A Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain and its application

Abstract

The invention discloses a Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain and its application. The strain (Δsdhα) is obtained by directly knocking out TgSDHα of Toxoplasma gondii through gene editing technology; after knocking out TgSDHα, the growth of Toxoplasma gondii is significantly affected, the proliferation rate of the parasite in vitro is slowed down, and the number of Toxoplasma gondii reproduction in vivo can be reduced; the Δsdhα strain has the characteristics of being able to be subcultured in vitro and almost non-toxic in vivo, and all mice infected with the Δsdhα strain survive; after immunizing animals with the Δsdhα strain, good immune protection can be generated, and the infection of wild-type Toxoplasma gondii can be resisted. The Δsdhα strain can be used to prepare vaccines against toxoplasmosis, providing safe and effective vaccine candidates for the prevention and treatment of toxoplasmosis. At the same time, TgSDHα can be used as a target for Toxoplasma drug design, providing more effective drug targets and gene resources for the prevention and treatment of toxoplasmosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain and its application. Background Art

[0002] Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, including humans. Toxoplasma infection often causes miscarriage and stillbirth in humans and animals, seriously endangering human health and livestock and poultry farming. Currently, only one Toxoplasma vaccine, Toxovax, is approved for the prevention of toxoplasmosis in sheep, but the vaccine has the risk of reversion to virulence and restoration of oocyst formation, which limits its widespread use. Therefore, there is an urgent need to develop a safe and effective anti-toxoplasmosis vaccine.

[0003] Metabolic pathways are an important treasure trove for vaccine target discovery and attenuated vaccine strain design. The existing uridine nutrient-deficient strains lacking the carbamoyl phosphate synthetase CPSII or orotidine-5'-monophosphate decarboxylase OMPDC genes can only grow well under culture conditions containing uracil, but the parasites cannot establish acute infection in mice, so their virulence is severely weakened, and they have great vaccine potential. In addition, studies have found that lactate dehydrogenase LDH-mediated lactic acid fermentation does not affect the growth of Toxoplasma gondii under aerobic conditions, but LDH gene knockout strains cannot reproduce in vivo and have almost no virulence, and have the potential to become attenuated live vaccines; these research advances show that constructing nutrient-deficient strains based on metabolic pathways is a feasible method for developing attenuated candidate vaccines. Although some encouraging progress has been made in the research of toxoplasmosis vaccines in recent years, the number of potential candidate vaccine strains is still very limited. Due to the genetic diversity of Toxoplasma, the cross-immunity protection of a single vaccine strain is often insufficient. Therefore, more vaccine design targets need to be discovered and more candidate vaccine strains need to be developed.

[0004] The tricarboxylic acid cycle (TCA) is considered to be the main energy supply pathway for Toxoplasma tachyzoites and is closely related to the growth and reproduction of the parasite. Succinate dehydrogenase (SDHα) in TCA catalyzes succinate to produce fumarate and is one of the hubs connecting oxidative phosphorylation and electron transfer. However, the role of SDHα in the growth and pathogenicity of Toxoplasma is still unclear, and the potential of SDHα gene-deficient strains as vaccine strains is also unclear. Therefore, it is still necessary to discover more anti-Toxoplasma vaccines or target genes to overcome the shortage of existing vaccines and gene resources for preventing Toxoplasma infection. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the insufficiency of existing vaccines and gene resources for preventing Toxoplasma infection, and to provide a Toxoplasma succinate dehydrogenase α subunit gene-deficient vaccine strain and its application.

[0006] The first object of the present invention is to provide a Toxoplasma gondii succinate dehydrogenase α subunit (TgSDHα) gene-deficient vaccine strain.

[0007] The second object of the present invention is to provide a method for constructing a TgSDHα-deficient vaccine strain.

[0008] The third object of the present invention is to provide the application of TgSDHα-deficient vaccine strain.

[0009] The fourth object of the present invention is to provide an anti-toxoplasmosis drug.

[0010] A fifth object of the present invention is to provide a vaccine against Toxoplasma gondii infection.

[0011] The sixth object of the present invention is to provide the application of Toxoplasma gondii succinate dehydrogenase α subunit.

[0012] The seventh object of the present invention is to provide the use of a reagent for deleting or knocking out the α-subunit of Toxoplasma succinate dehydrogenase.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0014] The present invention provides a Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain, which is obtained by directly knocking out the Toxoplasma succinate dehydrogenase α subunit (TgSDHα) gene through gene editing technology; the nucleotide sequence of TgSDHα is shown in SEQ ID NO.1.

[0015] The present invention studies the effect of the Toxoplasma succinate dehydrogenase α subunit (TgSDHα) gene on the growth and pathogenicity of the parasite, reveals the application of TgSDHα as a drug action target or a vaccine design target, and successfully constructs a TgSDHα-deficient parasite strain Δsdhα after knocking out the parasite TgSDHα. After knocking out the parasite TgSDHα, the growth of the parasite is significantly affected, the growth rate is slowed down, and the proliferation ability of Toxoplasma in vitro is weakened, and the number of reproduction in vivo is reduced. The parasite strain (Δsdhα) can still be cultured in vitro, has almost non-toxic characteristics in vivo, and all mice infected with the Δsdhα strain survive; after animal immunization with Δsdhα, the infection of wild-type Toxoplasma can be resisted, the strain has good immune protection, and has the advantage of weakened toxicity, and can be used as a Toxoplasma vaccine strain, and can be used to prepare anti-toxoplasmosis drugs, providing effective means and methods for preventing and treating toxoplasmosis.

[0016] Furthermore, through CRISPR / Cas9 gene editing technology, TgSDHα was directly knocked out in the parental DiCre strain to obtain a gene-deficient vaccine strain.

[0017] The present invention provides a method for constructing a TgSDHα gene-deficient vaccine strain, comprising the following steps:

[0018] (1) Construction of the TgSDHα gene CRISPR knockout plasmid pCas9-gRNA1-gRNA2-TgSDHα;

[0019] (2) Construction of homologous recombination template Tub::YFP::DHFR*-TgSDHα;

[0020] (3) The pCas9-gRNA1-gRNA2-TgSDHα plasmid and the homologous recombination template were co-electroplated into the parental Toxoplasma gondii strain, and the Toxoplasma gondii TgSDHα gene-deficient strain was obtained through drug screening and PCR identification.

[0021] Preferably, in step (1), pCas9-U6-sgMIC3 plasmid is used as a template to perform homologous recombination to obtain a backbone fragment, and then the homologous arms are connected to construct a pCas9-gRNA1-gRNA2-TgSDHα plasmid.

[0022] More preferably, the pCas9-U6-sgMIC3 plasmid is used as a template, and the specific gRNA primers of the TgSDHα gene are used for homologous recombination to construct pCas9-U6-5UTR-sgSDHα plasmid and pCas9-U6-3UTR-sgSDHα plasmid, respectively, and then used as templates to amplify the backbone and insert fragments, respectively, to construct the pCas9-gRNA1-gRNA2-SDHα plasmid.

[0023] Preferably, in step (2), the pTub-YFP-DHFR* plasmid is used as a template to amplify the homologous fragment.

[0024] Further preferably, the gRNA primer sequence of the TgSDHα gene in step (1) is as shown in SEQ ID NO.2-3.

[0025] More preferably, the sequences of primers for amplifying the Tub::YFP::DHFR*-TgSDHα homologous fragment in step (2) are as shown in SEQ ID NOs. 10-11.

[0026] The present invention provides application of a TgSDHα gene-deficient vaccine strain in preparing an anti-toxoplasmosis drug.

[0027] The invention provides an anti-toxoplasmosis drug, comprising a TgSDHα gene-deficient vaccine strain.

[0028] The invention provides a vaccine for resisting toxoplasma infection, comprising a TgSDHα gene-deficient vaccine strain.

[0029] The present invention provides the use of a Toxoplasma gondii succinate dehydrogenase alpha subunit TgSDHalpha gene as a target in screening or preparing an anti-toxoplasmosis drug.

[0030] The present invention also provides the use of a reagent for deleting or knocking out the Toxoplasma succinate dehydrogenase alpha subunit TgSDHalpha gene in constructing a gene-deleted vaccine strain or in preparing a drug for resisting Toxoplasma infection.

[0031] Preferably, the reagent for deleting or knocking out the Toxoplasma gondii succinate dehydrogenase α subunit TgSDHα gene is a vector or a recombinant bacterium containing a TgSDHα gene knockout plasmid.

[0032] The present invention has the following beneficial effects:

[0033] The present invention provides a Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain, which is obtained by directly knocking out the Toxoplasma gondii succinate dehydrogenase α subunit TgSDHα gene through gene editing technology. After knocking out the TgSDHα gene, the growth ability of the worm body will be affected and the growth rate of the worm body will be slowed down, but the Δsdhα worm strain can still be cultured in vitro and has almost non-toxic characteristics in vivo, and all the mice after inoculation still survive; after further animal immunization Δsdhα, the vaccine worm strain can produce good immune protection, can resist infection by wild-type Toxoplasma gondii, and can be used to prepare vaccines against toxoplasmosis to prevent Toxoplasma infection. At the same time, TgSDHα can be used as a target for Toxoplasma drug design, providing more effective drugs and gene resources for the prevention and treatment of toxoplasmosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of the construction of Toxoplasma Δsdhα.

[0035] Figure 2 These are the PCR results of the Δsdhα monoclonal strain.

[0036] Figure 3 These are the results of the plaque assay of the Δsdhα strain.

[0037] Figure 4 These are the results of the insect load experiment on the Δsdhα strain.

[0038] Figure 5 These are the results of the virulence experiment on the Δsdhα strain.

[0039] Figure 6 These are the results of the immune protection experiment when mice immunized with the Δsdhα strain were infected with the wild-type RH-Luc strain.

[0040] Figure 7 These are the results of the immune protection experiment when mice immunized with the Δsdhα strain were infected with the wild-type ME49 strain. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0043] Example 1 Construction of a Toxoplasma gondii gene knockout Δsdhα strain

[0044] 1. Source of insect strains

[0045] In this example, gene knockout was performed on the basis of the parent strain DiCre (kindly donated by Huazhong Agricultural University), and the succinate dehydrogenase α subunit gene (flavoprotein subunit of succinate dehydrogenase, TgSDHα, gene ID: TGGT1_215590) in the ToxoDB (www.toxodb.org) website was searched. The nucleotide sequence of TgSDHα is shown in SEQ ID NO.1.

[0046] 2. Construction of pCas9-gRNA1-gRNA2-TgSDHα plasmid

[0047] (1) Design and synthesize TgSDHα gene-specific gRNA primers through the gRNA website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html), as shown in Table 1 below, and construct gRNA-specific plasmids;

[0048] Table 1 Primers for amplifying pCas9-U6-5UTR-sgSDHα plasmid and pCas9-U6-3UTR-sgSDHα plasmid

[0049]

[0050] (2) Using pCas9-U6-sgMIC3 plasmid (kindly donated by Huazhong Agricultural University) as a template, the backbone fragments required for constructing TgSDHα-specific CRISPR / Cas9 plasmids were amplified, and the primers used were shown in Table 2. The backbone fragments were then connected with the TgSDHα-specific primers in Table 1, and the reaction was carried out at 37°C for 30 minutes according to the reaction system shown in Table 3 to construct pCas9-U6-5UTR-sgSDHα plasmids and pCas9-U6-3UTR-sgSDHα plasmids;

[0051] Table 2 Primers for amplifying the backbone fragments required for pCas9-U6-5UTR-sgSDHα plasmid and pCas9-U6-3UTR-sgSDHα plasmid

[0052]

[0053] Table 3 Reaction system

[0054]

[0055] (3) All the above products were added to the TCS-C01-100 competent cells of Qingke, first cooled on ice for 5 minutes, then heat-shocked in a 42°C water bath for 1 minute, cooled on ice again for 2 minutes, and then spread on Amp+ solid culture medium, cultured at 37°C for 16 hours, and 6 single colonies were picked and placed in liquid culture medium containing ampicillin for expansion culture. If the sequencing results of the bacterial liquid correspond to the expected plasmid map, it means that the pCas9-U6-5UTR-sgSDHα plasmid and the pCas9-U6-3UTR-sgSDHα plasmid were successfully constructed;

[0056] (4) Using the primers in Table 4, the insert fragment was amplified using primers of SEQ ID NO. 6-7 with the pCas9-U6-3UTR-sgSDHα plasmid as a template; using primers of SEQ ID NO. 8-9 with the pCas9-U6-5UTR-sgSDHα plasmid as a template, the backbone was amplified, and the PCR product of the amplified backbone was digested with Dpn I to remove the template DNA according to the system and reaction procedure in Tables 5 and 6;

[0057] Table 4 Primers for constructing pCas9-gRNA1-gRNA2-TgSDHα plasmid

[0058]

[0059]

[0060] Table 5 DpnⅠ digestion system

[0061]

[0062] Table 6 DpnⅠ digestion procedure

[0063]

[0064] (5) The PCR reaction solution of the amplified backbone and fragments was subjected to agarose gel electrophoresis, the target band fragment was cut out and placed in a 1.5 mL EP tube, and the target fragment was purified using the Novozyme product purification kit DC301-01;

[0065] (6) The purified backbone and fragment products were reacted at 50°C for 15 min according to the reaction system in Table 7, and then the pCas9-gRNA1-gRNA2-TgSDHα plasmid was successfully constructed by referring to the method in step (3).

[0066] Table 7 Reaction system

[0067]

[0068]

[0069] 2. Amplify the Tub::YFP::DHFR*-TgSDHα homologous fragment

[0070] Using the Tub-YFP-DHFR* plasmid as a template, the corresponding homologous fragments were amplified according to the primers in Table 8, and the amplified products were purified.

[0071] Table 8 Primers for amplifying Tub::YFP::DHFR*-TgSDHα homologous fragments

[0072]

[0073] 3. Construction of the Toxoplasma gondii knockout strain Δsdhα

[0074] (1) The type I parent strain DiCre was cultured in a T25 culture flask. When a large number of nanovesicles grew after about 2-3 days, the cells were washed with PBS for 2-3 times, and then all the attached cells were scraped off with a cell scraper and blown 10 times with a 5 mL syringe until the cells were blown away. Then all the DiCre tachyzoites were collected and centrifuged at 1000 rpm for 3 min; the supernatant was collected and filtered for purification, and centrifuged at 3000 rpm for 8 min; the supernatant was discarded, and 8 mL of Cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4 / KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, pH = 7.6) was added to resuspend the worms, and centrifuged again at 3000 rpm for 8 min; 300 μL of Cytomix was used to resuspend the worms;

[0075] (2) Take 1500 ng of Tub::YFP::DHFR*-TgSDHα homologous fragment and 7500 ng of pCas9-gRNA1-gRNA2-TgSDHα plasmid, and the resuspended insect body in step (1) and add them to a 1.5 mL EP tube and mix evenly. Transfer all to a 4 mm electric rotating cup and let it stand for 2 minutes;

[0076] (3) Place the electroporation cup containing the mixed solution in the previous step into the TBX electroporator and set the following conditions for electroporation: 1600V, 25μF, 50Ω, 4mm once; 1500V, 25μF, 50Ω, 4mm once; 1500V, 25μF, 50Ω, 4mm once. After the first two electroporations, take out the cup and shake it gently. After the last electroporation, let it stand for 2 minutes. Then add all the mixed solution in the electroporation cup to a T25 culture flask full of human fibroblast HFF cells for culture.

[0077] (4) When the electroporated strains are cultured until more vesicles can be seen, drug screening is started using DMEM medium containing 2% FBS with a 1 μM pyrimethamine concentration. After three generations of drug screening, the strains are relatively stable and the number of wild-type strains gradually decreases. At this time, single clone strain screening can be performed;

[0078] (5) Collect freshly escaped strains of the third generation of drug screening, filter and purify them according to (1), dilute them 10 times and count them, take an appropriate volume of the stock solution and dilute it to 20 mL of DMEM medium containing 2% FBS, mix it and add it to a 96-well plate at a rate of 200 μL / well so that there is only one Toxoplasma tachyzoite in each well, culture it in a constant temperature incubator at 37°C and 5% CO2 for 7 days, observe and select the single-spotted strain under a microscope, collect half of the tachyzoites in the well to extract DNA for strain identification, and subculture and expand the other half;

[0079] (6) TgSDHα gene knockout construction model Figure 1 As shown in Table 9, the monoclonal insect strains selected in the above steps were identified by PCR, and the results were as follows: Figure 2 As shown, the band sizes shown by PCR1 and PCR2 of the monoclonal insect strain are consistent with expectations, and there is no band in PCR3; while there are no bands in PCR1 and PCR2 of the wild-type DiCre, and the band amplified by PCR3 is also consistent with expectations, indicating that the TgSDHα gene has been knocked out and the Δsdhα monoclonal insect strain has been successfully constructed.

[0080] Table 9 PCR primers for identification of monoclonal strains of Δsdhα

[0081]

[0082] Example 2 In vitro plaque assay of Δsdhα strain

[0083] (1) When the DiCre and Δsdhα strains grew a large number of nanocysts in a T25 culture flask, fresh parasites were enriched and purified according to the method in Example 1, and 100 Toxoplasma tachyzoites / 200 μL / well were inoculated into a 6-well plate full of HFF cells, and cultured in a 37° C., 5% CO2 incubator for 6 days;

[0084] (2) After 6 days, each well was washed 2-3 times with PBS, fixed with 4% paraformaldehyde at 37°C for 20 min, and washed 2-3 times with PBS;

[0085] (3) Add 1 mL of 0.1% crystal violet to each well and stain for 30 min at 37°C, wash 2-3 times with PBS, and air-dry in a cool and dry place;

[0086] (4) Scan the image with a scanner.

[0087] The results are as follows Figure 3 As shown in the figure, under normal culture conditions, the wild-type strain will form visible empty plaques after 6 days of culture, while the Δsdhα strain will hardly form plaques. This result is consistent with the phenomenon observed during the experiment: the parent strain DiCre normally only needs 2-3 days to pass, while the Δsdhα strain needs 5-6 days to pass. In summary, after the worm body lacks TgSDH, the growth rate in vitro slows down, but the Δsdhα strain can still proliferate and can still be passed down and cultured.

[0088] Example 3 In vivo proliferation experiment of Δsdhα strain

[0089] (1) Serum-free DMEM medium was used to collect intracellular parasites of the Δsdhα strain and the type I strain DiCre;

[0090] (2) The collected insects were diluted 10-fold and counted. The Δsdhα and DiCre strains were counted at 1×10 4 Five 7-week-old female ICR mice (Guangzhou Ruige Biotechnology Co., Ltd.) were inoculated with the same doses by intraperitoneal injection. After inoculation, the mice were fed normally under the same conditions. After 5 days, the mice were killed and 4 mL of normal saline was injected into the peritoneal cavity. The mouse bodies were gently shaken to suspend the parasites in the peritoneal fluid as much as possible. The ascites were then withdrawn to collect the parasites in the peritoneal cavity. The tubes were centrifuged at 3500 rpm for 7 minutes, and the supernatant was discarded to leave 2 mL of liquid in a 15 mL centrifuge tube. The tubes were resuspended with a pipette tip, and 200 μL of liquid was drawn for gDNA extraction. Sterile saline was injected into the mice intraperitoneally, and an extract was used as a negative reference sample. Then, different concentrations of DiCre tachyzoites (0, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 pcs / 2mL) was added to the negative reference sample. 200 μL was taken from each mixed solution to extract genomic DNA, and then a standard curve was prepared. The load of mice was detected by real-time fluorescence quantitative PCR (qPCR), the detection primers are shown in Table 10, the qPCR reaction system is shown in Table 11, and the reaction procedure is shown in Table 12.

[0091] Table 10 Primers used for qPCR

[0092]

[0093] Table 11 qPCR reaction system

[0094]

[0095] Table 12 qPCR reaction program

[0096]

[0097] The DNA samples were tested using a real-time fluorescence quantitative PCR instrument (LC480). The sample Ct values ​​were substituted into the standard curve, the worm load of each mouse was calculated according to the formula, and a bar graph was drawn to statistically analyze the results.

[0098] Test results such as Figure 4 As shown, the type I strain DiCre continued to multiply in the mouse and its number increased significantly. In contrast, the Δsdhα strain hardly multiplied in the mouse, indicating that the loss of TgSDHα significantly affected the growth ability of the worm in vivo.

[0099] Example 4 Experiment on the immune protection of Δsdhα strain on mice

[0100] 1. In vivo virulence experiment of Δsdhα strain

[0101] DiCre and Δsdhα tachyzoites were collected and diluted using serum-free DMEM medium, and 10 7-week-old female ICR mice were inoculated with 100 DiCre tachyzoites / mouse. Similarly, 10 7-week-old female ICR mice were inoculated with 100 Δsdhα tachyzoites / mouse. The mental state and survival status of the mice were recorded every day after inoculation. Blood was collected from all surviving mice after 30 days, and the mouse serum was tested by IFA.

[0102] The results showed that all mice were serum positive. The mice inoculated with wild-type DiCre strain had some dead individuals on the 7th day after infection, and all died by the 9th day. Figure 5 As shown; all the mice inoculated with Δsdhα worms survived, indicating that the virulence of the Δsdhα worm strain in mice was weakened, significantly prolonging the survival time of mice.

[0103] 2. Immunoprotective effect of Δsdhα strain on mice

[0104] (1) Wash the extracellular tachyzoites with PBS, add serum-free DMEM medium to collect intracellular Toxoplasma gondii RH-Luc and ME49 strains (kindly donated by Huazhong Agricultural University), dilute the collected liquid 10 times and count it under a microscope using a cell counting plate, and dilute the tachyzoites after counting;

[0105] (2) In the above in vivo virulence experiment, 10 mice inoculated with the Δsdhα strain at a dose of 100 parasites / mouse were still alive one month after inoculation, and their serum was positive by IFA test. These 10 mice were evenly divided into two groups, each with 10 4 RH-Luc and ME49 tachyzoites were injected intraperitoneally per mouse, and 10 blank mice were injected intraperitoneally with 10 4 RH-Luc and ME49 tachyzoites / mouse were inoculated, and the mouse survival rate curve was drawn using prism8.

[0106] The results after infection with different strains of insects are as follows Figure 6 and Figure 7 As shown, all mice in the non-immunized group died within 8 days after infection with RH-Luc, 20% of mice immunized with Δsdhα died within 30 days, and the remaining 80% of mice showed no clinical symptoms; 50% of mice in the non-immunized group died within 30 days after infection with ME49, while all mice immunized with Δsdhα survived within 30 days after infection with ME49 and showed no clinical symptoms. This shows that Δsdhα immunization has good immune protection and can resist wild-type Toxoplasma infection to a certain extent.

[0107] In summary, the present invention provides a Toxoplasma gene-deficient vaccine strain, which is obtained by directly knocking out the Toxoplasma succinate dehydrogenase α subunit TgSDHα gene through gene editing technology. After knocking out the TgSDHα gene, Toxoplasma can still be cultured in vitro, has almost non-toxic characteristics in vivo, and all mice survived after vaccination; further, animal immunization with Δsdhα can produce good immune protection against infection with wild-type Toxoplasma. TgSDHα can be used as a target for Toxoplasma vaccine design and for the preparation of Toxoplasma gene-deficient vaccine strains, providing a safe and effective vaccine candidate for the prevention and treatment of toxoplasmosis, and providing more methods for the research and development of anti-toxoplasmosis vaccines.

[0108] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Toxoplasma gondii succinate dehydrogenase α subunit gene-deficient vaccine strain, characterized in that: The vaccine strain directly knocks out the succinate dehydrogenase α subunit of Toxoplasma gondii through gene editing technology Tg SDHα gene is obtained; TgSDHα The nucleotide sequence is shown in SEQ ID NO.

1.

2. The vaccine insect strain according to claim 1, characterized in that: Through CRISPR / Cas9 gene editing technology, DiCre Direct knockout TgSDHα Gene, to obtain gene-deficient vaccine strains.

3. The method for constructing the vaccine strain according to claim 1 or 2, characterized in that: The following steps are involved: (1) Construction TgSDHα Gene CRISPR knockout plasmid pCas9-gRNA1-gRNA2- TgSDHα ; (2) Construction of homologous recombination template Tub::YFP:: DHFR *- TgSDHα ; (3) Place pCas9-gRNA1-gRNA2- TgSDHα The plasmid and homologous recombination template were co-transfected into the parental Toxoplasma strain, and Toxoplasma was obtained through drug screening and PCR identification. TgSDHα Gene deletion strains.

4. The construction method according to claim 3, characterized in that: In step (1), pCas9-U6-sgMIC3 plasmid was used as a template to perform PCR amplification to obtain the backbone fragment, and then the homology arms were connected to construct pCas9-gRNA1-gRNA2- TgSDHα Plasmid.

5. The construction method according to claim 4, characterized in that: In step (2), pTub-YFP- DHFR* The plasmid was used as a template to amplify the homologous fragment.

6. Use of the gene-deficient vaccine strain according to claim 1 or 2 in the preparation of an anti-toxoplasmosis vaccine.

7. A vaccine against Toxoplasma gondii infection, characterized in that: Contains the gene-deficient vaccine strain according to claim 1 or 2.

8. Deletion or knockout of Toxoplasma gondii succinate dehydrogenase α subunit TgSDHα The use of a gene reagent in constructing a gene-deficient vaccine strain or in preparing a vaccine against Toxoplasma gondii infection is characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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