CN111218450A - Vaccine for preventing toxoplasma infection and preparation method and application thereof - Google Patents

Abstract

The invention provides a vaccine for preventing toxoplasma infection and a preparation method and application thereof, belonging to the technical field of immunology and molecular biology. The invention designs and synthesizes primers according to a Toxoplasma gondii ROP47 sequence published by GenBank, amplifies ROP47 gene by PCR, constructs recombinant plasmid pEGFP-C1-ROP47(pROP47), extracts recombinant plasmid containing target gene to obtain Toxoplasma gondii ROP47DNA vaccine, and prepares the combined vaccine of recombinant Toxoplasma gondii ROP47DNA and MPLA by taking monophosphoryl lipid A (MPLA) as an adjuvant, thereby effectively improving immune protective reaction of mice to Toxoplasma gondii and reducing the formation of encystment in brain tissue, and having good value of practical application.

Description

Vaccine for preventing toxoplasma infection and preparation method and application thereof

Technical Field

The invention belongs to the technical field of immunology and molecular biology, and particularly relates to a vaccine for preventing toxoplasma infection, and a preparation method and application thereof.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Toxoplasma gondii (t. gondii) is a prevalent parasitic disease in the world, an intracellular parasite. Toxoplasma gondii affects one third of the world's population to varying degrees. In recent years, work using DNA vaccines against Toxoplasma gondii has made significant progress.

Many Toxoplasma DNA are widely used as candidate genes for genetic vaccines. The toxoplasma clavuli protein (ROP) gene of toxoplasma expresses important proteins that play a decisive role in the life cycle of the parasite. In addition, these genes are considered to be excellent candidates for DNA vaccines against toxoplasma gondii. Researchers have demonstrated that genes like ROP1, ROP16, ROP18 of toxoplasma can cause cellular and humoral immune responses in mice. But does not achieve the desired full protective effect.

The reason for this is that toxoplasma has a complex life history, various forms, a wide host range, different immunogens of antigens with different properties, and different pathogenic mechanisms of the cyst and tachyzoite, so that although vaccine candidate antigens based on different single antigen components can be found, the antigen has few lymphocyte binding sites and is greatly restricted by major histocompatibility complex of the body, so that the toxoplasma is resistant to attack infection with different forms. In addition, the existing recombinant antigenic vaccines are generally poor in immunogenicity, and a common adjuvant is needed to induce a positive immune response. Therefore, the search for more and better DNA candidate antigens derived from different toxoplasma stages, in particular to antigen genes related to toxoplasma toxicity, and the matching of a proper adjuvant is a key for developing an anti-toxoplasma vaccine.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a vaccine for preventing toxoplasma gondii infection and a preparation method and application thereof. The invention designs and synthesizes a pair of primers according to Toxoplasma gondii ROP47 sequences (Gene ID, TGME49_252500) published by GenBank, amplifies ROP47 Gene by PCR, constructs recombinant plasmid pEGFP-C1-ROP47(pROP47), extracts recombinant plasmid containing target Gene to obtain Toxoplasma gondii ROP47DNA vaccine, and prepares the combined vaccine of recombinant Toxoplasma gondii ROP47DNA and MPLA by taking monophosphoryl lipid A (MPLA) as adjuvant, thereby effectively improving the immune protective reaction of mice to Toxoplasma gondii and reducing the formation of encystment in brain tissue, therefore, the invention has good value of practical application.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a nucleotide sequence for use in the prevention of Toxoplasma gondii infection, which comprises a nucleotide sequence selected from any one or more of the following 1) to 3):

1) the nucleotide sequence of the clavulan protein ROP47 shown in SEQ ID NO. 1;

2) a nucleotide sequence complementary to the nucleotide sequence in 1);

3) a nucleotide sequence obtained by modifying the nucleotide sequence shown in 1) or 2) above by substitution, deletion or addition of one or more bases.

Research indicates that ROP47 is important for chronic infection of Toxoplasma gondii, and based on bioinformatics analysis, the research of the invention indicates that ROP47 contains good B cell epitope and T cell epitope, and further indicates that the Toxoplasma gondii ROP47 gene has the potential to become an excellent DNA vaccine.

In a second aspect of the invention, there is provided a vector comprising a nucleotide sequence as described above.

In the third aspect of the invention, a recombinant plasmid is obtained by inserting ROP47 gene into a eukaryotic expression vector pEGFP-C1, and further, the recombinant plasmid is obtained by inserting ROP47 gene into Kpn I and BamH I sites in the eukaryotic expression vector pEGFP-C1 to construct pEGFP-C1-ROP47(pROP 47).

In a fourth aspect of the present invention, there is provided a protein, wherein the amino acid sequence is encoded by the above recombinant plasmid.

In a fifth aspect of the present invention, a vaccine is provided, which comprises the above nucleotide sequence, the above vector and/or the above recombinant plasmid, and further comprises a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier is an adjuvant, the adjuvant is a TLR agonist, and further preferably monophosphoryl lipid A (MPLA). Monophosphoryl lipid a is a lipid a derivative of attenuated bacterial Lipopolysaccharide (LPS). It is a potent immunostimulant effective in inducing both humoral antibody responses and cell-mediated immune responses. The monophosphoryl lipid A is used as an adjuvant of the recombinant Toxoplasma gondii ROP47DNA vaccine, so that the immunoprotective reaction of mice to Toxoplasma gondii can be effectively improved, and the formation of encystment in brain tissues is reduced.

In a sixth aspect of the present invention, there is provided a method for preparing the vaccine, the method comprising:

constructing recombinant Toxoplasma gondii ROP47 recombinant plasmid; the ROP47 recombinant plasmid is mixed with a pharmaceutically acceptable carrier.

Wherein, the specific method for constructing the recombinant plasmid of the recombinant Toxoplasma gondii ROP47 comprises the following steps: PCR amplification ROP47 gene using toxoplasma cDNA as template, and connecting the purified amplification product with pEGFP-C1 plasmid carrier to form recombinant plasmid.

Wherein, the primers for amplifying the ROP47 gene are shown as follows:

an upstream primer: 5'-ggGGTACCTCCGCAAGCTCCAGTT-3' (SEQ ID NO.2)

A downstream primer: 5'-cgGGATCCGATTCGGACCCGCAG-3' (SEQ ID NO.3)

The PCR conditions for amplifying the ROP47 gene were as follows:

denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles.

The pharmaceutically acceptable carrier is an adjuvant, the adjuvant is a TLR agonist, and the monophosphoryl lipid A is further preferable.

In a seventh aspect of the invention there is provided the use of any one of the above nucleotide sequences, vectors, recombinant plasmids, proteins and/or vaccines in a) to c) as follows:

a) prevention of toxoplasma infection;

b) enhancing the immunoprotective response of the vaccinee to toxoplasma;

c) controlling encystment formation of brain tissue during toxoplasma chronic infection.

The vaccinees include humans and non-human animals, which are mammals, and further include mice.

The invention has the beneficial technical effects that:

the invention reports a recombinant toxoplasma ROP47DNA vaccine for the first time, and simultaneously, the vaccine is matched with an adjuvant monophosphoryl lipid A to carry out injection immunization on a mouse, and experiments prove that the vaccine prepared by the invention can effectively improve the immune protective reaction of the mouse against the toxoplasma, is particularly suitable for controlling the formation of encysted brain tissue in the chronic infection process of the toxoplasma, and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1: b cell epitope and T cell epitope analysis of Toxoplasma clavulirus protein ROP 47.

FIG. 2: expression and identification of recombinant pROP47 plasmid protein; (a) detecting cells transfected with pROP47 under white light; (b) observing cells transfected with pROP47 under blue light; (c) cells transfected with pEGFP-C1 were observed under white light; (d) cells transfected with pEGFP-C1 were observed under blue light; (e) observing the untransfected cells under white light; (f) non-transfected cells were observed under blue light; (g) western blot analysis, untransfected cells (lane 1), pEGFP-C1 transfected cells (lane 2), cells transfected with pROP47 (lane 3).

FIG. 3: detecting specific IgG antibodies in the serum of the immunized mouse; (a) two days before each immunization, serum was collected and the results were detected by ELISA; (b) ELISA assay results for sera were collected six weeks after the last immunization.

FIG. 4: results of ELISA testing the IgG1 and IgG2a antibody levels of the immunized mice. Immune sera were collected 2 weeks (a) and 6 weeks (b) after the last immunization and tested by ELISA.

FIG. 5: and (3) a correlation diagram for measuring the content of the cell factors in splenocytes of the immunized mice. Wherein the cytokine to be detected in (a) is IFN-gamma; (b) the cytokine to be detected is IL-2; (c) the cytokine to be detected in (a) is IL-4, and the cytokine to be detected in (d) is IL-10.

FIG. 6: immunoprotection of the recombinant DNA vaccine against toxoplasma.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

As mentioned above, the search for more and better candidate DNA antigens derived from different toxoplasma stages, especially the antigen genes related to toxoplasma toxicity, and the matching of a proper adjuvant is a key for developing anti-toxoplasma vaccine.

In view of the above, the present invention provides a DNA vaccine for preventing human and animal toxoplasmosis recombinant toxoplasma gondii ROP47 gene, IEDB predicts dominant epitopes of both human and BALB/C mouse MHC molecule restricted toxoplasma gondii ROP 47T cells, DNAStar-Protean software is used to comprehensively analyze and predict dominant epitopes of the B cell of ROP47, ROP47 gene is amplified by PCR, and inserted into eukaryotic expression vector pEGFP-C1 between Kpn I and BamH I sites to construct recombinant plasmid pEGFP-C1-ROP47 (op pr 47), and recombinant plasmid containing target gene is extracted in large quantity to obtain toxoplasma gondii ROP47DNA vaccine. The vaccine of the invention can be used as an effective candidate vaccine for preventing toxoplasma gondii infection of human or animals, and particularly controlling the encapsulation formation in host brain tissues.

The recombinant toxoplasma ROP47DNA vaccine of the invention is used for intramuscular injection immunization of BALB/c mice. Immunogenicity of the vaccine was evaluated by measuring cellular and humoral immune response indices, and immunoprotection of the vaccine was evaluated by counting the number of cysts in the brain of mice after the PRU strain toxoplasma challenge experiment. The results show that compared with the control group, the recombinant toxoplasma ROP47DNA vaccine group can effectively reduce the formation rate of cerebral cysts of immunized mice after the toxoplasma PRU strain (type II) is attacked. The invention has the following outstanding effects: the vaccine can be used as an effective candidate vaccine for controlling encystation of brain tissue in the process of toxoplasma gondii chronic infection.

The vaccine of the invention, the immunogenicity of which is evaluated by the following modes: BALB/c mice were immunized 3 times intramuscularly with the DNA and MPLA combination vaccine at 2 week intervals. Blood was collected from the inner canthus vein of the immunized rat before the first immunization, 2 weeks after the 2 nd immunization, and 2 weeks after the third immunization, and serum was isolated to determine the IgG concentration in the blood sample. 4 weeks after the last immunization, spleens of immunized mice are taken aseptically, a splenic single cell suspension is prepared by grinding through a 200-mesh copper mesh, and the immunogenicity of the vaccine is evaluated by adding ROP47 composite epitope peptide to stimulate the culture and then detecting cytokines.

The vaccine of the invention, the mode for evaluating the immunoprotection of the vaccine is as follows: mice were challenged with 20 Toxoplasma II PRU worm strains per experimental mouse encysted 4 weeks after the last immunization. Mice were sacrificed after 8 weeks, brains were removed under sterile conditions and brain tissue homogenates were prepared by physical grinding. Microscopically the number of cysts counted. The immunoprotection of the vaccine was evaluated by comparing the encystment numbers of the brains of mice in the experimental and control groups.

The invention provides a recombinant toxoplasma ROP47DNA and MPLA combined vaccine, and experiments prove that the vaccine can effectively improve the immune protective response of mice to toxoplasma, and reduce the formation of encystment in brain tissues.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.

Example 1 Toxoplasma ROP47 epitope prediction

B cell dominant epitope of ROP47 was predicted by comprehensive analysis of DNAStar-Protean software; IEDB is used to predict the dominant epitope of Toxoplasma gondii ROP 47T cells with both human and BALB/C mouse MHC molecule restriction.

Example 2 construction of recombinant Toxoplasma gondii ROP47 plasmid

The ROP47 gene is amplified by using the prepared toxoplasma gondii cDNA as a template under the following reaction conditions: denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles. And (3) subjecting the amplified product to 1% agarose gel electrophoresis, cutting gel to recover a target fragment, recovering and purifying the PCR product and the pEGFP-C1 plasmid vector, performing enzyme digestion for 1h respectively, recovering and purifying the PCR purified product and the pEGFP-C1 plasmid vector, mixing the recovered fragments in a ratio of 5: 1, and connecting the fragments at 4 ℃ under the action of T4 ligase overnight. And (3) taking the connecting mixture to transform competent bacteria, uniformly coating the connecting mixture on an LK culture medium plate, culturing overnight, selecting a single colony to culture in an LK liquid culture medium, and extracting plasmids after 8 hours to obtain the recombinant plasmids containing the target genes.

Example 3 Toxoplasma recombinant Gene vaccine immunization of BALB/c mice

SPF-grade female BALB/c mice (6-8 weeks) were purchased from the university of Shandong laboratory animal center. 100 mice were randomly divided into 5 groups, and each group was injected intramuscularly in the hind leg with 100. mu.g of the recombinant vaccine pROP47 plus 20. mu.g of MPLA, and the other four groups were injected with 100. mu.g of pEGFP-C1, 100. mu.g of PBS, 20. mu.g of MPLA and 100. mu.g of pROP47, respectively. Mice were immunized three times in total, two weeks apart.

Example 4 determination of the level of humoral and cellular immunity in immunized mice

Blood is taken from the inner canthus of the mouse 2 days before each immunization and 6 weeks after the last immunization, blood samples are firstly kept still for 2 hours at room temperature, then are centrifuged for 15min at 4000rpm, and serum is collected. The post-ELISA method measures the level of total anti-Toxoplasma immunoglobulin (IgG) in the serum. The results are shown in FIG. 4. After two immunizations, antibody titer levels were significantly higher in the pROP47/MPLA vaccine immunized group compared to the other groups, with the highest level of Toxoplasma-specific IgG antibodies detected in the pROP47/MPLA immunized group of mice 6 weeks after the last immunization (P < 0.05).

Spleens from immunized mice were aseptically harvested 2 and 6 weeks after the last immunization and were ground through a 200 mesh copper mesh to prepare splenic single cell suspensions. After red blood cells were removed from spleen cells using a red blood cell lysis buffer, the cells were resuspended in 1640 medium containing 10% fetal bovine serum, and the concentration of the cell suspension was adjusted to 5X 10⁷Adding ROP47 composite epitope peptide to stimulate the content of cell factor in each cell/ml, and detecting the content of cell factor by flow cytometry after culture. As shown in FIG. 5, the cytokine content of the pROP47/MPLA vaccine immunized group was significantly increased (P) compared to the PBS, MPLA, pEGFP-C1 or pROP47 group (P)<0.05), and the highest level of IFN-. gamma.was detected in splenocytes from mice of the pROP47/MPLA immunized group (P)<0.01)。

Example 5 Toxoplasma recombinant DNA Gene vaccine protective Studies

2 weeks after the last immunization, the experimental mice were subjected to an insect challenge experiment. Mice were orally infected with 20 Toxoplasma gondii cysts suspended in 0.1 ml PBS. After 1 month of infection, all mice were sacrificed and the brains of the mice were isolated for physical grinding to prepare brain tissue homogenates. The brain tissue homogenates were mixed well, 10 μ l were counted under a microscope three times and the average value was taken, the encapsulation number of the mouse brain was 10 times the average value. As shown in fig. 6, the worm challenge experiment showed that: the minimum number of cysts was detected in the brains of mice in the pROP47/MPLA vaccine immunized group compared to the PBS, MPLA, pEGFP-C1 or pROP47 groups (P < 0.01).

In conclusion, the pROP47/MPLA gene vaccine can effectively induce the specific humoral and cellular immune response of the mouse. The vaccine can successfully improve humoral immunity and cellular immune response, effectively protect immune mice against attack of toxoplasma gondii cysts, and reduce the quantity of brain cysts of the immune mice. The vaccine can be used as an effective candidate vaccine for controlling the toxoplasma chronic infection, and is suitable for preventing the toxoplasma infection of people and animals.

The invention is not the best known technology.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong first medical university (Shandong province medical science institute)

<120> vaccine for preventing toxoplasma infection and preparation method and application thereof

<130>

<160>3

<170>PatentIn version 3.3

<210>1

<211>3076

<212>DNA

<213> Artificial sequence

<400>1

ggcaagcaaa gacgcttgag acgctagcgc cgtcagacgc agggtgacgg aacgaactgt 60

cgttgtgggt ctatttgtct cacttgccaa gacaaggcat gtctgatgtc acgtatcgct 120

agctgtaagt cggtagcact gtgcgctgca ggcaacgacg ctcgcctgct aggcccccgt 180

ttagtacgcg cagttgagcg agagcactgc tggcagaaag cacaacgctg attcgcccga 240

attgacagac acgcaactgg caaatccgtt ccttgaggga gaagctcgat tcgtcagagc 300

ttcctcttaa ggaccacagc cgggcctttg ccgagaaaat cccgcatttt gccggggcaa 360

tctcaagtgg atccgcgctg gaagcagggt tattgtctgt ttaacttttt gagggaagga 420

ccaacatgag gcgttcgagg tcaaaatacc atgtcggtac ttttgtcact gggtcagctg 480

ttcagcgact gacgctagtt ggaatgtatt ctgcagtgtg ggtcacttca gcacatagct 540

cgcctgtcaa ttctgcaact gtgtcccctt cgttcttttt cctgcattcc aagccgcctc 600

atggctcccc cccaaatgag aggaccactg gagagtccag tggctccaac gcctccatag 660

agttgatgga aatgcagcct acgcaaccag agtccctttc cccgaccgcg atggccgcac 720

gtcgcccgaa tgtaccacca cttcccgaag caagcgggct gccgtcgcgc ttgcctccat 780

ggcaggaaac gcccttttcg cctggaaagg gtgacacaca ggactctgca caagtgagca 840

ctccttcagg cactccacgc gtgcagccga taagttcccc gaagcgagaa gctctttttc 900

ggatgtcccc tcccccggta ccaaagcgtt ctctgcgcag gactctttcg caatatttcc 960

gtgcacggaa acattctggt gtcccaaatc cccgcgcctc tgcacaactg ccaacagccg 1020

atcgggagca agcagtgaac cggccgcaca gagcttcccg gctgatgaag agttctccgt 1080

cttctgcaag tcaccaggcg catggagcag cgcctcatcg gcggcgcgct gctacctcac 1140

cccagcctcg cacacgcaga gacccagcag gtccagttgg tcggacgctg aggaattgga 1200

ttagcgaccg attatcccgc gcatacagac atgtcaggag caggctgcgc acgggatatg 1260

ccagactgct ccggcaactt cattccatga cagagaaaat tcccacacca ccagtacagc 1320

tgcccggtca ggagtgcgta aacgttttgg gggctcatgt cgctcgacag gtggccgact 1380

ggaggcaaca aatgggacgc acctcgctcg agtttgcagt ggatgcagct tttcctcgcg 1440

ataaagatat ttcgttcaag tcatacatct caaggaaaag tgtcacttta cgcatgggga 1500

aggttttagg agggggtgca aggagcattg ttgtggaggt aacgcgagtt gataccggcg 1560

acagatgggc tttgaaagct ttcctggttc cacgggatgt cattggctcc cctggaaccc 1620

caccgccttg ggacgcaatc agcgaggaag cccatgcgat aaaattacta ccgcatacag 1680

ttccagctgt agtttatcga aacctgcggt ttctagtccc cgccgacgtg ttgatatacg 1740

agggagacgg aatggagaag ctttggaaca gacgcggaac gggctcaaac ttctacacaa 1800

cctatcttct tttcccaata gctcaaggtt ctctgttgga cgctgtcaga gcaatgttcc 1860

gagatgcgag acggggcaat gctgaggtgt ctgatggtga ggcagcacgc accctggccg 1920

cgaatgtgag tttttcgatt cagattatcc gcctcacggc actgcttcac agccacgggg 1980

ttgtgcatgg ggacttccaa acgaagagct tttttttgtg tcaagacggc tctctgttcc 2040

tcggtagagt ccgcagtctg acgcgagtag gagccccata tcgaagcccc agttccgcga 2100

cagacagtcc tcgctgcact cccccagagg tgctatcccc cccagaaaat gcggctttta 2160

cattttcgct gaatgcctgg gaacttggtt gcattctcta ccagctctgg tgtgagggtc 2220

ttccgtttgg tctgtccccg acacccgaga cacctgggaa aatcgtgctg cctcctgtca 2280

taacatcgcc ttatttgcag acaaagaaac gatctggctt agcgggcaca tcaacacgtc 2340

cagtcaagca tctggtgttc actggctgcg ttaaaaacat gcccgttgaa atgcgacaat 2400

tgattcgtcg cctgctccag gaagacccac acaaaaggtt actcccgact gagattcttc 2460

gggatcctgt tttccagaaa ctcgaagagc aatcgaggcc accggacgcc caggcttaga 2520

tggggttgac atgtttcgaa cccagtttcg ttctcggaag gttcttgccc gctattcagg 2580

gcatctgact cggtccagct ttgagtgcct tgtgcctgca cagtgttgaa attcgaaagc 2640

gtaggtgcac cgagatgtcc gtgtcactgt tctagccagt tctctcctga tgcttttcct 2700

cgaacctcca tacttgtacg atgttgatcc gcaaatacgt gtctattgaa agggttgcac 2760

acaaggtgtg tgtggagaga gtttgttttg caagctcgtg cttctgtatc acgcaaccac 2820

gcgggacacg ctccacagtg tggggcctct atggaggacg acatttttga ttgttgttga 2880

tcaattcatc caatagcaca aacaggaaag agttttttcg tttgctgctc gtcaacgcgc 2940

tcttctctcg ttgtgcctct cacctgcgtc ctcttgccct cgctccgctc ccaagtcttg 3000

cagaaatgtc aatttataac gaacttgtgg ctgagtagac aaatttagaa agctgggctg 3060

ctgaccatat taagcg 3076

<210>2

<211>24

<212>DNA

<213> Artificial sequence

<400>2

ggggtacctc cgcaagctcc agtt 24

<210>3

<211>23

<212>DNA

<213> Artificial sequence

<400>3

cgggatccga ttcggacccg cag 23

Claims (10)

1. A nucleotide sequence for use in the prevention of toxoplasma infection, which comprises a nucleotide sequence selected from any one or more of the following 1) to 3):

1) the nucleotide sequence of the clavulan protein ROP47 shown in SEQ ID NO. 1;

2) a nucleotide sequence complementary to the nucleotide sequence in 1);

3) a nucleotide sequence obtained by modifying the nucleotide sequence shown in 1) or 2) above by substitution, deletion or addition of one or more bases.

2. A vector comprising the nucleotide sequence of claim 1.

3. A recombinant plasmid is obtained by inserting ROP47 gene into eukaryotic expression vector pEGFP-C1; preferably, the recombinant plasmid is obtained by inserting ROP47 gene into eukaryotic expression vector pEGFP-C1 between Kpn I and BamH I sites.

4. A protein encoded by the recombinant plasmid of claim 3, wherein the protein has an amino acid sequence.

5. A vaccine comprising the nucleotide sequence of claim 1, the vector of claim 2 and/or the recombinant plasmid of claim 3, and a pharmaceutically acceptable carrier.

6. The vaccine of claim 5, wherein the pharmaceutically acceptable carrier is an adjuvant; preferably, the adjuvant is a TLR agonist; further preferred is monophosphoryl lipid a.

7. A method for preparing the vaccine of claim 5 or 6, wherein the method comprises:

constructing recombinant Toxoplasma gondii ROP47 recombinant plasmid; the ROP47 recombinant plasmid is mixed with a pharmaceutically acceptable carrier.

8. The preparation method of claim 7, wherein the specific method for constructing the recombinant Toxoplasma gondii ROP47 recombinant plasmid comprises the following steps: PCR amplification ROP47 gene using toxoplasma cDNA as template, and connecting the purified amplification product with pEGFP-C1 plasmid carrier to form recombinant plasmid.

9. The method of claim 7, wherein the primers for amplifying the ROP47 gene are as follows:

an upstream primer: 5'-ggGGTACCTCCGCAAGCTCCAGTT-3' (SEQ ID NO. 2);

a downstream primer: 5'-cgGGATCCGATTCGGACCCGCAG-3' (SEQ ID NO. 3);

alternatively, the PCR conditions for amplifying the ROP47 gene were as follows:

denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles;

the pharmaceutically acceptable carrier is an adjuvant, the adjuvant is a TLR agonist, and further preferably monophosphoryl lipid A.

10. Use of the nucleotide sequence according to claim 1, the vector according to claim 2, the recombinant plasmid according to claim 3, the protein according to claim 4 and/or the vaccine according to claim 5 or 6 in a) to c) as follows:

a) prevention of toxoplasma infection;

b) enhancing the immunoprotective response of the vaccinee to toxoplasma;

c) controlling encystment formation of brain tissue in the process of toxoplasma chronic infection;

the vaccinees include humans and non-human animals, which are mammals, and further include mice.

Images