, wherein the first part comprises: intercepting 1 st to 480 th bases of a Toxoplasma gondii P30 gene, carrying out codon optimization on the gene in order to improve the protein expression quantity, and mutating 17 bases in total, wherein the gene is called a P30 mutant gene; a second part: selecting a T cell epitope, splicing the two genes together to form a P30 fusion gene, inserting the gene into an escherichia coli expression vector for expression and purification to obtain a recombinant fusion protein, and adding an adjuvant to prepare the swine toxoplasma gondii genetic engineering subunit vaccine. Can be used for preventing sow abortion caused by toxoplasma infection. Preferably, the vaccine composition further comprises an inactive substance and a pharmaceutically acceptable salt, further preferably, the vaccine composition comprises an immunological adjuvant, and further preferably, the immunological adjuvant is 206 adjuvant.

In the prior art, toxoplasma vaccines have not been successfully marketed. Toxoplasma gondii has many types of types, each of which is classified into many types, either genotypes or serotypes, and there are various isolates under each serotype or genotype, like other bacteria or viruses. However, different genotypes of different isolates have differences, so that in the existing scientific research, the types of the antigen fragments which can be used for preparing the toxoplasma vaccine are very many, and the length of the antigen fragments is selected differently, so that the selection of the antigen fragments in different researches is random, whether good effects can be achieved or not needs to be verified according to the result of the final immune animal. In the invention, in the selection of countless antigen fragments, the toxoplasma gondii P30 antigen epitope mutant provided by the invention is selected, and the prepared vaccine can effectively protect animals through experimental verification and can be preferably used as a vaccine for preventing swine toxoplasmosis.

The invention has the beneficial technical effects that the toxoplasma gondii P30 epitope mutant-T cell epitope fusion protein, the vaccine for preventing abortion of pregnant sows caused by toxoplasmosis and the vaccine composition thereof are provided, compared with the prior art, the fusion protein can be expressed in a soluble way in bacterial liquid supernatant, does not form inclusion bodies, and has the characteristics of high expression quantity and high immunogenicity. The vaccine and the vaccine composition can effectively prevent abortion of pregnant sows caused by toxoplasmosis, and have high vaccine stability.

Drawings

FIG. 1 shows the result of the amplification of the fusion gene of Toxoplasma gondii P30 epitope mutant gene and T cell epitope gene.

FIG. 2 shows the SDS-PAGE result of the recombinant expression protein of the Toxoplasma gondii P30 fusion gene.

FIG. 3 shows the SDS-PAGE result of protein purification of recombinant expression of Toxoplasma gondii P30 fusion gene.

FIG. 4 Western blot result of recombinant expression protein of toxoplasma gondii P30 fusion gene.

FIG. 5 Toxoplasma gondii P30 recombinant fusion protein stimulated mouse RAW264.7 cells to produce TNF- α levels.

FIG. 6. Toxoplasma gondii P30 recombinant fusion protein stimulated IL-12 production by mouse dendritic cells.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the following examples, unless otherwise specified, all methods are conventional. The reagents, vectors, cells and cells used are commercial products well known in the art unless otherwise specified.

Example 1: construction of prokaryotic expression vector of toxoplasma recombinant protein

Primers were designed with reference to the open reading frame of the Toxoplasma gondii P30 gene sequence and the physical map of the prokaryotic expression vector pET28a and the restriction sites BamH I and Hind III were introduced.

Upstream: GGATCCatgtcggtttcgctgcacca

Downstream: AAGCTTtggctcctttggaaag

In E.coli, in general, proteins with relatively large molecular weight are expressed in the form of inclusion bodies, and codons in natural sequences are not favorable for expression in E.coli, so that codon optimization is required to be favorable for the preference of an E.coli expression system. Meanwhile, in the research process of the invention, the main epitope of the P30 gene is found to be mainly located in the first 480 nucleotides (namely, compared with the full-length P30 gene in the prior art, the invention provides a shorter nucleotide sequence containing the main epitope of the P30 gene and the protein obtained by the method is proved to have immunogenicity in the subsequent research), so that the method is subjected to codon optimization and 17-nucleotide point mutation by DNAstar software analysis. (it should be noted here that although the optimization of the sequence by software analysis is a conventional technical means, it is clear to those skilled in the art that the sequence obtained by software analysis is only a conjecture, and does not necessarily guarantee the soluble expression of the protein, and even can guarantee that the immunogenicity of the obtained protein can be maintained, so that the mutant sequence obtained by the present invention can be used for preparing the swine toxoplasmosis vaccine as a result of creative work). In the invention, a fusion gene of a toxoplasma gondii P30 gene mutant gene (namely, a toxoplasma gondii P30 epitope mutant gene) and a T cell epitope gene which are connected in series is artificially synthesized (the fusion gene of the toxoplasma gondii P30 epitope mutant gene and the T cell epitope gene is hereinafter referred to as a toxoplasma gondii P30 fusion gene). The gene sequence is SED ID NO.5, PCR amplification is carried out by taking the SED as a template, and PCR parameters are as follows: 3min at 95 ℃; 1min at 94 ℃; and (3) extending at 72 ℃ for 10min after 30 cycles at 59.5 ℃ for 40s and 72 ℃ for 1 min), cloning the PCR purified product to pGEM-T vector system, identifying positive clone bacteria through enzyme digestion and sequencing, carrying out double enzyme digestion on positive plasmid BamHI and Hind III, then connecting the purified target fragment to a prokaryotic expression vector pET28a, transforming the ligation product into E.coli DH5 alpha competent cells, screening recombinant plasmids, and carrying out double enzyme digestion identification through BamHI and Hind III to obtain the prokaryotic expression plasmid pET28 a-P30. The PCR size of the fusion gene was 528 bp. The results are shown in FIG. 1, in which the loading sequence of each lane is: marker (Marker, M): DL10000 marker; blank control; fusion of gene fragments.

Example 2: expression and purification of expressed proteins

(1) Extraction and solubility verification of expressed protein

The E.coli BL21(DE3) single colony transformed with recombinant plasmid pET-28a-P30 is used as an engineering strain of swine toxoplasma vaccine (BL21(DE3) -pET-P30), the E.coli BL21(DE3) single colony transformed with pET-28a empty vector is used as a blank control strain (BL21(DE3) -pET-28a), and the two strains are respectively inoculated in 8mL LB liquid culture medium and cultured with shaking overnight at 37 ℃. Transferring the seed solution to 400mL LB liquid culture medium for propagation at 37 ℃, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.1Mm/L when the OD600 of the bacterial solution is monitored to be 0.6-0.7, and carrying out induced expression for 16 hours under the optimal condition of 18 ℃. And (3) centrifugally collecting thalli, suspending the thalli by using 10mL of soluble protein lysate, adding 100 mu L of 0.1M PMSF, repeatedly freezing and thawing for 3 times (-80 ℃,1h/37 ℃ and 10min), then carrying out ultrasonic treatment, or adding 100 mu L of DNase, 100 mu L of 100 XDNase Buffer and 100 mu L of LRNase, carrying out water bath at 37 ℃ for 15min, and centrifugally taking supernatant to obtain the soluble protein (protein with activity). The two supernatants were subjected to SDS-PAGE to verify the expression of the target protein of the engineered vaccine strain, as shown in FIG. 2, the engineered strain of the swine toxoplasma vaccine expressed the target protein at around 19KD, while the blank control did not, as shown in FIG. 2: m, protein marker; 1, BL21(DE3) -pET-28a blank; 2, BL21(DE3) -pET-P30.

(2) Purification of fusion expressed proteins

Adding 2mL of 50% Ni-NTA suspension into 10mL of cracking supernatant containing the protein, and slowly shaking for 1-2h on a rotary shaker at the rotating speed of 200 rpm; the entire suspension was poured into a column with a bottom cap and a cap (QIAGEN), the bottom cap was opened, the column was gently pressed down with the cap, the cap was removed, the liquid was allowed to drip slowly by gravity, and the effluent suspension was collected for SDS-PAGE to determine whether any unbound protein was present. Washing the column filled with Ni-NTA with lysis solution, 1mL each time, detecting the concentration of protein at OD280 with a NanodROP1000spectrophotometer, and repeatedly washing until no protein is detected; after the washing liquid is changed, washing the Ni-NTA column by the same method, and detecting the protein concentration by using NANODROP; when the protein can not be detected, dissolving the protein by using a protein dissolving solution, loading 0.5mL of the protein into a column every time, collecting the solution flowing out every time into a new 1.5mL centrifuge tube, dissolving the protein which is the purified expression protein until the protein can not be detected by using NANODROP, detecting the protein purity by SDS-PAGE, detecting the protein concentration by using a BCA protein detection kit, wherein the concentration is 5.3mg/mL, and the result is shown in figure 3: m, protein marker; 1, recombinant P30 fusion protein (Toxoplasma gondii P30 epitope mutant-T cell epitope fusion protein).

Example 3: western blot identification

The western blot identification is carried out on the fusion protein induced and expressed by BL21(DE3) -pET-P30 engineering bacteria by using dog toxoplasma positive serum as a primary antibody, rabbit anti-dog IgG labeled by horseradish peroxidase as a secondary antibody and DAB as a substrate color developing agent. BL21(DE3) -pET-28a served as a negative control. The results are shown in FIG. 4, FIG. 4: m: pre-dyeing a Marker III; 1, BL21(DE3) -pET-P30; 2, BL21(DE3) -pET-28 a. .

Example 4: detection of immunogenicity of mouse macrophage by expression product

RAW264.7 cells were cultured at 0.5 × 10⁶Perml was inoculated into 24-well plates, 1mL per well, 5% CO₂And culturing at 37 ℃ for 24 h. Sucking out supernatant, dissolving prepared toxoplasma recombinant P30 mutant protein (toxoplasma P30 epitope mutant-T cell epitope fusion protein) inRAW264.7 cell culture solution, and 1 mL/well of 1. mu.g/mL, 0.1. mu.g/mL, 0.01. mu.g/mL, added to the cell culture plate, and equal volume of blank plasmid purified protein NR was used as a negative control in the experiment. Place the cell culture plate in 5% CO₂And culturing in an incubator at 37 ℃ for 48 hours, collecting supernatant, and detecting the TNF-a level by an ELISA experiment. The results showed that the extent of TNF-a production varied and that the amount of TNF-a increased with increasing protein concentration, ranging from 180pg to 520pg, and are shown in FIG. 5; the results show that the recombinant protein can stimulate the TNF-a production of RAW264.7 cells.

Example 5 detection of immunogenicity of expression products to mouse dendritic cells

Separating mouse dendritic cell, regulating cell concentration to 0.5 × 10⁶one/mL, seeded into 48 well tissue culture plates, 0.5mL per well. The treated recombinant protein (Toxoplasma gondii P30 epitope mutant-T cell epitope fusion protein) was added to the cell culture plate at a concentration of 1. mu.g/mL, 0.1. mu.g/mL, 0.01. mu.g/mL, respectively, per well. Place the cell culture plate in 5% CO₂And culturing at 37 ℃ in an incubator for 24 hours, collecting culture supernatant, and detecting the IL-12 level in the supernatant through an ELISA experiment. The results show that each group produces different IL-12 content and presents protein concentration dependence, the results are shown in FIG. 6, and the results show that the recombinant protein can stimulate dendritic immune cells of mice to produce a large amount of IL-12.

Example 6: preparation of vaccines

The purified P30 fusion protein antigen (Toxoplasma gondii P30 epitope mutant-T cell epitope fusion protein) is filtered by a 0.22 micron sterilization filter into an autoclaved bag body. Diluted to 600. mu.g/ml with sterile 0.01mol/L PBS (pH 7.4). Adjuvant 206 was filled into another emulsion tank and autoclaved at 121 ℃ for 40 minutes. The antigen protein is injected into the emulsification tank by using compressed air, and the ratio of the water phase to the oil phase is 50: 50. And (3) setting emulsification conditions: stirring was carried out at 30 ℃ and a stirring speed of 100rpm for 2 hours. The vaccine is qualified by aseptic inspection, the content of endotoxin is lower than 100 EU/first part, the emulsified vaccine is centrifuged for 1min at 12000rpm, and the layering phenomenon of the vaccine appears, which indicates that the stability of the vaccine is good.

Example 7 study of Toxoplasma recombinant subunit vaccine in mammalian applications

20 pigs (2-3 years old) pregnant for 1 month are divided into 2 groups, the first group is 10 pigs, the P30 experimental group (adjuvant is 206 adjuvant, 200 mug/pig, intramuscular injection) and the second group is 10 pigs, the negative control group (blank expression vector purified protein with equal volume amount, matched with 206 adjuvant, intramuscular injection) are provided, each group is injected three times and three weeks are provided. Week 10 intravenous injection of purified Toxoplasma trophozoite 10⁴The abortion protection status of the pigs was recorded daily for each individual, and the results are shown in table 1.

TABLE 1 protective Effect of recombinant Toxoplasma gondii P30 fusion protein on test animals (swine) after immunization

Grouping	Immunological pathways	Number of immunizations (times)	Rate of protection
				206 adjuvant vaccine group	Intramuscular injection	3	(9/10)90％
Negative control group	Intramuscular injection	3	(0/10)0％

The results show that 9 of the 10 pregnant sows did not suffer abortion and only 1 of the 10 pregnant sows did suffer abortion in the 206 vaccine group after challenge. After the negative control group had been challenged, 10 pregnant sows all had miscarriages.

By combining the experimental results, the swine toxoplasma gondii genetic engineering subunit vaccine provided by the invention can be used as a vaccine for preventing toxoplasmosis of pregnant sows.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention. None of the chemical agents used in the present invention are commercially available ski agents such as sigmaaldrich. The experimental techniques used are described in the handbooks of molecular cloning, and the like, unless otherwise specified.

Claims

1. A fusion protein is characterized by comprising a toxoplasma gondii P30 protein subunit and a T cell epitope, wherein the toxoplasma gondii P30 protein subunit is a toxoplasma gondii P30 epitope mutant, the amino acid sequence of the toxoplasma gondii P30 epitope mutant is the amino acid sequence shown in SEQ ID NO. 2, and the amino acid sequence of the fusion protein is the amino acid sequence shown in SEQ ID NO. 6.

2. The fusion protein of claim 1, wherein the amino acid sequence of the T cell epitope is the amino acid sequence set forth in SEQ ID NO. 4.

3. A nucleic acid molecule encoding the fusion protein of any one of claims 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleic acid sequence of said nucleic acid molecule is the nucleic acid sequence set forth in SEQ ID NO. 5.

5. A toxoplasma subunit vaccine and vaccine compositions thereof comprising the fusion protein of any one of claims 1 or 2.

6. Use of the fusion protein of any one of claims 1 or 2 for the preparation of a vaccine and compositions thereof for the prevention of abortion in pregnant sows due to toxoplasma infection.