An attenuated toxoplasma gondii vaccine strain and its preparation method and application in anti-tumor

Abstract

The present invention relates to the field of genetic engineering, and specifically to an attenuated Toxoplasma gondii vaccine strain, a preparation method thereof, and an application thereof in anti-tumor. The strain has the same safety as the traditional anti-tumor Toxoplasma vaccine ME49Δompdc / ldh1, and compared with ME49Δompdc / ldh1, ME49Δompdc / ldh1 / gra4 can mediate a stronger IFN-I response in the host, thereby further inhibiting tumor growth in both early prevention and late treatment modes.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to an attenuated Toxoplasma gondii vaccine strain and a preparation method thereof and application thereof in anti-tumor. Background Art

[0002] Tumor refers to a new organism formed when cells in local tissues lose normal regulation of their growth at the genetic level under the influence of physical, chemical or biological carcinogenic factors, resulting in cell gene mutations and abnormal proliferation. Tumors can be divided into benign tumors and malignant tumors according to their biological characteristics and degree of harm. Among them, cancer diseases caused by malignant tumors have caused great harm to human health and social and economic development. According to the latest global cancer burden data in 2020 released by the World Health Organization's International Agency for Research on Cancer, there were 4.57 million new cases of cancer in my country in 2020, and 8 people were diagnosed with cancer every minute. Traditional clinical treatment of cancer mainly relies on surgical resection, radiotherapy, chemotherapy and targeted drugs. In recent years, research on biological anti-tumor treatments such as CAR-T, oncolytic viruses and BCG has developed rapidly, and many therapies have entered the clinical trial stage. This type of treatment method is not only effective, but also safe and has fewer side effects. Its important role in improving the quality of life of patients and reducing the recurrence rate has been increasingly recognized and valued.

[0003] As a globally spread opportunistic pathogenic parasite, Toxoplasma gondii can infect almost all mammalian cells, survive and reproduce in cells. Toxoplasmosis caused by Toxoplasma infection has brought a heavy burden to human health, animal husbandry development and social and economic development. Compared with the serious threat of wild-type Toxoplasma, the attenuated Toxoplasma transformed by scientists through gene editing technology is another biological vaccine that can effectively play an anti-tumor function in recent years in addition to CAR-T, oncolytic virus and BCG. Among them, we previously obtained an attenuated strain by knocking out the ompdc and ldh1 genes in the wild-type II type Toxoplasma ME49 by referring to CN108434447A, but the strain cannot continue to reproduce in the host because it cannot synthesize uracil. Although studies have reported that the strain can inhibit the growth of host tumors to a certain extent, its anti-tumor effect is limited. Therefore, the present invention hopes to further transform the strain and propose an attenuated Toxoplasma strain with good safety and better ability to induce the host to resist tumor growth, so as to prepare a tumor biotherapy vaccine. Summary of the invention

[0004] In view of the above problems, the present invention further performs gene editing on the basis of ME49Δompdc / ldh1 to provide a new attenuated Toxoplasma vaccine. The vaccine can highly activate the body's type I interferon response, thereby having a better therapeutic effect on a variety of subcutaneous transplanted tumors such as mouse melanoma and lung cancer, and can effectively inhibit melanoma lung metastasis. In addition, the preparation process of the vaccine is relatively simple and the economic cost is low, providing a new research and application idea for tumor biotherapy.

[0005] The technical solution adopted by the present invention is:

[0006] In the first aspect: The present invention provides an attenuated Toxoplasma vaccine strain ME49Δompdc / ldh1 / gra4, which is a type II Toxoplasma ME49 strain that simultaneously lacks the orotidine-5-phosphate decarboxylase gene ompdc, the lactate dehydrogenase 1 gene ldh1 and the Toxoplasma dense granule protein 4 gene gra4, and the nucleotide sequences of the ompdc, ldh1 and gra4 are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

[0007] This strain has the same safety as the traditional anti-tumor Toxoplasma vaccine ME49Δompdc / ldh1, and compared with ME49Δompdc / ldh1, ME49Δompdc / ldh1 / gra4 can mediate a stronger IFN-I response in the host, thereby further inhibiting tumor growth in both early prevention and late treatment modes.

[0008] In a second aspect, the present invention also provides a method for preparing the attenuated Toxoplasma gondii vaccine strain ME49Δompdc / ldh1 / gra4, comprising the following steps:

[0009] Preparation of GRA4-5'utr::DHFR::GRA4-3'utr homology template:

[0010] Using type II Toxoplasma gondii ME49 genomic DNA as a template, primers were designed according to the 5' homology arm and 3' homology arm of the gra4 gene, and PCR amplification was performed to obtain the gra4-5'utr fragment and the gra4-3'utr fragment; using pLinker-AID-3xHA-DHFR-LoxP as a template, DHFR-specific primers were used to amplify the Loxp-DHFR-Loxp fragment by PCR; gra4-5'utr, gra4-5'utr and Loxp-DHFR-Loxp were connected by seamless connection technology to obtain the gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr fragment;

[0011] Preparation of pSAG1-Cas9-sgGRA4 plasmid for targeted knockout of gra4:

[0012] Using the pSAG1-Cas9-sgMIC3 plasmid as a template, the designed gra4-specific sgRNA was used to replace the sgMIC3 fragment in the template to obtain the pSAG1-Cas9-sgGRA4 plasmid for targeted knockout of the gra4 gene;

[0013] Electroporation to construct target insect strain:

[0014] The 5H-Loxp-DHFR-Loxp-3H fragment and pSAG1-Cas9-sgGRA4 plasmid were integrated into the ME49Δompdc / ldh1 genome by electroporation, and the GRA4-deficient ME49Δompdc / ldh1 / gra4 strain was obtained by drug screening and PCR identification.

[0015] Preferably, the nucleotide sequences of the primer pairs used to amplify the gra4-5'utr fragment are shown as SEQ ID No.6 and SEQ ID No.7.

[0016] Preferably, the nucleotide sequences of the primer pair used to amplify the gra4-3'utr fragment are shown as SEQ ID No.8 and SEQ ID No.9.

[0017] Preferably, the nucleotide sequences of the primer pairs used to amplify the Loxp-DHFR-Loxp fragment are shown as SEQ ID No.10 and SEQ ID No.11.

[0018] Furthermore, ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech, China) was used for seamless ligation to obtain the gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr fragment.

[0019] Preferably, the nucleotide sequences of the primer pair sgRNA for targeted knockout of gra4 are shown as SEQ ID No.4 and SEQ ID No.5.

[0020] Furthermore, the gra4 sgRNA was inserted into the pSAG1-Cas9-sgMIC3 template using the Q5 site-directed mutagenesis (New England Biolabs) site-directed mutagenesis kit to obtain the pSAG1-Cas9-sgGRA4 plasmid for targeted knockout of gra4.

[0021] Preferably, the transformation of the recombinant fragment and plasmid of the present invention uses E. coli DH5 competent cells.

[0022] Preferably, the specific fragment and plasmid are transfected into the ME49Δompdc / ldh1 strain using an electroporator from BioRAD Corporation, USA.

[0023] Preferably, 1 M pyrimethamine or 30 μM chloramphenicol is used to screen for recombinants after transfection.

[0024] Preferably, the nucleotide sequences of the primers for PCR identification of the target insect strain are shown as SEQ ID No. 12-17.

[0025] In a third aspect, the present invention provides the use of ME49Δompdc / ldh1 / gra4 vaccine strain in the preparation of drugs or vaccines for preventing, treating, alleviating or improving mammalian tumors.

[0026] Preferably, the tumor cell is a B16-F10 melanoma cell line, or an LLC lung cancer cell line.

[0027] Further preferably, the tumor is a subcutaneous implanted tumor or a lung metastatic melanoma.

[0028] Preferably, the mammal is a human or a mouse.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention utilizes CRISPR / Cas9 technology, uses ME49Δompdc / ldh1 uracil-deficient Toxoplasma gondii as the parent strain, knocks out the dense granule protein gra4 of Toxoplasma gondii, and constructs the ME49Δompdc / ldh1 / gra4 strain. The strain has the same safety as the traditional anti-tumor Toxoplasma vaccine ME49Δompdc / ldh1, and compared with ME49Δompdc / ldh1, ME49Δompdc / ldh1 / gra4 can mediate a stronger IFN-I response in the host, thereby further inhibiting tumor growth in both early prevention and late treatment modes. Therefore, the present invention provides a new idea and means for anti-tumor biological therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of constructing the gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr fragment using homologous recombination technology;

[0032] Figure 2 Schematic diagram of constructing the pSAG1-Cas9-sgGRA4 fragment using CRISPR / Cas9;

[0033] Figure 3 This is the PCR identification result of Toxoplasma gondii ME49Δompdc / ldh1 / gra4 strain;

[0034] Figure 4 The results of the toxicity test of ME49Δompdc / ldh1 / gra4 strain on mice;

[0035] Figure 5 The results of the Toxoplasma gondii load experiment in the spleen of mice acutely infected with the ME49Δompdc / ldh1 / gra4 strain;

[0036] Figure 6 The expression results of Ifnb and Isg56 genes in the peritoneal cavity and spleen cells of mice after acute infection with ME49Δompdc / ldh1 / gra4 strain;

[0037] Figure 7 The results are for the concentration of IFN-β in the serum of mice after acute infection with the ME49Δompdc / ldh1 / gra4 strain;

[0038] Figure 8 Schematic diagram of the anti-tumor treatment mode of ME49Δompdc / ldh1 / gra4 strain;

[0039] Fig. 9 This is the growth curve of subcutaneous tumor in mice after early prevention and late treatment of ME49Δompdc / ldh1 / gra4 strain;

[0040] Fig.10 This is the survival curve of mice after early prevention and late treatment of ME49Δompdc / ldh1 / gra4 strain;

[0041] Fig.11 This is a gross image of the lungs in the mouse melanoma lung metastasis model after early prevention and late treatment with the ME49Δompdc / ldh1 / gra4 strain. DETAILED DESCRIPTION

[0042] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limiting effect on the scope of protection claimed in the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention. 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.

[0043] Example 1: Construction of Toxoplasma gondii ME49Δompdc / ldh1 / gra4 strain:

[0044] (1) Parental insect strain ME49Δompdc / ldh1

[0045] ME49Δompdc / ldh1 is a type II strain of Toxoplasma of the family Sporozoan, which is consistent with wild-type ME49 and has the GRA4 gene. The code of the GRA4 gene (SEQ ID NO.3) is: TGME49_310780. The nucleotide sequences of the deleted ompdc and ldh1 genes are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0046] For the specific preparation method of ME49Δompdc / ldh1 strain, please refer to patent publication CN108434447A.

[0047] (2) Construction of pSAG1-Cas9-sgGRA4 plasmid for targeted knockout of gra4

[0048] Using the pSAG1-Cas9-sgMIC3 plasmid as a template, the MIC3 target-specific sgRNA was replaced with the gra4 gene-specific sgRNA using New England Biolabs' Q5 site-directed mutagenesis to obtain the pSAG1-Cas9-sgGRA4 plasmid. The specific steps are as follows:

[0049] ① Use the CHOPCHOP (http: / / chopchop.cbu.uib.no) website to design sgRNA primer pairs specifically targeting gra4. The sequences are shown in Table 1:

[0050] Table 1 Sequences of sgRNA primer pairs targeting gra4

[0051]

[0052] ② Prepare the PCR reaction system according to the instructions of the Q5 point mutation kit:

[0053] Components volume Q5 Hot Start High-Fidelity 2x Master Mix 6.25μL 10 μM forward primer 0.625μL 10 μM reverse primer 0.625μL pSAG1-Cas9-sgMIC3 0.5μL Molecular cloning water 2.4μL

[0054] ③ Place the PCR tubes containing the above components in the PCR instrument and perform the reaction according to the following procedure:

[0055]

[0056] ④ Add 1 μL Dpn1 to the reaction product and place it in a PCR instrument for digestion at 37°C for 30 minutes.

[0057] ⑤KLD reaction: Prepare the PCR reaction system according to the following table:

[0058] Components volume ④Intermediate products 0.5μL 2×KLD Reaction Buffer 2.5μL 10×KLD Enzyme Mix 0.5μL Molecular cloning water 1.5μL

[0059] ⑥ Place the PCR tube with the above components in the PCR instrument and react at 25℃ for 15 minutes. The obtained product can be used for the next step of transformation.

[0060] ⑦ Transformation of the recombinant product: Take out the E. coli DH5α competent cells stored at -80℃, place them on ice to melt; add the recombinant product to the DH5α competent cells, mix well, and place on ice for 30 minutes; heat shock in a 42℃ water bath for 75 seconds, and ice bath for 2 minutes; add 400μL of antibiotic-free LB liquid culture medium, and resuscitate and culture at 37℃220r / min for 45 minutes; centrifuge, discard 300μL of the supernatant, resuspend, take 100μL and spread it on the same Amp(+) LB plate, invert and culture at 37℃ for 10-12h; pick a single colony, place it in 1-3ml Amp(+) LB liquid culture medium, and culture it at 37℃, 220r / min for 10-12h, and take 500μL of the bacterial solution for sequencing verification.

[0061] ⑧Transfer the correctly sequenced pSAG1-Cas9-sgGRA4 bacterial solution into 10 mL Amp(+) liquid culture medium, culture at 37°C, 220 r / min for 12-16 h, extract the plasmid using Omega's Endo-free Plasmid Mini Kit II, and store at -20°C.

[0062] (3) Preparation of gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr homologous template

[0063] ① Use the ToxoDB website to determine the specific location of the gra4 gene, and then determine the 5' homology arm (gra4-5'utr) and 3' homology arm (gra4-3'utr) of the gra4 gene to be knocked out through its genomic sequence information.

[0064] ② According to the instructions of ClonExpress MultiS One Step Cloning Kit of Novizan, primers for amplifying gra4-5', gra4-3'utr and loxp-DHFR fragments were designed. The specific sequences are shown in Table 2:

[0065] Table 2 Primer pair sequences for amplifying gra4-5', gra4-3'utr and loxp-DHFR fragments

[0066]

[0067] ③Using PureLink from Thermo Fisher Scientific TM The genomic DNA of ME49Δompdc / ldh1 strain was extracted using a genomic DNA mini kit and used as a template. The 2×Taq Plus Master Mix high-fidelity enzyme from Novezan was combined with the above primers to amplify the gra4-5' and gra4-3'utr fragments. The plasmid containing the DHFR gene was used as a template to amplify the loxp-DHFR fragment.

[0068] ④ The above PCR product was subjected to agarose gel electrophoresis, and the purified target fragment was recovered using Tiangen Company's agarose gel DNA recovery kit.

[0069] ⑤Use ClonExpress MultiS One Step Cloning Kit from Novizan to seamlessly connect gra4-5', gra4-3'utr and loxp-DHFR fragments in sequence and clone them into pUC19 vector.

[0070] ⑥Using the ligation product in ⑤ as a template, use gra4-5' forward primer and gra4-3'utr reverse primer to perform large-scale amplification to obtain gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr homologous fragments.

[0071] ⑦ The above PCR products were subjected to agarose gel electrophoresis, and the purified target fragments were recovered using Tiangen Company's agarose gel DNA recovery kit.

[0072] (4) Preparation of ME49Δompdc / ldh1 / gra4 strain

[0073] Using the electroporator from BioRAD, USA, the 5H-Loxp-DHFR-Loxp-3H fragment and the pSAG1-Cas9-sgGRA4 plasmid were integrated into the ME49Δompdc / ldh1 genome by electroporation, and the GRA4-deficient ME49Δompdc / ldh1 / gra4 strain was obtained by drug screening and PCR identification. The specific steps are as follows:

[0074] ①Use HFF cells to culture Toxoplasma ME49Δompdc / ldh1 tachyzoites (250μmol / L uracil should be added to the culture medium). When 30%-50% of the parasites have escaped from the cells, discard the original culture medium and add 6mL of fresh DMEM containing 2% FBS. Scrape the cells with a cell scraper and repeatedly blow the suspension 8-10 times with a 5mL syringe. Filter the purified parasites with a sterile 3μm filter membrane, centrifuge at room temperature for 10min at 1500r / min, discard the supernatant, resuspend and wash the parasites with 6-8mL cytomix, and centrifuge at 1500r / min for 10min. Then discard the supernatant and resuspend the precipitate with 250μL cytomix.

[0075] ② Add gra4-5'utr-Loxp-DHFR-Loxp-gra4-3'utr (1500ng) and pSAG1-Cas9-sgGRA4 plasmid (7500ng) in a 4mm electroporation cup at a ratio of 1:5, take 250μL of the Toxoplasma tachyzoite suspension in step ① and add it to the electroporation cup, mix thoroughly, and the total volume does not exceed 350μL. Avoid bubbles and put it into the electroporator for electrotransfection. The conditions are as follows: 1600v, 25μF, 50Ω, 4mm electroporation 2-3 times.

[0076] ③ The electroporated worms were added to new HFF cells for normal culture (250 μmol / L uracil was added to the culture medium). At the same time, a non-transfected control group was set up for culture observation and comparison.

[0077] ④ After 48 hours of culture, the medium of the experimental group and the control group was changed, and culture medium containing a final concentration of 1 μM pyrimethamine (or 30 μM chloramphenicol) was added for drug screening.

[0078] ⑤ After 3-5 generations of drug screening and the PCRs identification library is positive, the worms are placed in a 96-well plate containing host cells for monoclonal screening and cultured at 37°C for 10-12 days (250 μmol / L uracil needs to be added to the culture medium), and 1-2 (calculated amount) tachyzoites are inoculated in each well; the remaining worms can be used to extract genomic DNA, and primers can be used to detect the homologous arm integration efficiency and gene knockout efficiency.

[0079] ⑥ After culturing in a 96-well plate for 10-12 days, observe whether a single clone exists. Use a sterile pipette tip to scrape the host cells containing the single clone from the well, add them to a 24-well plate containing host cells, and continue culturing. When 70% of the host cells in the 24-well plate are lysed, 150 μl of the worms in the well can be transferred to a new 24-well plate for culturing, and the remaining worms are used to extract genomic DNA for PCRs to identify the single clone.

[0080] ⑦ The primers, PCR system and procedures used for identification are as follows. Figure 1 Shown is a schematic diagram of constructing the ME49Δompdc / ldh1 / gra4 strain. Figure 2 Schematic diagram of constructing the pSAG1-Cas9-sgGRA4 fragment using CRISPR / Cas9. Figure 3 This is the PCR identification result of Toxoplasma gondii ME49Δompdc / ldh1 / gra4 strain.

[0081] The primer sequences used for identification of ME49Δompdc / ldh1 / gra4 strain are shown in Table 3:

[0082] Table 3 Primer sequences for identification of ME49Δompdc / ldh1 / gra4 strain

[0083]

[0084]

[0085] Use the 2×Taq Plus Master Mix kit from Novezan for PCR identification and prepare the following reaction system according to the instructions:

[0086] Components volume 2×Taq Plus Master Mix II 6.25μL 10 μM forward primer 0.5μL 10 μM forward primer 0.5μL Genomic DNA (250ng / μL) 1μL Molecular cloning water 4.25μL

[0087] The reaction procedure is as follows:

[0088]

[0089] ⑧Transfer the positively identified insect strains into new HFF cell T25 culture flasks for further culture expansion. 250 μmol / L uracil should be added to the culture medium.

[0090] Example 2: Virulence experiment of ME49Δompdc / ldh1 / gra4 strain on mice

[0091] ①Use HFF cells to culture Toxoplasma ME49Δompdc / ldh1 / gra4 tachyzoites (250μmol / L uracil should be added to the culture medium). When 30%-50% of the parasites have escaped from the cells, discard the original culture medium and add 6mL of fresh DMEM containing 2% FBS. Scrape the cells with a cell scraper and repeatedly blow the suspension 8-10 times with a 5mL syringe. Filter the parasites with a sterile 3μm filter membrane, centrifuge at 1500r / min at room temperature for 10min, discard the supernatant, resuspend with 1mL PBS, count, and adjust the tachyzoite concentration to 2×10 6 / 200μL.

[0092] ② Take 6-8 week old wild-type C57BL / 6 mice, inject each mouse with 200 μL of fixed volume ME49Δompdc / ldh1 / gra4 tachyzoite suspension, and set up a control group, inject each mouse with 200 μL of fixed volume ME49Δompdc / ldh1 tachyzoite suspension, 4 mice in each group, observe for 15 consecutive days, and record the changes in mouse weight. The results are as follows Figure 4 As shown, neither the ME49Δompdc / ldh1 / gra4 nor the ME49Δompdc / ldh1 strains affect the normal growth of mice.

[0093] Example 3: Detection of Toxoplasma gondii load in the abdominal cavity and spleen of mice infected with ME49Δompdc / ldh1 / gra4 strain.

[0094] ① Collect wild-type ME49 (ME49wt), ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites according to the method of step ① of Example 2.

[0095] ② Wild-type C57BL / 6 mice were intraperitoneally injected with 2×10 6 ME49 (ME49wt), ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites.

[0096] ③ On the 15th day, the mice were killed, and the RNA of their spleen cells was extracted using the TRIZOL method. The RNA was reversed into cDNA using the StraScript reverse transcription kit of Genstar Company, and the expression of the Toxoplasma-specific ITS-1 gene in their spleen cells was further detected by qPCR. The primers used to detect the expression of the ITS-1 gene are shown below. The results are as follows Figure 5 As shown, on the 15th day after acute infection with the two strains, the expression of the Toxoplasma-specific ITS-1 gene was not detected in the spleen of mice. The above results indicate that ME49Δompdc / ldh1 / gra4 cannot continue to reproduce in vivo and is safe for biological treatment.

[0097] The primer sequences used for ITS-1 detection are as follows:

[0098] ITS-1:

[0099] Forward primer: 5'-AATATTGGAAGCCAGTGCAGG-3' (SEQ No. 18)

[0100] Reverse primer: 5'-CAATCTTTCACTCTCTCTCAA-3' (SEQ No. 19)

[0101] Example 4: Comparison between ME49Δompdc / ldh1 / gra4 and ME49Δompdc strains in inducing IFN-I gene expression in mice.

[0102] ① Collect ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites according to the method of step ① of Example 2.

[0103] ② Wild-type C57BL / 6 mice were intraperitoneally injected with 2×10 6 ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites.

[0104] ③ On the 15th day, the mice were killed, and the TRIZOL method was used to extract RNA from their spleen cells. The RNA was reversed into cDNA using the StraScript reverse transcription kit from Genstar, and qPCR was used to detect the expression of mouse Ifnb and Isg15 genes in their spleen cells. The primers used to detect the expression of Ifnb and Isg15 genes are shown below. The results are as follows: Figure 6 As shown, on the 15th day after acute infection with the two strains, the expression levels of Ifnb and Isg15 genes in the spleen of mice infected with ME49Δompdc / ldh1 / gra4 were significantly increased compared with mice infected with ME49Δompdc / ldh1 strain. The above results indicate that ME49Δompdc / ldh1 / gra4 can enhance the expression of host IFN-I related genes, and this strain has great potential for tumor treatment.

[0105] The primer sequences used for Ifnb detection are as follows:

[0106] Forward primer: 5′-TCACCTACAGGGCGGACTTC-3′ (SEQ No. 20);

[0107] Reverse primer: 5'-TACTCTCTGCTCGGACCACCATC-3' (SEQ No. 21).

[0108] The primer sequences used for Isg15 detection are as follows:

[0109] Forward primer: 5′-GGTGTCCGTGACTAACTCCAT-3′ (SEQ No. 22);

[0110] Reverse primer: 5'-CTGTACCACTAGCATCACTGTG-3' (SEQ No. 23).

[0111] Example 5: Comparison between ME49Δompdc / ldh1 / gra4 and ME49Δompdc strains in inducing IFN-I cytokine secretion in mice.

[0112] ① Collect ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites according to the method of step ① of Example 2.

[0113] ② Wild-type C57BL / 6 mice were intraperitoneally injected with 2×10 6 ME49Δompdc / ldh1 and ME49Δompdc / ldh1 / gra4 tachyzoites.

[0114] ③On the 15th day, peripheral blood was collected by removing the eyeballs and centrifuged (5000g×10min) to obtain peripheral blood serum, and then the mice were killed. The IFN-β protein level in the serum of mice infected with the two strains was detected using the mouse IFN-β ELISA detection kit (Cat. No.: DY8234-05) of R&D system. The results are shown in the figure. Figure 7 As shown, on the 15th day after acute infection with the two strains, the protein level of IFN-β in the peripheral blood of mice infected with ME49Δompdc / ldh1 / gra4 was significantly increased compared with mice infected with ME49Δompdc / ldh1 strain. The above results indicate that ME49Δompdc / ldh1 / gra4 can induce a stronger IFN-I response in the host, and the strain has great potential for tumor treatment.

[0115] Example 6: The inhibitory effect of ME49Δompdc / ldh1 / gra4 strain on B16-F10 subcutaneous tumors.

[0116] (1) Construction of B16-F10 subcutaneous tumor model

[0117] ① Depilate the right abdominal area of ​​C57 / BL6 mice to completely expose the skin;

[0118] ② Collect B16 / F-10 cells that are in the logarithmic growth phase and in good condition. Before collection, wash the cells with PBS buffer to remove the culture medium and serum;

[0119] ③ The washed and evenly resuspended tumor cell suspension in PBS buffer was subcutaneously injected into the depilated area (2×10 6 cells / mouse).

[0120] ④ Starting from the seventh day after inoculation, the volume of the subcutaneous tumor was measured using a vernier caliper, and the length (L) and width (W) measurements were used for calculation: tumor volume (V) = 0.52 × L × W 2 .

[0121] (2) The difference in anti-tumor effect between the ME49Δompdc / ldh1 / gra4 strain in early prevention and late treatment modes.

[0122] ① The Toxoplasma gondii mouse treatment model in this study includes two types: "early prevention" and "late treatment". Figure 8 shown.

[0123] “Early prevention” model: 2×10 6 ME49Δompdc / ldh1 / gra4 strain.

[0124] “Late rescue” model: 2×10 6 ME49Δompdc / ldh1 / gra4 strain.

[0125] ② Mouse subcutaneous tumor growth curve: On days 7-30 after inoculation, subcutaneous tumors were measured using a vernier caliper, and the length (L) and width (W) measurements were taken for calculation: tumor volume (V) = 0.52 × L × W 2 The results are as follows Fig. 9 As shown, under both the "early prevention" and "late treatment" vaccination modes, the ME49Δompdc / ldh1 / gra4 strain was able to better inhibit tumor growth, and among the mice vaccinated with the ME49Δompdc / ldh1 / gra4 strain in the early stage, about 50% of the mice were able to completely resist the occurrence of tumors.

[0126] ③ Survival curve of tumor-bearing mice: Starting from the day of tumor implantation, the survival of mice was observed and recorded. In accordance with animal welfare and ethical requirements, when the tumor volume of mice exceeded 2000mm 3 When the tumor ulcerated or became infected, the experiment was terminated and euthanasia was performed, and the time was recorded as the survival endpoint of the mouse. Fig.10 As shown, compared with the control treatment, both "early prevention" and "late treatment" vaccination modes can significantly prolong the survival time of mice, and no mice died within 30 days.

[0127] Example 7: The inhibitory effect of ME49Δompdc / ldh1 / gra4 strain on B16-F10 lung metastasis.

[0128] (1) Construction of B16-F10 lung metastasis model

[0129] ① Wild-type C57BL / 6 mice aged 6-8 weeks were injected with 2×10 6 B16 / F-10 cells in logarithmic growth phase and in good condition.

[0130] (2) The therapeutic effect of the ME49Δompdc / ldh1 / gra4 strain in inhibiting melanoma lung metastasis in both “early prevention” and “late treatment” modes.

[0131] The processing method is the same as that of Example 6-(2)-①.

[0132] (3) Observation of mouse lung tumors

[0133] ① On the 18th day after tail vein injection, the mice were killed by dislocation and dissected, and their intact lungs were removed.

[0134] ② Soak the lungs in 4% paraformaldehyde solution.

[0135] ③After soaking for a week, when the lungs turn white, observe the growth and metastasis of melanoma. Fig.11 As shown, the number and volume of lung tumors in mice treated with the ME49Δompdc / ldh1 / gra4 strain were significantly lower than those in the control group, indicating that the ME49Δompdc / ldh1 / gra4 strain also has a better therapeutic effect in inhibiting the melanoma lung metastasis model.

[0136] In summary, it can be seen that the present invention knocks out the gra4 gene on the basis of the ME49Δompdc / ldh1 strain, so that the strain can be prevented from continuing to reproduce in vivo and has the safety of biological treatment. It has a specific enhanced IFN-I response and can be used for the treatment of tumors, especially B16-F10 melanoma.

[0137] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An attenuated Toxoplasma gondii vaccine strain ME49Δ ompdc / ldh1 / gra4 , characterized in that, The vaccine strain is a type II ME49 Toxoplasma gondii strain that lacks ompdc, ldh1 and gra4. ompdc , ldh1 , gra4 The nucleotide sequences are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively.

2. The attenuated Toxoplasma gondii vaccine strain ME49Δ according to claim 1 ompdc / ldh1 / gra4 The preparation method is characterized in that The steps include: Preparation of GRA4-5'utr::DHFR::GRA4-3'utr homology template: Using the genomic DNA of type II Toxoplasma gondii ME49 as a template, primers were designed based on the 5' homology arm and 3' homology arm of the gra4 gene, and PCR amplification was performed to obtain gra 4-5' UTR fragment and gra 4-3' utr fragment; pLinker-AID-3xHA-DHFR-LoxP was used as a template and DHFR-specific primers were used to amplify the Loxp-DHFR-Loxp fragment by PCR; seamless ligation technology was used to gra 4-5' utr, gra 4-5' utr and Loxp-DHFR-Loxp were connected to obtain gra 4-5' utr-Loxp-DHFR-Loxp- gra 4-3' utr fragment; Targeted knockout gra 4. Preparation of pSAG1-Cas9-sgGRA4 Plasmid: Using the pSAG1-Cas9-sgMIC3 plasmid as a template, the designed gra 4. Specific sgRNA replaces the sgMIC3 fragment in the template to obtain targeted knockout gra pSAG1-Cas9-sgGRA4 plasmid containing 4 genes; Electroporation to construct target insect strain: The 5H-Loxp-DHFR-Loxp-3H fragment and pSAG1-Cas9-sgGRA4 plasmid were integrated into ME49Δ by electroporation. ompdc / ldh1 In the genome, GRA4-deficient ME49Δ was identified by drug screening and PCR ompdc / ldh1 / gra4 Insect strains.

3. The preparation method according to claim 2, characterized in that: For amplification gra The nucleotide sequences of the primer pairs for the 4-5' utr fragment are shown in SEQ ID No.6 and SEQ ID No.7; gra The nucleotide sequences of the primer pair for the 4-3' utr fragment are shown in SEQ ID No.8 and SEQ ID No.9; The nucleotide sequences of the primer pair used to amplify the Loxp-DHFR-Loxp fragment are shown in SEQ ID No.10 and SEQ ID No.

11.

4. The preparation method according to claim 2, characterized in that: Targeted knockout gra4 The nucleotide sequences of the primer pair sgRNA are shown in SEQ ID No.4 and SEQ ID No.

5.

5. The preparation method according to claim 2, characterized in that: The recombinants were screened after transfection using 1 μM pyrimethamine or 30 μM chloramphenicol.

6. The preparation method according to claim 2, characterized in that: The nucleotide sequences of the primer pairs for PCR identification of target insect strains are shown in SEQ ID No. 12 to 17.

7. The attenuated Toxoplasma gondii vaccine strain ME49Δ according to claim 1 ompdc / ldh1 / gra4 Application in the preparation of drugs or vaccines for preventing, treating, alleviating or improving mammalian tumors, wherein the tumor is a subcutaneous tumor of B16-F10 melanoma cells and a melanoma lung metastasis caused by B16-F10 melanoma cells.

8. The use according to claim 7, characterized in that: The mammal is a human or a mouse.

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