Construction method and application of toxoplasma gondii gra5 gene knockout strain

Abstract

The application belongs to the technical field of biological products, and discloses a construction method of a Toxoplasma gondii gra5 gene knockout worm strain and application thereof. The construction method comprises the following steps: preparation of a GRA5-5UTR:DHFR:GRA5-3UTR homologous template, construction of a CRISPR plasmid for targeted excision of GRA5, and transformation of a worm strain, so as to obtain the Toxoplasma gondii gra5 gene knockout worm strain. The Toxoplasma gondii gra5 gene knockout worm strain obtained by the construction method has good safety, can inhibit the formation of cysts, can induce the body to produce effective immune protection, and can be used for preparing a Toxoplasma gondii attenuated live vaccine for the prevention and treatment of toxoplasmosis. In addition, the Toxoplasma gondii gra5 gene knockout worm strain can also be used as an antitumor preparation to kill tumor cells and inhibit tumor growth.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological products, and particularly relates to a construction method of a Toxoplasma gondii gra5 gene knockout strain and application thereof. BACKGROUND

[0002] Toxoplasmosis is a very important zoonosis parasitic disease caused by Toxoplasma gondii (Toxoplasma gondii, simply referred to as T. gondii) which is a specific intracellular parasite. T. gondii is an opportunistic pathogenic parasite that can infect the nucleated cells of all warm-blooded animals, including humans. About one-third of the world's population is infected with T. gondii, among which, if a pregnant woman is infected with T. gondii, it may cause miscarriage, stillbirth, premature birth, and teratogenicity and other serious consequences. In addition, pregnant livestock infected with T. gondii mainly shows miscarriage or stillbirth, resulting in a huge economic loss due to the decrease in the quality and quantity of agricultural products such as meat and milk. Therefore, the prevention and control of T. gondii and toxoplasmosis are of great significance to human health and the development of animal husbandry.

[0003] T. gondii has a complex population structure, and these different virulence strains have different proliferation capacity and pathogenicity in animals, which inevitably brings challenges to the prevention and control of toxoplasmosis. In addition, T. gondii has multiple transmission routes in humans or animals, among which the important route is to be infected by ingesting raw meat or uncooked meat containing pseudocysts or tissue cysts. Although pyrimethamine and sulfadiazine can be used for the treatment of tachyzoite infection, they have no therapeutic effect on bradyzoites in tissue cysts. Therefore, it is urgent to develop a safe and long-acting vaccine against T. gondii infection for the prevention and control of toxoplasmosis.

[0004] Therefore, the present application hopes to provide a preparation method of a T. gondii attenuated strain with good safety, which can induce the body to produce effective immune protection and inhibit the formation of cysts, so as to be used for preparing a live attenuated T. gondii vaccine. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a construction method of a T. gondii gra5 gene knockout strain and application thereof. The T. gondii gra5 gene knockout strain obtained by using the construction method has good safety, can inhibit the formation of cysts, induce the body to produce effective immune protection, and can be used for preparing a live attenuated T. gondii vaccine for the prevention and control of toxoplasmosis. In addition, the T. gondii gra5 gene knockout strain can also be used as an anti-tumor preparation to kill tumor cells and inhibit tumor growth.

[0006] The present application provides a construction method of a T. gondii gra5 gene knockout strain, comprising the following steps:

[0007] Preparation of a GRA5-5UTR::DHFR::GRA5-3UTR homologous template: taking the genomic DNA of the Toxoplasma strain as a template, primers are designed according to the 5' homologous arm and the 3' homologous arm of the gra5 gene to amplify the GRA5-5UTR fragment and the GRA5-3UTR fragment; taking a plasmid containing the DHFR sequence as a template, primers are designed to amplify the DHFR fragment; the GRA5-5UTR fragment, the GRA5-3UTR fragment and the DHFR fragment are connected by using a seamless connection technology to obtain the GRA5-5UTR::DHFR::GRA5-3UTR homologous template;

[0008] Construction of a CRISPR plasmid for targeted excision of GRA5: taking the CRISPR plasmid as a template, a PCR reaction is performed by using an sgRNA specific to the gra5 gene to achieve site-directed mutagenesis; the product after the PCR reaction is subjected to KLD connection; the product after the KLD connection is transformed into a competent cell to obtain the CRISPR plasmid for targeted excision of GRA5;

[0009] Transformation of the strain: the CRISPR plasmid for targeted excision of GRA5 and the GRA5-5UTR::DHFR::GRA5-3UTR homologous template are co-electrotransformed into the Toxoplasma strain, and a Toxoplasma gra5 gene knockout strain is obtained through drug detection and PCR identification.

[0010] It is pointed out in the present application that the gra5 gene is an important virulence-related gene of Toxoplasma, and the deletion of the gene can cause a severe cyst formation defect of Toxoplasma in the body and significantly reduce the virulence, thereby playing a good immune protection role and effectively preventing and treating toxoplasmosis. Experiments also show that the constructed Toxoplasma gra5 gene knockout strain also has a significant inhibitory effect on breast cancer and other tumors, thereby playing an anti-tumor role.

[0011] Preferably, the nucleotide sequences of the primers for amplifying the GRA5-5UTR fragment are shown in SEQ ID NO. 6-7.

[0012] Preferably, the nucleotide sequences of the primers for amplifying the DHFR fragment are shown in SEQ ID NO. 8-9.

[0013] Preferably, the nucleotide sequences of the primers for amplifying the GRA5-3UTR fragment are shown in SEQ ID NO. 10-11.

[0014] Preferably, the seamless connection is performed by using a HiFi DNA Assembly Cloning Kit. The seamless connection is performed by using a HiFi DNA Assembly Cloning Kit.

[0015] Preferably, the nucleotide sequence of the GRA5-5UTR::DHFR::GRA5-3UTR homologous template is shown as SEQ ID NO. 12.

[0016] Preferably, the site-directed mutagenesis is performed by using a Q5 site-directed mutagenesis kit.

[0017] Preferably, the sgRNAs specifically targeting the gra5 gene are sgRNA-GRA5-Fw and sgRNA-GRA5-Rv, and the nucleotide sequences thereof are shown as SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0018] Preferably, the competent cell is Trans1-T1 competent cell.

[0019] Preferably, the primers used in the PCR identification include gra5-KO1 shown as SEQ ID NO. 13-14, gra5-KO2 shown as SEQ ID NO. 15-16, and gra5-KO3 shown as SEQ ID NO. 17-18.

[0020] The application also provides a use of the Toxoplasma gondii gra5 gene knockout strain in preparing a Toxoplasma gondii vaccine, wherein the Toxoplasma gondii gra5 gene knockout strain is obtained by using the above construction method.

[0021] The application also provides a use of the Toxoplasma gondii gra5 gene knockout strain in preparing an anti-tumor preparation, wherein the Toxoplasma gondii gra5 gene knockout strain is obtained by using the above construction method.

[0022] Preferably, the tumor is breast cancer.

[0023] More preferably, the breast cancer is triple-negative breast cancer.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The application uses the CRISPR / Cas9 technology, takes the wild strain of Toxoplasma gondii as the parent strain, knocks out the gra5 gene of Toxoplasma gondii, and realizes the Toxoplasma gondii gra5 gene knockout strain. The knockout strain constructed in the application can significantly reduce the amount of Toxoplasma gondii in the abdominal cavity fluid of mice, and most of the Toxoplasma gondii will be cleared after infecting the mice. Compared with other reported gene knockout strain vaccines, the knockout strain provided in the application has a serious cyst formation defect, and compared with the wild-type strain, the knockout strain will not cause chronic infection, which will make it have a safer and more extensive application prospect.

[0026] In addition, the mice immunized with the knockout strain can provide good protection against the wild strain of Toxoplasma and tissue cyst re-infection, effectively resist the re-infection of Toxoplasma, and have the potential to become a live attenuated vaccine against Toxoplasma. At the same time, the experiment shows that the knockout strain has a good therapeutic effect on 4T1 breast tumors, and the knockout strain has a good potential to become an anti-tumor biological agent. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A schematic diagram for constructing the ME49Δgra5 strain using CRISPR / Cas9 technology.

[0028] Figure 2 PCR identification results of the Toxoplasma ME49Δgra5 monoclonal strain.

[0029] Figure 3 Toxicity experiment results of the ME49Δgra5 strain on mice.

[0030] Figure 4 The abdominal cavity Toxoplasma load experiment results of mice acutely infected with the ME49Δgra5 strain.

[0031] Figure 5 Cyst formation experiment of the ME49Δgra5 strain in mice.

[0032] Figure 6 Detection of Toxoplasma load in brain tissue of mice chronically infected with the ME49Δgra5 strain.

[0033] Figure 7 Immunoprotection experiment results of the ME49Δgra5 strain on mice acutely infected with wild strain of Toxoplasma tachyzoites.

[0034] Figure 8 Immunoprotection effect of the ME49Δgra5 strain on mice acutely infected with Toxoplasma tissue cysts.

[0035] Figure 9 Treatment inoculation procedure and time of the ME49Δgra5 strain.

[0036] Figure 10 Tumor growth curve of tumor-bearing mice treated with the ME49Δgra5 strain.

[0037] Figure 11 Tumor weight of tumor-bearing mice treated with the ME49Δgra5 strain.

[0038] Figure 12 Survival curve of tumor-bearing mice treated with the ME49Δgra5 strain. DETAILED DESCRIPTION

[0039] In order to make the skilled in the art more clearly understand the technical solutions described in the present application, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present application, and do not constitute a limitation on the scope of protection required by the present application. Any modification, substitution, combination made without deviating from the spirit and principles of the present application is included within the scope of protection of the present application.

[0040] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0041] Example 1: Construction of Toxoplasma ME49Δgra5 strain

[0042] (1) Parental strain ME49

[0043] ME49 wild strain is a type II strain of the Sporozoea family of Toxoplasma, which has a dense granule protein 5 gene, and the dense granule protein 5 gene sequence (TGME49_286450) is shown in SEQ ID NO. 1.

[0044] (2) Construction of pSAG1::CAS9-U6::sgGRA5 plasmid

[0045] Using pSAG1::CAS9-U6::sgUPRT plasmid as a template, and using NEB's Site-Directed Mutagenesis Kit, the sgRNA specific to the UPRT target site was replaced with sgRNA specific to the gra5 gene target, thereby obtaining the pSAG1::CAS9-U6::sgGRA5 plasmid, and the specific steps are as follows:

[0046] ① Use the E-CRISP design website (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) to design sgRNA primers specific to the gra5 gene sequence:

[0047] Upstream primer sgRNA-GRA5-Fw: 5'-ACGCCCACAAAAATTAAAGCGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 2);

[0048] Downstream primer sgRNA-GRA5-Rv: 5'-AACTTGACATCCCCATTTAC-3' (SEQ ID NO. 3).

[0049] ② Prepare the following reaction system in a sterile PCR tube:

[0050]

[0051] 3 PCR reaction cycle parameters are as follows:

[0052]

[0053] 4 KLD reaction: the product after the above PCR reaction is subjected to KLD connection, and the reaction system is as follows:

[0054]

[0055] oscillate and mix, and react at room temperature for 5 min.

[0056] 5 transformation reaction: all KLD reaction products are transformed into Trans1-T1 (100 μL) competent cells, plated on Amp + solid medium, placed at 37°C for inverted culture for 12-16 h, and single colonies are picked up in 1 mL Amp+ liquid medium, cultured at 37°C / 250 rpm for 6-12 h, and 500 μL bacterial liquid is taken for sequencing analysis. The sequencing primer is M13 reverse primer. If the sequencing result shows that the target sequence is completely replaced successfully, the plasmid construction is successful.

[0057] 6 the sequencing correct pSAG1::CAS9-U6::sgGRA5 plasmid is transferred to 12 mL Amp + liquid medium, cultured at 37°C / 250 rpm for 12-16 h, and the plasmid is extracted by using the endotoxin-free plasmid extraction kit (Endo-free Plasmid Mini Kit II) of Omega company, and stored at -20°C.

[0058] (3) preparation of GRA5-5UTR: :DHFR: :GRA5-3UTR homologous template

[0059] 1 according to the specific position of the gra5 gene in ToxoDB website, the 5' homologous arm (GRA5-5UTR) and 3' homologous arm (GRA5-3UTR) of the gra5 gene to be knocked out are determined through the genomic sequence information, and the gene sequence is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0060] 2 according to the method introduced in the instruction manual of NEB company HiFi DNA Assembly Cloning Kit connection kit, the primers of GRA5-5UTR, GRA5-3UTR and DHFR drug screening tag sequence fragments are designed, and the primer information is shown in table 1.

[0061] Table 1 Construction of the gra5-5UTR::DHFR::gra5-3UTR homologous template primer sequence

[0062]

[0063]

[0064] (3) Collecting the genomic DNA of the wild type strain of Toxoplasma gondii ME49 as a template, using high-fidelity Pfu enzyme and the designed primers to amplify the GRA5-5UTR and GRA5-3UTR fragments, and using the plasmid containing the DHFR sequence as a template to amplify the DHFR fragment.

[0065] (4) Gel recovery of the target fragment: using the Omega Gel Extraction Kit to purify and recover the PCR amplified fragment of step (3).

[0066] (5) The PCR fragment after gel recovery was used HiFi DNA Assembly Cloning Kit to perform seamless ligation, maintaining 50°C on a PCR instrument for 1 h, to obtain the PCR product after ligation of the three fragments.

[0067] (6) Using the ligation product obtained in (5) as a template, using the upstream primer of GRA5-5UTR and the downstream primer of GRA5-3UTR to amplify the GRA5-5UTR::DHFR::GRA5-3UTR fragment of homologous recombination in large quantities, and the gene sequence is shown in SEQ ID NO. 12.

[0068] The PCR reaction system and parameter settings are as follows:

[0069] PCR reaction system

[0070]

[0071]

[0072] PCR reaction parameters

[0073]

[0074] (7) Gel purification and recovery of the PCR target fragment obtained in the above step (5): using the Omega Gel Extraction Kit to purify and recover the GRA5-5UTR::DHFR::GRA5-3UTR homologous recombination fragment, storing at -20°C, and after sequencing correctly, the next step can be performed.

[0075] (4) Obtaining the Toxoplasma gondii ME49 Δgra5 strain

[0076] ① Take ATP 15 mg, glutathione 18 mg, dissolved in 12.5 mL cytomix buffer, filter sterilization with 0.22 μm filter for standby.

[0077] ② Collect the well-grown ME49 wild strain speed sporozoites described in step (1), use a 5 mL syringe to suck 6-7 times repeatedly, filter with a filter with a pore size of 3 μm, transfer to a 15 mL centrifuge tube, centrifuge at 2500 rpm / min at room temperature for 10 min, discard the supernatant, resuspend with 2 mL of the prepared electrotransfer buffer, take 10 μL to the cell counting plate for counting.

[0078] ③ According to the counting result, take 2×10 7 speed sporozoites in a new 15 ml centrifuge tube, centrifuge at 2500 rpm / min at room temperature for 10 min, discard the supernatant, add 350 μL of electrotransfer buffer and resuspend.

[0079] ④ Prepare the electrotransfer plasmid system:

[0080]

[0081] ⑤ Mix the suspensions of the above ③ and ④, transfer to a BIO-RAD 4 mm shock cup, mix well. Place the shock cup in the BIO-RAD electrotransformation instrument, set the voltage to 1500 V, the capacitance to 25 μF, and the resistance to ∞ Ω, and shock once.

[0082] ⑥ After the shock is completed, stand for 15 min, then transfer it to a T25 culture bottle with HFFs and culture and observe, and after 48 h, replace the complete culture medium with 3 μM allopurinol drug screening medium and continue to culture and observe.

[0083] ⑦ After 3 generations of drug screening, extract a portion of the genomic DNA of the worm strain for PCR identification to preliminarily determine whether the gra5 gene knockout worm strain has been successfully constructed. The PCR identification primer sequences used are shown in Table 2. Figure 1 The schematic diagram for constructing the ME49Δgra5 worm strain is shown, the band of gra5-KO1 indicates that the 5' homologous arm has been successfully integrated into the Toxoplasma genome, the band of gra5-KO2 indicates that the 3' homologous arm has been successfully integrated into the Toxoplasma genome, and the absence of band of gra5-KO3 indicates that the gra5 gene no longer exists, if there is a band of gra5-KO3, it indicates that the gra5 gene still exists, at this time, if both gra5-KO1 and gra5-KO2 have bands, it indicates that the worm strain being screened is a mixed strain of knockout and wild type.

[0084] Table 2 PCR identification primer sequences of ME49Δgra5 worm strain

[0085]

[0086] ⑧ After 5 generations of drug screening, the worms were split and filtered, centrifuged at 2500 rpm / min for 10 min, resuspended in 2 mL of culture medium, and 10 μL was taken for counting. The worms were diluted in multiples according to the number of 3 worms per well. The amount of worms required for 100 wells was taken and supplemented with pyrimethamine drug screening medium to 15 mL. Finally, the worms were inoculated into a 96-well plate filled with HFF cells for monoclonal screening and culture.

[0087] ⑨ After 8 days of culture, observe under a microscope and select single colonies. Scrape the selected colonies and transfer them to a 24-well plate filled with HFF cells. Continue to expand the culture for 5 days. Scrape half of the colonies to extract genomic DNA for PCR identification. Using the primers listed in Table 2, prepare the following PCR reaction system:

[0088] PCR reaction system

[0089]

[0090] PCR reaction parameters

[0091]

[0092] ⑩PCR product identification: The above PCR products were loaded into 1% agarose gel, set at 160V, and electrophoresed for 30 minutes. The PCR identification results were observed using a gel imaging system. Figure 2 As shown, it shows that the ME49Δgra5 monoclonal strain has been successfully constructed. The correctly identified monoclonal strain was transferred to a new HFF cell T25 culture flask for continued expansion and culture.

[0093] Example 2: Toxicity test of ME49Δgra5 strain on mice

[0094] ① Collect ME49 wild-type or ME49Δgra5 parasites cultured in HFF cells, discard the original culture medium, replace with 5 mL of new culture medium, scrape the cells from the culture surface with a disposable cell scraper, repeatedly aspirate with a 5 mL syringe 7-8 times to break the parasites, filter through a filter with a pore size of 3 μm into a 15 mL centrifuge tube, centrifuge at 2500 rpm / min at room temperature for 10 min, discard the supernatant, resuspend thoroughly with 2 mL of PBS buffer, take 10 μL for counting, and dilute according to the required parasite dose.

[0095] ② Six-week-old female BALB / c mice were intraperitoneally inoculated with ME49Δgra5 strain at a dose of 1×10 3 In the control group, each mouse was injected intraperitoneally with 1×10 3 The ME49 wild strain tachyzoites were divided into groups of 6 mice each. The mice were observed for 30 days and their survival was recorded.Figure 3 As shown in Figure 2, it can be seen that the survival rate of mice infected with the knockout strain within 30 days was 100%, while all the mice infected with the ME49 wild strain died within 10 days, indicating that the virulence of the Toxoplasma gra5 gene knockout was significantly reduced.

[0096] Example 3: Detection of intraperitoneal Toxoplasma load in mice infected with the ME49Δgra5 strain

[0097] ①Prepare two Toxoplasma suspensions according to step ① in Example 2.

[0098] ②Intraperitoneally inject 6-week-old female BALB / c mice with 1 x 10 3 Toxoplasma tachyzoites of the ME49Δgra5 strain or the wild strain, 3 mice per group. After 7 days of infection, the mice were sacrificed, and 5 mL of PBS buffer was injected intraperitoneally. After mixing, the mouse peritoneal fluid was collected, 1 mL of the peritoneal fluid was taken for genomic DNA extraction, and the genomic extraction method was performed according to the DNeasy Blood & Tissue Kits kit instructions. The primer sequences used for qPCR are shown in Table 3.

[0099] Table 3 qPCR primer sequences

[0100]

[0101] ③The standard curve was prepared by gradient dilution of Toxoplasma ME49 wild strain tachyzoites from the peritoneal fluid of blank mice, 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , and 10 1 , and the genomic DNA was extracted for quantitative PCR to draw the standard curve.

[0102] ④The Toxoplasma load in the peritoneal fluid of mice was calculated using the standard curve, and the results are shown in Figure 4 . It is shown that the Toxoplasma load in the peritoneal fluid of mice infected with the knockout strain was significantly lower than that of mice infected with the wild strain, indicating that the proliferation of the Toxoplasma ME49Δgra5 strain in the mouse body was inhibited, and most of the parasites could be cleared by the host.

[0103] Example 4: Encystment of the ME49Δgra5 strain in mice

[0104] ①Prepare two Toxoplasma suspensions according to step ① in Example 2.

[0105] ② Male SV129 mice aged 6-8 weeks were intraperitoneally inoculated with 100 tachyzoites of the ME49Δgra5 strain. Each mouse in the control group was intraperitoneally injected with 100 tachyzoites of the ME49 wild strain. There were 3 mice in each group. After 75 days of observation, the mice were killed by cervical dislocation. The brain tissue was homogenized and the volume was fixed with PBS buffer. 1 / 4 of the brain homogenate was taken and stained with DBA dye according to the relevant instructions (https: / / vectorlabs.com / fluorescein-dolichos-biflorus-agglutinin.html) and the cysts were counted. The results of the whole brain cyst count are as follows: Figure 5 As shown, no cysts were detected in the brain tissue of mice infected with this knockout strain.

[0106] ③ Take a portion of the remaining brain homogenate and refer to TRIzol TM Extract total RNA from brain tissue according to the instructions II. Instructions for the All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit: Total RNA was reverse transcribed into cDNA, and then the copy number of the B1 gene in brain tissue was quantitatively detected by qPCR using the primers in Table 3 and the standard curve in step ③ of Example 3. Figure 6 The results showed that the copy number of the Toxoplasma gondii B1 gene in the brain tissue of mice infected with the knockout strain was significantly lower than that of mice infected with the wild-type strain, and was similar to the level of the uninfected group. These results indicate that the ME49Δgra5 strain has severe cyst formation defects in mice.

[0107] Example 5: Immunoprotective effect of ME49Δgra5 strain on mice acutely infected with Toxoplasma gondii tachyzoites

[0108] The ability of mice immunized with the ME49Δgra5 strain to resist reinfection with ME49 wild-type tachyzoites was evaluated by immune protection experiments.

[0109] Prepare the tachyzoite suspension of Toxoplasma gondii ME49Δgra5 strain according to step ① in Example 2. 3 BALB / c mice were immunized with ME49Δgra5 strain tachyzoites for 30 days. After 30 days, the immunized and non-immunized mice were reinfected with 1×10 3 The ME49 wild strain tachyzoites were divided into 14 groups, and the observation was continued for 30 days to draw the survival curve.

[0110] The results are as follows Figure 7As shown, the unimmunized mice all died within 11 days after infection with the ME49 wild strain, while the survival rate of the mice immunized in advance was as high as 100% after re-infection with the wild strain, indicating that the ME49Δgra5 strain vaccine has a good protective effect on acute infection of mice caused by the wild strain of Toxoplasma.

[0111] Example 6: Immune protection effect of ME49Δgra5 strain on acute infection of mice with Toxoplasma tissue cysts

[0112] The ability of the mice immunized with the ME49Δgra5 strain to resist re-infection with ME49 tissue cysts was evaluated by an immune protection experiment.

[0113] First, prepare the Toxoplasma ME49 wild strain tachyzoite suspension according to step ① in Example 2, then intraperitoneally inject a number of BALB / c mice, 100 ME49 wild strain tachyzoites per mouse, two months later, kill the chronically infected mice, take the brain tissue for homogenization, and count the number of cysts formed under a microscope, then dilute according to the number of cysts required by the following experiment.

[0114] 6-week-old female BALB / c mice were first immunized with 1×10 3 ME49Δgra5 strain tachyzoites for 30 days, and 30 days later, each mouse was re-inoculated with 20 fresh ME49 cysts obtained in the above step by oral gavage, 5 mice in each group, and then continued to be monitored for 30 days.

[0115] The results are shown in Table 2. Figure 8 As shown, it can be found that the unimmunized mice all died within 10 days after gavage, while the survival rate of the immunized mice after gavage of the cysts was as high as 100%, indicating that the ME49Δgra5 strain vaccine also has a protective effect on infection with Toxoplasma tissue cysts. Therefore, the ME49Δgra5 strain provided by the present application has the potential to become an attenuated live vaccine against Toxoplasma.

[0116] Example 7: Inhibition effect of ME49Δgra5 strain on 4T1 breast tumor

[0117] (1) Construction of mouse 4T1 breast cancer model

[0118] ① Collect 4T1 cells growing well in a T75 culture flask for the construction of a 4T1 breast cancer mouse model.

[0119] ②Discard the original culture medium, add 5 mL PBS buffer to wash twice, discard, add 3 mL 0.25% trypsin, shake evenly to make it fully cover the culture surface, digest at 37°C for 2 min, after most of the cells shrink and fall off, gently tap the culture bottle wall to make it completely separate into single cells, add 3 mL complete medium containing serum to terminate digestion, transfer to a 15 mL centrifuge tube, centrifuge at 800 rpm / min for 5 min.

[0120] ③Discard the supernatant, wash the cells twice with 5 mL PBS buffer, then resuspend thoroughly with 5 mL PBS buffer.

[0121] ④Take 10 μL of the cell suspension to a cell counting plate, adjust the cell concentration to 1×10 6 / mL with PBS buffer, and the total volume of the cell suspension should exceed the required injection amount and be injected as soon as possible.

[0122] ⑤6-week-old female BALB / c mice, shave the abdomen 1 day in advance, anesthetize the mice with isoflurane through the respiratory tract, disinfect the mouse abdomen with 75% alcohol, blow and mix the 4T1 cell suspension, then use a 1 mL syringe to suck 100 μL of the cell suspension, the needle head is inclined upward, gently penetrate the mouse skin, slowly inject 100 μL of the 4T1 cell suspension (1×10 5 per mouse) subcutaneously on the right side of the abdomen, and slowly withdraw the needle after completing the injection. Observe the mouse state and tumor growth every other day.

[0123] (2) Intratumoral injection of T. gondii ME49Δgra5 strain for treatment

[0124] Prepare the T. gondii ME49Δgra5 strain tachyzoite suspension according to step ① in Example 2, and give intratumoral injection of 1×10 5 ME49Δgra5 strain tachyzoites for treatment on the 9th, 11th, and 13th days after tumor inoculation, with PBS treatment as a control. The treatment scheme and time for tumor-bearing mice are intended as shown in Figure 9 .

[0125] ① Tumor growth curve: After the start of treatment, measure the length and width of the tumor every other day with a vernier caliper, calculate the tumor volume of each mouse according to the tumor volume calculation formula: volume (mm 3 ) = (length × width 2 ) / 2, draw the tumor growth curve for each group of 7 mice, and euthanize the mice when the tumor volume is greater than 1500 mm 3 . Figure 10As shown in Figure 6, it can be found that the tumor growth rate of ME49Agra5 strain treatment group is slower than that of PBS treatment control group, and the volume growth is significantly slower. On the 27th day after tumor inoculation, the tumor volume of PBS negative control treatment group is about 5 times that of ME49Agra5 strain treatment group. This indicates that ME49Agra5 strain can significantly inhibit the growth of 4T1 tumor in mice.

[0126] ②Tumor weight: On the 12th day after the first treatment, 6 mice in each group were sacrificed, the tumor tissue was stripped, and the tumor weight was measured. The results are shown in Figure 7. Figure 11 As shown in Figure 7, it can be seen that the tumor weight of ME49Agra5 strain intratumoral treatment group is significantly lower than that of PBS treatment control group.

[0127] ③Survival curve of tumor-bearing mice: After the treatment of tumor-bearing mice, their survival was observed every day. As long as either of the two events of tumor-bearing mice tumor volume greater than 1500mm 3 or tumor-bearing mice died, it was considered that the mouse reached the death endpoint on that day. The survival time was recorded until the 50th day after tumor inoculation, and the survival curves of two groups of tumor-bearing mice were drawn according to the recorded survival time, 7 tumor-bearing mice in each group. The results are shown in Figure 8. Figure 12 As shown in Figure 8, the survival rate of tumor-bearing mice in ME49Agra5 strain treatment group can reach more than 70% on the 50th day after tumor inoculation, while all tumor-bearing mice in PBS control group died within 33 days.

[0128] In summary, in situ injection of ME49Agra5 strain tachyzoite can inhibit the growth of 4T1 breast tumor in mice, significantly reduce the tumor burden of 4T1 breast tumor in mice, and significantly prolong the survival time of tumor-bearing mice. Therefore, ME49Agra5 strain has the potential to become an anti-breast tumor biological agent.

[0129] The above embodiments of the present application are described in detail in combination with the drawings, but the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a Toxoplasma gondii gra5 gene knockout strain, characterized in that: The following steps are involved: Preparation of GRA5-5UTR::DHFR::GRA5-3UTR homology template: Using genomic DNA of a Toxoplasma gondii strain as a template, primers were designed based on the 5' homology arm and 3' homology arm of the gra5 gene to amplify the GRA5-5UTR fragment and the GRA5-3UTR fragment; using a plasmid containing the DHFR sequence as a template, primers were designed to amplify the DHFR fragment; the GRA5-5UTR fragment, GRA5-3UTR fragment, and DHFR fragment were connected using seamless ligation technology to obtain the GRA5-5UTR::DHFR::GRA5-3UTR homology template; Construction of a CRISPR plasmid for targeted excision of GRA5: Using the CRISPR plasmid as a template, a PCR reaction was performed using an sgRNA specifically targeting the gra5 gene to achieve site-directed mutagenesis; the product after the PCR reaction was subjected to KLD ligation; the KLD ligation product was transformed into competent cells to obtain a CRISPR plasmid for targeted excision of GRA5; Transformation of the parasite: The CRISPR plasmid targeting GRA5 and the GRA5-5UTR::DHFR::GRA5-3UTR homologous template are co-transformed into the Toxoplasma gondii strain, and the Toxoplasma gondii gra5 gene knockout strain is obtained through drug testing and PCR identification; The Toxoplasma gondii strain is Toxoplasma gondii ME49; Wherein, the nucleotide sequences of the primers used to amplify the GRA5-5UTR fragment are shown in SEQ ID NO.6-7; The nucleotide sequences of the primers used to amplify the GRA5-3UTR fragment are shown in SEQ ID NOs. 10-11; The nucleotide sequence of the GRA5-5UTR::DHFR::GRA5-3UTR homologous template is shown in SEQ ID NO.12; the sgRNAs specifically targeting the gra5 gene are sgRNA-GRA5-Fw and sgRNA-GRA5-Rv, and their nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The construction method according to claim 1, characterized in that The nucleotide sequences of the primers used to amplify the DHFR fragment are shown in SEQ ID NO. 8-9.

3. The construction method according to claim 1, characterized in that The primers used for PCR identification include: gra5-KO1 as shown in SEQ ID NO.13-14, gra5-KO2 as shown in SEQ ID NO.15-16, and gra5-KO3 as shown in SEQ ID NO.17-18.

4. The use of a Toxoplasma gondii gra5 gene knockout strain in the preparation of a Toxoplasma gondii vaccine, characterized in that: The Toxoplasma gondii gra5 gene knockout strain is obtained by the construction method described in any one of claims 1-3.

5. Use of a Toxoplasma gondii gra5 gene knockout strain in the preparation of an anti-tumor preparation, characterized in that: The Toxoplasma gondii gra5 gene knockout strain is obtained by the construction method described in any one of claims 1 to 3, and the tumor is breast cancer.

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