Toxoplasma gondii gene deletion strain ME49 delta TGZS and application thereof in preparation of gene engineering inactivated vaccine

Abstract

The invention discloses a toxoplasma gondii gene deletion strain ME49 [delta] TGZS and an application thereof in preparation of a gene engineering inactivated vaccine. The toxoplasma gondii gene deletion strain ME49 [delta] TGZS is prepared by taking a toxoplasma gondii strain ME49 as a parent strain and knocking out a TGZS gene in the toxoplasma gondii strain ME49; the nucleotide sequence of the TGZS gene is as shown in SEQ ID NO: 1. The strain can maintain normal polypide growth and replication capacity under an in-vitro culture condition, after inactivation treatment, the strain loses proliferative activity but maintains a complete polypide morphological structure, and the strain is compatible with an aluminum hydroxide adjuvant to prepare a vaccine preparation. According to the vaccine, by activating the specific immune response of a terminal host cat, the sexual reproduction cycle of the toxoplasma gondii in the intestinal epithelial cells of the cat is effectively blocked, and the formation and discharge of oocysts are remarkably inhibited, so that the propagation chain of the toxoplasma gondii is controlled from the source. The invention provides a new effective means for prevention and control of toxoplasmosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of veterinary biological products, and particularly relates to a Toxoplasma gondii gene deletion strain ME49Δ TGZS and application thereof in preparing a genetically engineered inactivated vaccine. BACKGROUND

[0002] Toxoplasma gondii is a obligate intracellular parasite of the Apicomplexa phylum, which can infect almost all warm-blooded animals and cause human and animal shared toxoplasmosis. The pathogen is widely distributed in the world, and according to epidemiological investigations, about one-third of the world's population is infected, which poses a serious threat to human health and livestock production.

[0003] In intermediate hosts (including humans, pigs, sheep and other mammals), Toxoplasma gondii mainly performs asexual reproduction of tachyzoites and bradyzoites; and in the intestinal epithelial cells of the terminal host cats, Toxoplasma gondii completes the sexual reproduction cycle, including the stages of gametogony and sporogony, Toxoplasma gondii has a complex life cycle and a wide host range. In intermediate hosts (including humans, pigs, sheep and other mammals), Toxoplasma gondii only performs asexual reproduction; and in the intestinal epithelial cells of the terminal host cats, Toxoplasma gondii completes the sexual reproduction cycle and finally forms sporulated oocysts with strong infectivity. This unique biological characteristic makes cats the key medium for environmental transmission of Toxoplasma gondii.

[0004] Oocyst shedding is the core link of Toxoplasma gondii transmission. Infected cats can shed 10^6-10^7 unsporulated oocysts per day, and these oocysts sporulate in the external environment to form mature infectious oocysts containing two sporocysts, each sporocyst containing 4 sporozoites. Mature oocysts have strong resistance to the environment and can survive in the soil for several months or even years, causing large-scale transmission through contaminated water sources, feed and environment.

[0005] Currently, the prevention and control of Toxoplasma gondii mainly relies on chemical treatment, and the combination of pyrimethamine and sulfonamide drugs is commonly used in clinical treatment. However, this treatment strategy has significant limitations: first, chemotherapy drugs cannot completely eliminate bradyzoite cysts in tissues, and the recurrence rate is high after drug withdrawal; second, the treatment is only for infected individuals and cannot prevent infection; most importantly, existing drugs cannot stop the sexual reproduction process in the intestinal tract of cats and cannot block the formation and shedding of oocysts, so they cannot control environmental pollution from the source.

[0006] Vaccine immunization is an ideal strategy for preventing Toxoplasma gondii. Existing veterinary Toxoplasma gondii vaccines are mainly based on live vaccines or subunit vaccines, which are designed for the asexual reproduction stage of intermediate hosts. However, these vaccines have obvious defects: (1) Immunogenicity limitation: mainly induces immune response against bradyzoite antigens, lacks cross-protection against sexual reproduction stage antigens; (2) Incomplete transmission blocking: although can reduce tissue cyst formation, cannot prevent gametogony and oocyst discharge in the intestinal tract of felines; (3) Host specificity limitation: vaccines for intermediate hosts lack protective effect on feline end hosts.

[0007] Therefore, developing a new type of vaccine capable of specifically blocking the sexual reproduction process of Toxoplasma gondii and targeting feline end hosts has important theoretical significance and practical value for controlling Toxoplasma gondii transmission from the source and ensuring public health safety. SUMMARY

[0008] The present application provides a Toxoplasma gondii gene deletion strain ME49 TGZS and its application in preparing a genetically engineered inactivated vaccine. TGZS The present application constructs a Toxoplasma gondii gene deletion strain ME49 TGZS and develops a genetically engineered inactivated vaccine for cats targeting the blocking of Toxoplasma gondii sexual reproduction transmission, which is a new type of vaccine preparation for feline end hosts and can specifically block the sexual reproduction of Toxoplasma gondii, thereby solving the following technical problems: (1) The existing vaccine is mainly designed for the asexual reproduction stage of intermediate hosts, and cannot effectively block the sexual reproduction process in the intestinal tract of feline end hosts; (2) The immune response induced by the vaccine lacks effective inhibition of oocyst formation and discharge; (3) The existing vaccine cannot control the environmental transmission of Toxoplasma gondii from the source.

[0009] To achieve the above-mentioned purposes, the technical solutions designed by the present application are as follows: The present application provides a Toxoplasma gondii gene deletion strain ME49 ΔTGZS , wherein the deletion strain ME49 ΔTGZS is a Toxoplasma gondii ME49 strain as a parent worm strain, and a TGZS gene in the Toxoplasma gondii ME49 strain is knocked out by gene editing technology, and the nucleotide sequence of the TGZS gene is shown in SEQ ID NO: 1.

[0010] The present application provides a construction method of the above-mentioned Toxoplasma gondii gene deletion strain ME49 ΔTGZS , comprising the following steps: (1) Collect Toxoplasma gondii bradyzoites, and prepare a worm suspension using Cytomix buffer; (2) co-transfect the homologous template of TGZS-5UTR::DHFR::TGZS-3UTR and the pSAG1-Cas9-U6-sgTGZS plasmid DNA, mix with the worm suspension, and then add to the electroporation cup for electroporation; (3) after electroporation, add worm culture solution for flushing, transfer the liquid in the electroporation cup to the host cells for culture; (4) after the worms escape from the host cells, lyse the cells to release the worms, take the worms and add to the host cells, use ethyl pyrimidine for 3-5 generations of drug screening, and perform single clone worm screening; (5) after the single clone worms appear, take the host cells containing the single clone worms and add to the host cells for continuous culture; (6) when the host cells are lysed, the single clone worms in the host cells are used for DNA extraction, PCR identification, and the correct identification result is obtained to obtain the Toxoplasma gene deletion strain ME49 ΔTGZS .

[0011] Further, in the step (1), the amount of worms in the worm suspension is 1×10 7 ~3.3×10 7 / mL.

[0012] Further, in the step (2), the mass-volume ratio of the transfected DNA and the worm suspension is 1:25-30 μg / μL, and in the transfected DNA, the DNA molar ratio of the homologous template and the pSAG1-Cas9-U6-sgTGZS plasmid is 1:5.

[0013] Further, in the step (2), the preparation method of the pSAG1-Cas9-U6-sgTGZS plasmid is as follows: S1. The gRNA online design website is used to design the target site of the gene TGZS, and the gRNA primer is designed according to the designed target sequence, wherein the gRNA primer is: gRNA-TGZS-F: 5'-GCGAGTAGGCGGCAAACCACGTTTTAGAGCTAGAAATAGC-3', as shown in SEQ ID NO: 2; gRNA-TGZS-R: 5'-AACTTGACATCCCCATTTAC-3', as shown in SEQ ID NO: 3.

[0014] S2. Q5 super fidelity DNA polymerase, gRNA primer and plasmid pSAG1::CAS9-U6::sgUPRT are used for PCR amplification, and the PCR amplification product is digested to obtain a digestion product; S3. The reaction product of the digestion product was transformed into E. coli DH5α competent cells, which were cultured and screened to obtain plasmid pSAG1-Cas9-U6-sgTGZS.

[0015] Further, in step (2), the preparation method of the TGZS-5UTR::DHFR::TGZS-3UTR homologous template is as follows: 1) The 5' homologous arm fragment was obtained by PCR amplification using primer U5TGZS-F and U5TGZS-R and taking the Toxoplasma gondii ME49 genomic DNA as a template; wherein, U5TGZS-F is: 5'-CGACGGCCAGTGAATTCGAGACAACGGGAGCTCTGAACAC-3', as shown in SEQ ID NO: 4, U5TGZS-R is: 5'-GATGTCTTCTGCGCGGGTTGAGTGGCCGCTTCATCCGTC-3', as shown in SEQ ID NO: 5; 2) The 3' homologous arm fragment was obtained by PCR amplification using primer U3TGZS-F and U3TGZS-R and taking the Toxoplasma gondii ME49 genomic DNA as a template; wherein, U3TGZS-F is: 5'-GCCACAAGTTCAGCGTGTCCACGCTGGGTTTCCGAAGTG-3', as shown in SEQ ID NO: 6, U3TGZS-R is: 5'-GCTATGACCATGATTACGCCACTCCTCAGAAAGCGATGTG-3', as shown in SEQ ID NO: 7; 3) The pUC19 vector fragment was obtained by PCR amplification using primer pUC19-F and pUC19-R and taking the pUC19 plasmid as a template; wherein, primer pUC19-F is: 5'-CTGGCGTAATCATGGTCATAGC-3', as shown in SEQ ID NO: 8, primer pUC19-R is: 5'-CGAACACTGGCCGTCG-3', as shown in SEQ ID NO: 9; 4) The DHFR fragment was obtained by PCR amplification using primer loxP-DHFR-F and loxP-DHFR-R and taking the plasmid with DHFR as a template; wherein, LoxP-DHFR-F is 5'-CAACCCGCGCAGAAGACATC-3', as shown in SEQ ID NO: 10, LoxP-DHFR-R is 5'-GGACACGCTGAACTTGTGGC-3', as shown in SEQ ID NO: 11; 5) The 5' homologous arm fragment, the DHFR fragment, the 3' homologous arm fragment and the pUC19 vector fragment are connected to obtain a plasmid pTGZS-5UTR::DHFR::TGZS-3UTR; 6) The plasmid pTGZS-5UTR::DHFR::TGZS-3UTR is used as a template, and primers U5TGZS-F and U3TGZS-R are used for PCR amplification to obtain a TGZS-5UTR::DHFR::TGZS-3UTR homologous template.

[0016] The application also provides a Toxoplasma gondii gene deletion strain ME49 ΔTGZS In the preparation of an inactivated vaccine for targeted blocking of the sexual reproduction transmission of Toxoplasma gondii.

[0017] The application also provides a Toxoplasma gondii gene deletion strain ME49 ΔTGZS An inactivated vaccine preparation, wherein the inactivated vaccine preparation is prepared from the inactivated gene deletion strain ME49 ΔTGZS Mixed with an aluminum hydroxide adjuvant at a volume ratio of 1:1; The concentration of the aluminum hydroxide adjuvant is 0.3 mg / mL.

[0018] The application also provides a Toxoplasma gondii gene deletion strain ME49 ΔTGZS A method for preparing an inactivated vaccine preparation, wherein the method mixes the inactivated ME49 ΔTGZS At a volume ratio of 1:1, and the vaccine preparation is packaged.

[0019] Further, the concentration of the inactivated vaccine ME49 ΔTGZS In the vaccine preparation is 10 6 Worms / mL.

[0020] The beneficial effects of the application are: (1) Strong targeting: the vaccine of the application is based on the gene deletion strain ME49 Figure 1 After the deletion of specific genes, the strain exhibits unique biological characteristics in the sexual reproduction stage, and can induce specific immune responses against the sexual reproduction stage of Toxoplasma gondii.

[0021] (2) High safety: The inactivated vaccine form is adopted to avoid the potential risk of virulence return of live vaccine, and the construction of gene deletion strain further reduces the biological safety hazard.

[0022] (3) Significant immune effect: The vaccine can activate the specific immune response of the terminal host cat, effectively block the sexual reproduction cycle of Toxoplasma gondii in the intestinal epithelial cells of cats, and significantly inhibit the formation and discharge of oocysts.

[0023] (4) Complete transmission blocking: By inhibiting oocyst discharge, the environmental transmission of Toxoplasma gondii is controlled from the source, significantly reducing the risk of Toxoplasma gondii transmission mediated by terminal host cats.

[0024] (5) Mature preparation process: The vaccine preparation method is simple and controllable, suitable for large-scale production, providing a new effective means for the prevention and control of toxoplasmosis. BRIEF DESCRIPTION OF DRAWINGS

[0025] ΔTGZS is a schematic diagram for the construction of Toxoplasma gondii gene deletion strain ME49, showing the construction strategy of the target gene knockout vector and the homologous recombination process; Figure 2 ΔTGZS is a PCR identification result diagram of Toxoplasma gondii gene deletion strain ME49, in which M is a DNA molecular weight marker, lanes 1, 3, and 5 are PCR amplification products of gene deletion strain ME49, and lanes 2, 4, and 6 are PCR amplification products of wild-type ME49 strain; ΔTGZS Figure 3 ΔTGZS is a comparison of the growth characteristics of Toxoplasma gondii gene deletion strain ME49 and wild-type strain ME49 under in vitro culture conditions; Figure 4 ΔTGZS is a comparison of the growth characteristics of Toxoplasma gondii gene deletion strain ME49 and wild-type strain ME49, and inactivated ME49 after inactivation treatment; Δ TGZS Figure 5 ΔTGZS is a schematic diagram of the integrity of Toxoplasma gondii gene deletion strain ME49 after inactivation treatment; Figure 6 ΔTGZS is a body temperature measurement result diagram of cats during immunization with Toxoplasma gondii gene deletion strain ME49 inactivated vaccine. Figure 7

[0026] ΔTGZS is a body weight measurement result diagram of cats during immunization with Toxoplasma gondii gene deletion strain ME49 inactivated vaccine. Figure 8

[0027] TGZS ​​​​​​​​​Figure 1 is a statistical result chart of the oviposition time and the number of oocysts per gram (OPG) of the immune group and the control group after the tissue cyst infection. DETAILED DESCRIPTION

[0028] The application will be further described in detail below with reference to specific examples, so that those skilled in the art can understand.

[0029] Example 1 Toxoplasma gene deletion strain ME49 TGZS Construction I. Toxoplasma maternal strain ME49 Toxoplasma strain ME49 is a type II strain of the genus Toxoplasma in the family Toxoplasma in the order Coccidia, and the knockout gene ΔTGZS plays a key regulatory role in the sexual reproduction stage of Toxoplasma, and the nucleotide sequence is shown as SEQ ID NO: 1.

[0030] II. Construction of pSAG1-Cas9-U6-sgTGZS plasmid 1. The target site of the target gene TGZS was designed using the gRNA online design website (E-CRISP Design), and the gRNA primer was designed according to the designed target sequence, and the primer was as follows: The upstream primer gRNA-TGZS-F is: 5'-GCGAGTAGGCGGCAAACCACGTTTTAGAGCTAGAAATAGC-3'; The downstream primer gRNA-TGZS-R is: 5'-AACTTGACATCCCCATTTAC-3'.

[0031] 2. The PCR reaction system was prepared in a sterile PCR tube, and the template DNA was pSAG1::CAS9-U6::sgUPRT plasmid.

[0032] Table 1 PCR reaction system Table 2 PCR reaction program 4. After the PCR was completed, 1 μL of DpnI was directly added to the PCR product, and the digestion was performed at 37°C for 30 min in the PCR instrument to obtain the digestion product.

[0033] 5. A new sterile PCR tube was taken, and the KLD enzyme reaction system was prepared according to the parameters in Table 3, and the reaction was performed at 25°C for 15 min in the PCR instrument to obtain the reaction product.

[0034] Table 3 KLD enzyme reaction system 6. Transform all reaction products into *E. coli* DH5α competent cells, plate them on LB agar plates containing 100 μg / mL Amp, and incubate overnight at 37°C with the plates inverted until single colonies grow. Pick a single colony and place it in 5 mL of LB liquid medium containing 100 μg / mL Amp, and incubate at 37°C with shaking at 180 rpm for 10–12 h until the culture becomes turbid. Take 500 μL of the culture for sequencing analysis using the M13 reverse primer. The sequence of the M13 reverse primer is: CAGGAAACAGCTATGAC; If the sequencing results show that the target sequence has been completely replaced, the plasmid pSAG1-Cas9-U6-sgTGZS has been successfully constructed. The pSAG1-Cas9-U6-sgTGZS plasmid is extracted using an endotoxin-free plasmid extraction kit, and the concentration is measured for later use.

[0035] III. Preparation of homologous templates of TGZS-5UTR::DHFR::TGZS-3UTR 1. Based on the Gene ID of the gene to be knocked out, TGZS, the locus of the gene is determined on ToxoDB, and the 5' homologous arm (TGZS-5UTR) and 3' homologous arm (TGZS-3UTR) are determined by the genome sequence in which it is located. 2. Design seamless ligation amplification primers for the 5' homologous arm, 3' homologous arm, DHFR drug screening tag, and pUC19 vector according to the method described in the multi-fragment seamless ligation kit instructions; 3. Using the designed primers (as shown in Table 4), the 5' homologous arm and 3' homologous arm fragments were amplified from the Toxoplasma gondii ME49 genomic DNA using the high-fidelity enzyme (Phanta). The DHFR fragment was amplified from the plasmid containing DHFR (purchased from http: / / www.addgene.org). The pUC19 vector fragment (purchased from http: / / www.addgene.org) was linearized using the designed specific primers. The above target fragments were recovered to obtain the 5' homologous arm fragment, 3' homologous arm fragment, DHFR fragment, and pUC19 vector fragment, respectively. The concentration of the recovered products was determined using NanoDrop2000.

[0036] Table 4 Primers for constructing plasmids 4. Prepare the following reaction system based on the concentration of the recovered product: 5x CE MultiS Buffer: 2.0 μL; Exnase™ MultiS: 1.0 μL; TGZS-5UTR: 10 ng; TGZS-3UTR: 10 ng; DHFR: 30 ng; pUC19: 25 ng; add sterile deionized water (nuclease free) to a total reaction volume of 10 μL.

[0037] After thoroughly mixed, the reaction was carried out at 37°C for 30 min, and then placed on ice for 5 min to obtain the ligation product.

[0038] 5. 10 μL of the ligation product was transformed into 100 μL of E. coli DH5α competent cells, and then spread on LB plates containing 100 μg / mL Amp, and incubated at 37°C overnight to obtain single colonies. A single colony was picked and inoculated into a sterile PV glass bottle containing 5 mL of LB liquid medium containing 100 μg / mL Amp, and incubated at 37°C / 180 rpm for 10 h to obtain a bacterial suspension. Sequencing analysis was performed using M13 forward primer and M13 reverse primer, respectively. The sequence of the M13 forward primer was GTAAAACGACGGCCAGT. If the sequencing result is correct, the plasmid pTGZS-5UTR::DHFR::TGZS-3UTR is successfully constructed, and the correct bacterial suspension is extracted for plasmid, and stored at -20°C.

[0039] 6. PCR amplification of the pTGZS-5UTR::DHFR::TGZS-3UTR plasmid was performed using high-fidelity enzyme, and the amplification primers were U5TGZS-F upstream primer and U3TGZS-R downstream primer. The target fragment (TGZS-5UTR::DHFR::TGZS-3UTR) was recovered by gel cutting, and the concentration was determined and reserved. The TGZS-5UTR::DHFR::TGZS-3UTR homologous template was obtained.

[0040] Four, Toxoplasma gondii Figure 1 Gene deletion strain ME49 TGZS obtained 1. Fresh T. gondii ME49 tachyzoites were collected, and host cell debris was removed by filtration with a sterile 3 μm pore size filter membrane, and centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the worm pellet was resuspended in Cytomix buffer (120 mmol / L KCI, 0.15 mmol / L CaCl2, 10 mmol / L K2HPO4, 25 mmol / L HEPES, 2 mmol / L EGTA, 5 mmol / L MgCl2, 2 mmol / L ATP, 5 mmol / L glutathione, pH=7.6) to obtain a worm suspension, wherein the worm amount was about 3.3 x 107 tachyzoites / mL.

[0041] 2. Take 250-300 μL of the insect suspension into an electroporation cuvette, add the pSAG1-Cas9-U6-sgTGZS plasmid from step 2 and the TGZS-5UTR::DHFR::TGZS-3UTR homologous template from step 3 as the transfection DNA, wherein the molar ratio of plasmid to homologous template DNA is 5:1, the total amount of transfection DNA is 10 μg, and the total volume of the electroporation reaction is 350 μL.

[0042] 3. Use a pipette to mix the transfected DNA and the worm suspension evenly in a 4mm electroporation cuvette and remove air bubbles; place the cuvette in the Bio-Rad electroporator and set the conditions to 1600V, 25μF, 50Ω, and 4mm, and electroporate twice.

[0043] 4. Immediately add 1 mL of worm culture medium (DMEM + 2% FBS + double antibiotics, the double antibiotics being 100 μg / mL penicillin and 50 μg / mL streptomycin) to the electroporation cuvette, gently rinse, and then transfer the liquid to fresh host cells HFF-1 for culture (T25). At the same time, a control group ME49 without DNA transfection is set up, and culture observation is carried out simultaneously.

[0044] 5. After 60-80% of the transfected parasites escape from the host cells HFF-1, the cells are lysed to release the parasites from the host cells; 500-2000 μL of parasites are added to fresh host cells HFF-1 (T25) and screened using 1 μM pyrimethamine.

[0045] 6. After 3 days of treatment with pyrimethamine, parasite proliferation was observed in the host cells. When 50% of the parasites escaped from the host cells, the cells were lysed to release the parasites. 500-1000 μL of the parasites were added to fresh host cells HFF-1 and then screened with 1 μM pyrimethamine.

[0046] 7. After 3-5 generations of drug screening, the parasites were placed in 96-well plates containing host cells HFF-1 for monoclonal screening (using two 96-well plates), with two (calculated) tachyzoites inoculated into each well; the remaining parasites could be used for genomic DNA extraction, and the primers in Table 5 and TGZS The method shown was used to detect homologous arm integration efficiency and gene knockout efficiency. The presence of a band in PCR1 indicates this. TGZS The 5' homologous arm integrates into the Toxoplasma gondii genome, and the presence of a band on PCR2 indicates this. TGZS The 3' homologous arm was integrated into the Toxoplasma gondii genome, and the knockout gene was detected by PCR 3-band detection. Figure 2 Whether it is still in the genome, if the control group ME49 has a PCR3 band while the transfection experimental group does not, then it indicates that... Figure 2 Successfully knocked out, the result is as followsΔTGZS .

[0047] Table 5 PCR identification primers of monoclonal Toxoplasma strains 8、After 8~11 days of culture in 96-well plates, observe whether there is a monoclonal, use a sterile gun head to scrape the host cells containing the monoclonal worms from the hole and add to a 24-well plate containing host cells HFF-1 for continued culture; when 70% of the host cells in the 24-well plate are lysed, 150 μL of the worms in the hole are transferred to a new 24-well plate for culture, and the remaining monoclonal worms are used for DNA extraction for identification of the clone.

[0048] 9、PCR1, PCR2 and PCR3 are used to detect the selected monoclonal worms, if PCR1 and PCR2 have specific bands and PCR3 has no target band, it indicates that the monoclonal strain of Toxoplasma TGZS gene deletion strain ME49ΔTGZS; the strain determined as ME49ΔTGZS is further cultured to a T25 culture flask; the gene deletion strain in the T25 culture flask is detected again by PCR1, PCR2 and PCR3 for verification.

[0049] Table 6 PCR reaction system Table 7 PCR reaction conditions 10、PCR product identification: after amplification, take 10 μL of the PCR product, mix with 2 μL of 6×nucleic acid loading buffer, spot, 1.0% agarose gel, 1×TAE buffer, 110~120V, electrophoresis for 30 minutes, and observe by gel imaging system.

[0050] The PCR identification results are shown in Table 6: Figure 3 The Toxoplasma TGZS gene deletion strain ME49ΔTGZS is successfully constructed in this example.

[0051] Example 2 In vitro culture property verification of the gene deletion strain ME49 ΔTGZS ​Gene-deleted strain ME49ΔTGZS and wild-type ME49 were cultured simultaneously under normal conditions. After 30%–50% of the parasites overflowed from the host cells, they were scraped off using a Pasteur pipette and repeatedly pipetted 8–10 times with a syringe. After filtration, the parasites were counted, and 105 parasites were inoculated into 24-well plates containing HFF cells. The plates were incubated at 37°C for 1 hour. Uninvaded parasites were washed away with PBS, and the plates were cultured for another 24 hours. After 24 hours, the plates were fixed and stained with 4% paraformaldehyde. The number of parasites in the vesicles of the gene-deleted strain ME49ΔTGZS and wild-type ME49 was observed and counted under a fluorescence microscope. The number of vesicles in 1, 2, 4, 8, and 16 parasites was counted, with at least 100 vesicles counted each time. Each experiment was performed in triplicate, and the proportion of vesicles in different replicated parasites was calculated.

[0052] The results are as follows ΔTGZS As shown: Gene deletion strain ME49 ΔTGZS Compared with the wild-type ME49 strain, the number of worms in the vesicles was mainly 4 and 8. The replication rate of the knockout strain was slightly slower, but the overall replication capacity was not significantly different.

[0053] Example 3: Feline gene-deleted strain ME49 for targeted blocking of sexual reproduction transmission of Toxoplasma gondii. Figure 4 inactivated vaccine preparations (1) The gene deletion strain ME49 prepared in Example 1 was used. Figure 5 The parasites were cultured in large quantities in HFF cells and harvested when the infection rate reached 80-90%.

[0054] (2) The insect body was scraped by cell scraping and blown out of the larval vesicle by syringe. After filtration by filter, the purified insect body was obtained by centrifugation and washed three times with sterile PBS buffer to remove host cell debris and culture medium residue.

[0055] (3) The purified insect bodies were suspended in a diluent of PBS with a formaldehyde concentration of 0.02% / 10. 6 In each tachyzoite, inactivation was performed at 37°C for 24 hours. A plaque assay was used to ensure complete loss of reproductive activity in the parasites, and the results were as follows: ΔTGZS IFA testing ensured that the insects were inactivated while maintaining their morphological integrity, as shown in the results. ΔTGZS .

[0056] (4) Use aluminum hydroxide as an adjuvant. Shake the adjuvant well before use to ensure that the colloid is evenly dispersed.

[0057] (5) Vaccine preparation: The original concentration of aluminum hydroxide adjuvant was 1 mg / mL. It was diluted to 0.3 mg / mL with 0.9% physiological saline, and then mixed with 10... 6 Gene-deleted strain ME49 Figure 6The vaccine preparation is mixed at a volume ratio of 1:1 and then packaged (each vaccine has a total volume of 1 mL).

[0058] Example 4: Validation experiment of a vaccine for preventing feline toxoplasmosis infection (1) Immunization of feline toxoplasmosis and safety experiments in cats. ① Toxoplasma gondii-negative cats were selected as experimental subjects using MIC17A-iELISA and TXA-iELISA methods.

[0059] Immunization group: Two subcutaneous immunizations were administered according to the following protocol. Immunization schedule: Immunization was administered on day 0 and day 21, respectively; Immunization dosage: Calculated based on the cat's weight, the dosage for each immunization is 10g. 6 Gene-deleted strain ME49 Figure 7 ; Administration method: The prepared vaccine was administered via subcutaneous injection. Control group: A control trial was conducted using the same time intervals and administration method. Control group: Injected on day 0 and day 21, synchronously with the immunization group; Control dose: The dosage for each injection was the same as that of the immunization group; Control reagent: Use an equal volume of PBS buffer instead of the vaccine for subcutaneous injection.

[0060] ② Each cat was kept in a separate cage. After the initial immunization, the body temperature and weight of the immunized cats were measured every 3 days. Clinical symptoms during the immunization period were observed and recorded for 30 days. After 30 days, the measurement data were statistically analyzed. The body temperature and weight measurement results are as follows: △TGZS and Figure 8 As shown.

[0061] The results showed that no adverse reactions such as fever, weight loss, depression, or diarrhea occurred in the cats after vaccination, indicating that the vaccine (inactivated gene-deleted strain ME49) used to prevent feline toxoplasmosis infection is effective. ​ It is safe for cats.

[0062] (2) Immunoprotective efficacy of vaccines used to prevent feline toxoplasmosis infection in cats: On day 30 after the last immunization, cats in the immunized group and the control group were orally administered Toxoplasma gondii tissue cysts, 500 cysts per cat, 3 cats per group. The number of Toxoplasma gondii oocysts per gram of feces in the experimental cats was continuously measured and recorded from day 1 to day 20.

[0063] The results are as follows ​As shown: In the control group, 3 cats shed Toxoplasma gondii oocysts from days 5 to 13 after viral challenge; no Toxoplasma gondii oocysts were shed in the immunized group. The results indicate that the amount of oocysts shed in the vaccine-immunized group was 100% less than that in the control group. This demonstrates that the inactivated gene-deleted strain vaccine ME49△TGZS for the prevention of feline Toxoplasma gondii infection provides good protection against wild-type strain infection in cats, effectively blocking the transmission risk of Toxoplasma gondii caused by oocyst shedding in cats, and has the potential to become a sub-inactivated Toxoplasma gondii vaccine.

[0064] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Toxoplasma gondii gene-deleted strain ME49 ΔTGZS Its features are: The deletion strain ME49 ΔTGZS Using Toxoplasma gondii strain ME49 as the parent strain, gene editing technology was used to knock out the Toxoplasma gondii strain ME49. TGZS Genes, the ones mentioned TGZS The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The Toxoplasma gondii gene-deleted strain ME49 according to claim 1 ΔTGZS The construction method is characterized by: Includes the following steps: (1) Collect ME49 tachyzoites of Toxoplasma gondii and prepare a suspension of the parasites using Cytomix buffer; (2) Co-transfect DNA with the homologous template TGZS-5UTR::DHFR::TGZS-3UTR and pSAG1-Cas9-U6-sgTGZS plasmid, mix with the insect suspension and add to the electroporation cuvette for electroporation. (3) After electroporation, insect culture medium is added for rinsing, and the liquid in the electroporation cup is transferred to the host cells for culture; (4) After the parasite escapes from the host cell, the cell is lysed to release the parasite, the parasite is added to the host cell, and pyrimethamine is used for 3 to 5 generations of drug screening, and monoclonal parasite screening is performed. (5) After monoclonal worms appear, take host cells containing monoclonal worms and add them to host cells for continued culture; (6) When the host cell is lysed, the monoclonal parasites in the host cell are used for DNA extraction and PCR identification. If the identification result is correct, the Toxoplasma gondii gene deletion strain ME49 is obtained. ΔTGZS .

3. The construction method according to claim 2, characterized in that: In step (1), the amount of worms in the worm suspension is 1×10⁻⁶. 7 ~3.3×10 7 Tachyzoites / mL.

4. The construction method according to claim 2, characterized in that: In step (2), the mass-to-volume ratio of transfected DNA to insect suspension is 1:25~30 μg / μL, and the molar ratio of homologous template and pSAG1-Cas9-U6-sgTGZS plasmid in transfected DNA is 1:

5.

5. The construction method according to claim 2 or 4, characterized in that: In step (2), the preparation method of pSAG1-Cas9-U6-sgTGZS plasmid is as follows: S1. The TGZS gene target site was designed using an online gRNA design website, and gRNA primers were designed based on the designed target sequence. The gRNA primers are as follows: gRNA-TGZS-F: 5'-GCGAGTAGGCGGCAAACCACGTTTTAGAGCTAGAAATAGC-3'; gRNA-TGZS-R: 5'-AACTTGACATCCCCATTTAC-3'; S2. PCR amplification was performed using Q5 superfidel DNA polymerase, gRNA primers and plasmid pSAG1::CAS9-U6::sgUPRT. The PCR amplification products were digested to obtain digestion products. S3. The digestion products were reacted with KLD enzyme, and the reaction products were transformed into Escherichia coli DH5α competent cells for culture and screening to obtain plasmid pSAG1-Cas9-U6-sgTGZS.

6. The construction method according to claim 2 or 4, characterized in that: In step (2), the preparation method of the homologous template TGZS-5UTR::DHFR::TGZS-3UTR is as follows: 1) Using Toxoplasma gondii ME49 genomic DNA as a template, PCR amplification was performed using primers U5TGZS-F and U5TGZS-R to obtain the 5' homologous arm fragment; among which, U5TGZS-F is: 5'-CGACGGCCAGTGAATTCGAGACAACGGGAGCTCTGAACAC-3', U5TGZS-R is: 5'-GATGTCTTCTGCGCGGGTTGAGTGGCCGCTTCATCCGTC-3'; 2) Using Toxoplasma gondii ME49 genomic DNA as a template, PCR amplification was performed using primers U3TGZS-F and U3TGZS-R to obtain the 3' homologous arm fragment; among which, U3TGZS-F is: 5'-GCCACAAGTTCAGCGTGTCCACGCTGGGTTTCCGAAGTG-3', U3TGZS-R is: 5'-GCTATGACCATGATTACGCCACTCCTCAGAAAGCGATGTG-3'; 3) Using the pUC19 plasmid as a template, PCR amplification was performed using primers pUC19-F and pUC19-R to obtain the pUC19 vector fragment; among which, Primer pUC19-F is: 5'-CTGGCGTAATCATGGTCATAGC-3'. Primer pUC19-R is: 5'-CGAACACTGGCCGTCG-3'; 4) Using a plasmid containing DHFR as a template, PCR amplification was performed using primers loxP-DHFR-F and loxP-DHFR-R to obtain the DHFR fragment; among which, loxP-DHFR-F is: 5'-CAACCCGCGCAGAAGACATC-3', loxP-DHFR-R is: 5'-GGACACGCTGAACTTGTGGC-3'; 5) Ligate the 5' homologous arm fragment, DHFR fragment, 3' homologous arm fragment and pUC19 vector fragment to obtain plasmid pTGZS-5UTR::DHFR::TGZS-3UTR; 6) Using plasmid pTGZS-5UTR::DHFR::TGZS-3UTR as a template, PCR amplification was performed using primers U5TGZS-F and U3TGZS-R to obtain the homologous template TGZS-5UTR::DHFR::TGZS-3UTR.

7. The Toxoplasma gondii gene-deleted strain ME49 according to claim 1 ΔTGZS Application in the preparation of inactivated vaccines that target and block the sexual reproduction of Toxoplasma gondii.

8. A gene-deleted parasite strain ME49 for cats ΔTGZS The characteristics of inactivated vaccine formulations are: The inactivated vaccine formulation is derived from the inactivated gene-deleted strain ME49. ΔTGZS It is prepared by mixing with aluminum hydroxide adjuvant at a volume ratio of 1:1; The concentration of aluminum hydroxide adjuvant was 0.3 mg / mL.

9. The feline gene-deleted parasite strain ME49 as described in claim 8 ΔTGZS A method for preparing an inactivated vaccine formulation, characterized in that: The method involves reacting an aluminum hydroxide adjuvant at a concentration of 0.3 mg / mL with inactivated ME49. ΔTGZS Vaccine preparations mixed at a volume ratio of 1:1 and then packaged.

10. The preparation method according to claim 9, characterized in that: The vaccine formulation contains the inactivated vaccine ME49. Δ TGZS The concentration is 10 6 1 insect body / mL.

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