A live attenuated vaccine of Toxoplasma gondii lacking the TgAROM gene

Abstract

The present invention belongs to the field of genetic engineering technology and specifically discloses a method for Tg The attenuated Toxoplasma vaccine contains the AROM gene missing. Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom , Tg The nucleotide sequence of the AROM gene is shown in SEQ ID NO. 1. The present invention has found that the direct knockout of Toxoplasma gondii Tg The AROM gene had no significant effect on the intracellular growth of Toxoplasma gondii; Δarom The insect strain can be subcultured normally in vitro, but Δarom The worms do not reproduce in animals and have almost no toxicity; vaccination Δarom The insect strain can produce good immune protection; in summary, Δarom The strain has the potential to become a Toxoplasma gondii vaccine.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a method for Tg AROM gene-containing live attenuated Toxoplasma vaccine. Background Art

[0002] Toxoplasmosis is a zoonotic parasitic disease caused by the apicomplexan protozoan Toxoplasma gondii, which seriously endangers human health and the sustainable development of animal husbandry. Toxoplasma gondii is mainly transmitted orally. People or animals are generally infected by drinking water contaminated with Toxoplasma oocysts, or accidentally eating raw or undercooked meat containing tissue cysts. People or animals with normal immunity are generally latently infected with Toxoplasma, and often have no obvious clinical symptoms. However, people and animals with low or defective immunity are seriously endangered by Toxoplasma infection, and even life-threatening. Toxoplasma infection can cause toxoplasmosis eye disease and encephalopathy, and cause miscarriage, malformation or stillbirth in pregnant women or livestock.

[0003] There is no ideal drug or vaccine for toxoplasmosis. Commonly used drugs such as pyrimethamine and sulfadiazine are only effective against Toxoplasma tachyzoites in the acute infection period, but have no obvious effect on Toxoplasma bradyzoites or tissue cysts in the chronic infection period. They also have toxic side effects and are prone to drug resistance after long-term use. Therefore, it is particularly urgent to construct a Toxoplasma vaccine with good immunogenicity and protection.

[0004] Chinese patent application number CN202311771384.2 discloses the Toxoplasma gondii attenuated vaccine strain RH Δgra47 and its application, belonging to the technical field of vaccine preparation. Tg GRA47 gene, obtained Tg GRA47 gene-deficient attenuated vaccine strain RH of Toxoplasma gondii Δgra47 , missing TgGRA47 The permeability of the parasite vesicle membrane (PVM) of Toxoplasma gondii is reduced, causing the parasite vesicle (PV) to enlarge and become bubble-shaped, slowing down the in vitro growth ability and in vivo toxicity of Toxoplasma gondii. Δgra47It is a potential live attenuated vaccine strain of Toxoplasma gondii, which can provide effective immune protection for acute and chronic infection of Toxoplasma gondii. In addition, Chinese patent application number CN201810127167.2 discloses a live attenuated vaccine for preventing Toxoplasma infection, which contains a Toxoplasma vaccine strain that lacks both the orotidine-5-phosphate decarboxylase gene and the lactate dehydrogenase 1 gene. The live attenuated vaccine provided has the advantage of being almost non-toxic, can improve the immunity of animals to Toxoplasma, can be used to prevent Toxoplasma infection, and because the vaccine strain removes the drug resistance screening tag, it has the potential to prepare a genetically engineered vaccine against Toxoplasma. Although encouraging progress has been made in the development of Toxoplasma gene knockout vaccine strains, due to the genetic diversity of Toxoplasma, the cross-immune protection of a single vaccine strain is often insufficient. Therefore, more vaccine design targets still need to be explored.

[0005] Shikimic acid is an important metabolite in plants and microorganisms. It is important for many biosynthetic processes, including the synthesis of folic acid, aromatic amino acids and ubiquinone. This is because the precursor substance of these pathways, chorismate, must be synthesized by the shikimic acid pathway. The shikimic acid pathway is only present in plants, fungi, bacteria and parasitic protozoa, but not in mammals, making the enzymes in the shikimic acid pathway promising as potential targets for drug and vaccine design. Although in most organisms, the seven steps of the shikimic acid pathway are encoded in the form of individual monofunctional enzymes, the five central steps in fungi and protozoa are encoded in the form of pentafunctional complexes (AROMs). In Toxoplasma gondii, the enzymes of steps 2-6 of the shikimic acid pathway are annotated as a protein ( Tg AROM).

[0006] Therefore, we can focus on the shikimate pathway Tg AROM uses gene editing technology to construct an attenuated Toxoplasma gondii vaccine strain with good immunogenicity and immune protection. Its application has important value and significance for human health and the development of animal husbandry. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a missing Tg The live attenuated Toxoplasma vaccine with the AROM gene can prevent and treat toxoplasmosis.

[0008] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0009] A missing Tg AROM gene is missing in the live attenuated vaccine against Toxoplasma gondii. Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom , Tg The nucleotide sequence of the AROM gene is shown in SEQ ID NO.1.

[0010] Furthermore, the deletion Tg The live attenuated Toxoplasma gondii vaccine containing the AROM gene also contains a medically acceptable vaccine adjuvant.

[0011] Furthermore, the missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom The parental RH strain was knocked out by CRISPR / Cas9 gene editing technology Tg AROM gene obtained.

[0012] Compared with the prior art, the present invention finds that the direct knockout of Toxoplasma gondii TgAROM It has no effect on the intracellular growth of Toxoplasma gondii and can be cultured normally in vitro. TgAROM Knockout strains do not reproduce in vivo, have almost no virulence, and are immunized TgAROM The missing strain is extremely safe for the host and shows good immune protection effect, indicating that Δarom The strain has the potential to become a Toxoplasma gondii vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Construction of a knockout gene for the AROM gene of Toxoplasma gondii Δarom Schematic diagram of the strategy for insect strains.

[0014] Figure 2 for Δarom Figure 1. PCRs identification results of monoclonal insect strains.

[0015] Figure 3 for Δarom Figure 2. Results of plaque proliferation of the insect strain in vitro.

[0016] Figure 4 for Δarom Figure 1. Results of the virulence gradient test of the insect strains in mice.

[0017] Figure 5 for Δarom Figure 2. Results of the in vivo insect load test of the insect strain.

[0018] Figure 6 For vaccination Δarom Figure 3. Results of immune protection test after ME49 infection. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0020] Unless otherwise specified, the reagents and raw materials used in the following examples are commercially available.

[0021] This example first constructs the missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom , the pattern diagram is as follows Figure 1 As shown, specifically, the type I parent Toxoplasma gondii RH ( DiCre ) strain (kindly donated by Huazhong Agricultural University) was used as the starting strain, and Toxoplasma gondii type I strains were searched from the ToxoDB database (www.toxodb.org). Tg The nucleotide sequence of the AROM gene (gene ID: TGGT1_307040, shikimate dehydrogenase substrate binding domain-containing protein) is as follows:

[0022]

[0023]

[0024]

[0025] Note: Capital letters represent exons and lowercase letters represent introns.

[0026] Then proceed through the following steps:

[0027] 1. pSAG1-Cas9-5UTR-sg Tg AROM and pSAG1-Cas9-3UTR-sg Tg Construction of AROM plasmid

[0028] (1) Design targeted Tg The sgRNA sequences of the 5' and 3' non-coding regions of AROM were designed, and gRNA primers were designed according to the designed sequences. The primer sequences are shown in Table 1.

[0029] Table 1 Construction Tg Primers for AROM-specific CRISPR / Cas9 plasmids

[0030]

[0031] (2) Using pSAG1-Cas9- Tg U6-sg Tg UPRT (purchased from Addgene) plasmid was used as a template. Tg The construction of AROM-specific CRISPR / Cas9 plasmids, PCR amplification reaction system and procedures are shown in Tables 2 and 3.

[0032] Table 2 PCR amplification reaction system

[0033]

[0034] Table 3 PCR amplification reaction program

[0035]

[0036] After the reaction, the above products were detected and identified by agarose gel electrophoresis, and the target fragments were cut and recovered (Novozyme Biotech Co., Ltd., DC301-01, Nanjing), and the concentration of the recovered products was determined by NanoDrop2000 and stored at -20°C for future use;

[0037] (3) The above products were connected using a single fragment seamless cloning kit (Novozymes Biotech Co., Ltd., C112-01, Nanjing). The reaction system was prepared as shown in Table 4.

[0038] Table 4 Single fragment ligation reaction system

[0039]

[0040] The optimal amount of linear cloning vector = [number of base pairs of fragment × 0.02] ng, the optimal amount of insert fragment amplification product = [number of base pairs of fragment × 0.04] ng, gently mix the reaction system and react at 37°C for 30 min, and wait for the transformation of the plasmid.

[0041] (4) Add 50 μL of the above products to TSC-C01-100 Trelief ®5α Chemically Competent Cell (Beijing Qingke Biotechnology Co., Ltd., TSC-C01-100, Beijing), incubate on ice for 5 min, heat shock at 42°C for 1 min, spread the bacteria evenly on an ampicillin-resistant plate, and incubate at 37°C for 12 h; take a single clone and place it on LB / Amp + Liquid culture medium expansion and sequencing, if the sequencing results show that the target sequence has been completely replaced, that is, pSAG1-Cas9- Tg U6-5UTR-sg Tg AROM and pSAG1-Cas9- Tg U6-3UTR-sg Tg The AROM plasmid was constructed successfully.

[0042] 2. Dual gRNA- Tg Construction of AROM CRISPR / Cas9 plasmid

[0043] (1) Use the primers in Table 5 to generate the Cas9- Tg U6-3UTR-sg Tg AROM plasmid amplification of temp-gRNA-3UTR- Tg AROM fragment, from pSAG1-Cas9- Tg U6-5UTR-sg Tg AROM plasmid amplified Vec-gRNA-5UTR- Tg AROM fragments and the above fragments were excised and recovered (Novozyme Biotech Co., Ltd., DC301-01, Nanjing) and the concentration was detected (Thermo Fisher Scientific, NanoDrop2000, USA);

[0044] Table 5 Amplification of temp-gRNA-3UTR- Tg AROM and Vec-gRNA-5UTR- Tg AROM fragment

[0045]

[0046] (2) As above, the two fragments recovered above were connected using a single fragment seamless cloning kit (Novozyme Biotech Co., Ltd., C112-01, Nanjing) to construct Tg AROM-specific dual gRNA CRISPR / Cas9, PCR reaction system is shown in Table 4, and plasmid transformation, plate culture and single colony sequencing are performed as before; if the sequencing results show temp-gRNA-3UTR- Tg AROM fragment inserted into Vec-gRNA-5UTR- Tg AROM plasmid, indicating dual gRNA- Tg AROMCRISPR / Cas9 plasmid has been successfully constructed.

[0047] 3. Amplification of Ko- AROM -YFP- DHFR *Homologous fragments

[0048] pTub-YFP- DHFR *Plasmid as template, pTub-YFP- DHFR *The nucleotide sequence of the plasmid is:

[0049]

[0050] The primers shown in Table 6 were used to perform PCR amplification using Phanta high-fidelity enzyme (Novozyme Biotech Co., Ltd., P501-d1, Nanjing). After amplification, the Ko- AROM -YFP- DHFR *The homologous fragments were recovered by gel recovery and stored at -20℃ for a long time.

[0051] Table 6 Amplification of Ko- AROM -YFP- DHFR *Fragment Primer

[0052]

[0053] 4. Build is missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom

[0054] (1) Collect freshly escaped Toxoplasma gondii DiCre Tachyzoites, the supernatant was filtered with a 3 μm filter to remove cell debris, and centrifuged at 3000 rpm for 8 min at room temperature; the worms in the pellet were resuspended in 4 mL Cytomix (120 mM KCl, 0.15 mMCaCl2, 10 mM K2HPO4 / KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, pH = 7.6), and centrifuged at 3000 rpm for 8 min at room temperature; 250 μL cytomix was used to resuspend the worm pellet;

[0055] (2) Add 50 μL cytomix and 7500 ng of dual gRNA- Tg AROM CRISPR / Cas9 plasmid and 1500 ng Ko- AROM -YFP- DHFR * Homologous fragments, and add all the resuspended worms in the above step (1) into the EP tube, mix thoroughly and add all into the 4 mm electroporation cup;

[0056] (3) Place the electroporation cup in a BTX electroporator (BTX, ECM630, USA) and perform an electroporation at 1600 V, 25 μF, 50 Ω, 4 mm for one time; and 1500 V, 25 μF, 50 Ω, 4 mm for two times. After standing for 2 min, add all the liquid in the electroporation cup to human fibroblast HFF cells (purchased from ATCC cell bank) in DMEM medium containing 2% FBS and culture them.

[0057] (4) When larger vacuoles are visible in the cells, discard the original culture medium and add 5 mL of 2% FBS DMEM medium containing 1 μM pyrimethamine for drug screening;

[0058] (5) After 2-4 rounds of pyrimethamine screening and culture, single clones were selected in 96-well plates using the limiting dilution method. The 96-well plates were placed in a 37°C incubator for 7 days and then observed under a fluorescence microscope and an optical microscope for the presence of single clones. The host cells in the wells corresponding to the single clones were scraped from the wells using a sterile pipette tip and added to a 24-well plate containing HFF cells for expanded culture. When most of the parasites escaped, an appropriate amount of tachyzoites were aspirated for DNA extraction (Tiangen Biochemical Technology Co., Ltd., DP348, Beijing) and PCR identification.

[0059] (6) Use the primers in Table 7 to perform PCR identification on the monoclonal insect strain. The PCR reaction system and procedure are the same as those in Tables 2 and 3. The results are as follows: Figure 2 As shown in the figure, the PCR1 and PCR2 of the monoclonal strain have bands of the target size, while PCR3 has no target band, indicating that the monoclonal strain has been successfully cloned. DiCre Knockout Tg AROM gene and acquired deletion Tg Toxoplasma gondii vaccine strains containing AROM genes Δ arom .

[0060] Table 7 Δarom Primers for PCR identification of monoclonal insect strains

[0061]

[0062] Further, in order to verify the above embodiment constructed missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom Whether it can be used for vaccine preparation, in vitro and in vivo experiments are conducted respectively:

[0063] 1. Δarom In vitro plaque assay of insect strains

[0064] (1) Place HFF cells in a 6-well plate. When the cells are fully grown, add 100 tachyzoites per well to the parent plate. DiCre The insect strain and Example 1 were constructed Δarom The insect strain was cultured in a 37°C cell culture incubator for 7 days;

[0065] (2) Discard the supernatant, wash three times with PBS, add 1 mL of 4% paraformaldehyde to each well, and fix the cells for 20 min.

[0066] (3) After washing three times with PBS, add 1 mL of 0.1% crystal violet to each well and stain for 20 min;

[0067] (4) Discard the staining solution, wash once with PBS, dry at room temperature, and take photos for analysis (Scan Marker i600, MICROTEK, Shanghai Zhongjing Technology Co., Ltd.).

[0068] Plaque results Figure 3 As shown, DiCre and Δarom There was almost no difference in the size and number of plaques between the strains, indicating that the absence TgAROM It does not affect the growth of Toxoplasma gondii under normal, standard culture conditions.

[0069] 2. Toxoplasma gondii Δarom In vivo toxicity gradient experiments in mice

[0070] (1) Using HFF cells to culture the parent in vitro DiCre Insect strains and Δarom When there are large parasite vesicles, discard the culture medium in the original culture bottle, add PBS to wash, remove the escaped parasites and residual culture medium, and add fresh DMEM diluent without FBS;

[0071] (2) Use a disposable cell scraper to scrape off the cells together with the parasites, and use a 5 mL syringe to repeatedly blow up the suspension 8-10 times to lyse the cells and allow the parasites to escape from the cells. Use a sterile 3 μm pore size filter membrane to filter and purify the parasites. After the parasites are diluted, they are counted using a cell counting plate (WATSON BIO LAB, 177-112C, Japan);

[0072] (3) Intraperitoneal inoculation of 7-week-old female ICR mice (purchased from Guangzhou Ruige Biotechnology Co., Ltd.), Δarom Tachyzoites were 10 3 , 10 4 , 10 5 , 10 6 insects / mice, press 10 at the same time 3 Tachyzoites / mouse inoculation DiCre The worm strain was used as the control group, with 6 mice in each group. The survival of the mice was observed and recorded every day for 30 days. During this period, orbital blood was collected from the surviving mice, and the indirect immunofluorescence test (IFA) was used to detect whether the mice were successfully infected. Uninfected mice were discarded, and the mortality rate was plotted into a Kaplan-Meier survival plot and analyzed using prism8 software;

[0073] The results are as follows Figure 4 As shown, each dose ΔaromAll mice infected with the worm strain survived within 30 days, with a survival rate of 100%. DiCre All mice infected with the parasite strain died within 9 days, with a survival rate of 0, indicating that Toxoplasma gondii was absent. TgAROM Almost non-toxic.

[0074] 3. Toxoplasma gondii Δarom In vivo insect load test

[0075] (1) DiCre and Δarom Tachyzoites were calculated based on 1×10 4 worms / mouse, intraperitoneally inoculated 7-week-old female ICR mice (purchased from Guangzhou Ruige Biotechnology Co., Ltd.), 5 mice per group;

[0076] (2) Five days after infection, the mice were euthanized and 5 mL of normal saline was injected intraperitoneally. The ascites was collected and centrifuged at 3500 r / min for 6 min at room temperature. The supernatant was discarded and the volume was fixed to 2 mL. 1 / 10 of the resuspension was taken to extract gDNA.

[0077] (3) 5 mL of normal saline was injected into the peritoneal cavity of blank mice, and then removed to form a negative control solution. 0, 10, and 10 2 , 10 3 , 10 4 , 10 5 , 10 6 Toxoplasma gondii parent DiCre Tachyzoites were added to 2 mL of negative control solution, mixed and 1 / 10 of the mixed solution was taken to extract gDNA for the preparation of the standard curve;

[0078] (4) Real-time quantitative PCR (qPCR) was used to detect the amount of worms in the mouse ascites. The detection primers were shown in Table 8. The qPCR reaction system was prepared in a 96-well plate as shown in Table 9. The reaction procedure was shown in Table 10.

[0079] Table 8 Primers used for qPCR

[0080]

[0081] Table 9 qPCR reaction system

[0082]

[0083] Table 10 qPCR reaction procedure

[0084]

[0085] The above samples were placed in LC480 (Roche LC480, Switzerland) for detection, and the amount of worms in the ascites of each mouse was calculated. The data results were presented in a bar graph.

[0086] The results are as follows Figure 5 As shown, vaccination Δarom The ascites load of the worm strain was significantly lower than that of the control group DiCre The number of mice carrying parasites was lower than the detection threshold, indicating that Toxoplasma gondii was absent. TgAROM Does not reproduce in the body.

[0087] 4. Toxoplasma gondii Δarom Experiment on the immune protection of insect strains on mice

[0088] (1) Δarom Tachyzoites were calculated based on 1×10 2 Five 7-week-old female ICR mice were intraperitoneally inoculated with a dose of tachyzoites / mouse in advance, and five blank mice were raised as controls. All groups of mice were raised under the same conditions for 30 days.

[0089] (2) 30 days after immunization, 1×10 2 indivual Δarom Tachyzoite mice (5) and unimmunized control ICR mice (5) were inoculated with 1×10 4 The death of mice was observed and recorded. After 30 days, the survival rate of mice was statistically analyzed using prism8 software.

[0090] The results are as follows Figure 6 As shown, unimmunized control mice were infected with 1×10 4 All ME49 strains died within 10 days, with a survival rate of 0, while 1×10 2 indivual Δarom The mice immunized with tachyzoites did not die within 30 days, and the survival rate was 100%, indicating that the vaccination Δarom The mice infected with the strain acquired resistance to the wild strain. Δarom The insect strain has the potential to become a genetically engineered vaccine.

[0091] In summary, through the Tg AROM gene was found to be directly knocked out TgAROM It has almost no effect on the growth of Toxoplasma gondii in vitro. Δarom The insect strain can be cultured normally in vitro, and animal experiments have found that Δarom The strain has almost no toxicity in vivo and has been found to have a strong immune protection against Toxoplasma infection. Δ aromTachyzoites of Toxoplasma gondii can be mixed with a medically acceptable vaccine adjuvant to produce a vaccine lacking Tg AROM gene-containing live attenuated Toxoplasma vaccine.

[0092] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A missing Tg AROM gene attenuated live vaccine of Toxoplasma gondii, characterized in that: Contains missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom , the missing Tg Toxoplasma gondii vaccine strains containing AROM genes Δarom The CRISPR / Cas9 gene editing technology was used to knock out the type Ⅰ parent Toxoplasma gondii DiCre of insect strain Tg AROM gene obtained, Tg The AROM gene is derived from the Toxoplasma genome database available at www.toxodb.org. Tg The gene ID corresponding to the AROM gene is TGGT1_307040.

2. The deletion according to claim 1 Tg AROM gene attenuated live vaccine of Toxoplasma gondii, characterized in that: It also contains a medically acceptable vaccine adjuvant.

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