CN111265659A - Toxoplasma gondii nano-material subunit vaccine and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Toxoplasma gondii nanomaterial subunit vaccine and a preparation method and application thereof. The vaccine is to coat the Toxoplasma gondii recombinant protein PSMB1 with chitosan to form nanoparticles. The recombinant protein TgPSMB1 comes from the proteasome subunit 1 of Toxoplasma gondii, and its amino acid sequence is shown in SEQ ID NO. It was then coated with the nanomaterial chitosan into a new form of vaccine. After evaluating the immune protection efficacy of the vaccine, it is found that the vaccine can prolong the survival time of infected mice, indicating that it can provide partial immune protection, and the present invention can be used to prevent animals from being infected with toxoplasmosis.

Description

A kind of Toxoplasma gondii nanomaterial subunit vaccine and its preparation method and application

technical field

The invention relates to the technical field of biological veterinary drugs, and relates to a Toxoplasma gondii nanomaterial subunit vaccine and a preparation method and application thereof.

Background technique

It has been more than 100 years since Toxoplasma gondii was first discovered. As an obligate intracellular parasite, it can infect almost all warm-blooded vertebrates, including mammals, birds, poultry, livestock, and even humans. , can cause serious public health problems. About 20% of people in the world are infected with Toxoplasma gondii, which generally does not show clinical symptoms after infection, but may cause serious harm such as miscarriage, stillbirth and even congenital malformations of newborns to pregnant women. Toxoplasma gondii mainly infects pigs in livestock, and is characterized by high fever, dyspnea and reproductive failure. In recent years, the incidence of toxoplasmosis in pig farms has shown an upward trend, and the mortality rate has exceeded 50%, which has caused serious problems to the pig industry. huge economic loss. At present, although pyrimethamine combined with sulfadiazine is used to control the reproduction of Toxoplasma gondii, the drug has many side effects and poor tolerance. . Therefore, the prevention and control of the disease is still based on vaccine prevention as the most effective and important means. Currently, there is only one live attenuated veterinary vaccine "Toxovax" approved for the prevention of Toxoplasma gondii infection in sheep and goats. However, live attenuated vaccines may show virulence recovery and spread, and may even induce severe disease in some immunocompromised individuals, limiting their application. In recent years, studies on DNA vaccines of Toxoplasma gondii have shown that DNA vaccines can induce Th1 immune responses to a certain extent and improve the survival rate of mice, but no vaccines have been approved for clinical use. There are also concerns about the safety of DNA vaccines.

The recombinant protein-based subunit vaccine of the present invention can effectively avoid the biosafety risks caused by using pathogens to produce vaccines, and at the same time has the advantages of safe use, stable properties and low production costs. Chitosan (CS) nano-adjuvant can accelerate, prolong or enhance antigen-specific immune response, and is often combined with vaccines to form vaccine preparations. Nanoadjuvant refers to molecules with particle diameter < 1,000nm, which have good biocompatibility and unique physicochemical properties. Compared with traditional adjuvants, they have targeting, sustained release, safety and high efficiency. advantage. Chitosan is derived from the shells of marine arthropods such as shrimps and crabs. It has structural characteristics similar to glycosaminoglycans. It has strong hydrophilicity and can be used for the association and delivery of unstable macromolecular compounds. The aqueous solution of chitosan is positively charged, which enables it to combine with negatively charged polymers, macromolecules and polyanions in aqueous solution, making chitosan a promising drug carrier material. Therefore, the construction of a nanomaterial subunit vaccine of Toxoplasma gondii with good immune protection is of great value and significance for human health and the efficient development of animal husbandry.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Toxoplasma gondii nanomaterial subunit vaccine for the above-mentioned deficiencies of the prior art.

Another object of the present invention is to provide a preparation method of the Toxoplasma gondii nanomaterial subunit vaccine.

The object of the present invention can be realized through the following technical solutions:

Application of the recombinant protein TgPSMB1 shown in SEQ ID NO. 1 in the preparation of a vaccine for preventing Toxoplasma gondii infection.

A Toxoplasma gondii nanomaterial subunit vaccine, the Toxoplasma gondii nanomaterial subunit vaccine is made of a chitosan-coated recombinant protein TgPSMB1, which is derived from Toxoplasma gondii proteasome subunit 1 , and its amino acid sequence is shown in SEQ ID NO.1.

The Toxoplasma gondii nanomaterial subunit vaccine is preferably mainly prepared by the following methods:

(1) Preparation of recombinant protein TgPSMB1;

(2) Coat the recombinant protein TgPSMB1 with chitosan: take 2 mg/mL chitosan and add 2 mg/mL sodium tripolyphosphate dropwise to it under stirring, continue to stir for 15-20 min after the dropping is completed, keep In the stirring state, dropwise add the recombinant protein TgPSMB1 to be coated, continue to stir for 15-20min after the dropwise addition is completed, transfer the above-mentioned liquid to a centrifuge tube, perform ultrasonication at 4°C, and set the power of the ultrasonic crusher to 40-50W, Ultrasonic 4s～8s, interval 4s～5s, the total ultrasonic time is 3～5min, transfer the liquid in the centrifuge tube to the ultracentrifuge tube, centrifuge at 40,000r/min for 50min at 4℃, the liquid is divided into supernatant and jelly sample Precipitate, separate out the precipitation, resuspend the precipitation with deionized water, transfer the liquid to a vial after resuspending, and freeze-dry to obtain the Toxoplasma gondii nanomaterial subunit vaccine freeze-dried powder.

The preparation method of the Toxoplasma gondii nanomaterial subunit vaccine of the present invention comprises the following steps:

(1) Preparation of recombinant protein TgPSMB1;

(2) Coat the recombinant protein TgPSMB1 with chitosan: take 2 mg/mL chitosan and add 2 mg/mL sodium tripolyphosphate dropwise to it under stirring, continue to stir for 15-20 min after the dropping is completed, keep In the stirring state, dropwise add the recombinant protein TgPSMB1 to be coated, continue to stir for 15-20min after the dropwise addition is completed, transfer the above-mentioned liquid to a centrifuge tube, perform ultrasonication at 4°C, and set the power of the ultrasonic crusher to 40-50W, Ultrasonic 4s～8s, interval 4s～5s, total ultrasonic time is 3～5min, transfer the liquid in the centrifuge tube to the ultracentrifuge tube, centrifuge at 30,000～40,000r/min at 4℃ for 50min, the liquid is divided into supernatant and jelly Sample precipitation, separate out the precipitation, resuspend the precipitation with deionized water, transfer the liquid to a vial after resuspending, and freeze-dry to obtain the Toxoplasma gondii nanomaterial subunit vaccine freeze-dried powder.

The method of step (1) is preferably: extracting the total RNA of Toxoplasma gondii and reverse transcribing it into cDNA, using PCR primers shown in SEQ ID NO. The vector pET-32a is connected to form the recombinant expression plasmid pET-32a-PSMB1, and then the prepared plasmid is transferred into Escherichia coli BL21 (DE3) for expression, and the expressed recombinant protein PSMB1 is purified by His protein purification column to obtain the recombinant protein PSMB1.

Step (2) The specific method for preparing the nanomaterial subunit vaccine rTgPSMB1-CS is preferably: take 2 mg/mL chitosan solution and add 2 mg/mL sodium tripolyphosphate dropwise to it under stirring, and the stirring speed is 450～500r/ min, the volume ratio of chitosan solution and sodium tripolyphosphate is 4-6:1; after the dropwise addition is completed, continue stirring for 15-20min, drop 4-5mg of the target protein to be coated into it, and continue after the dropwise addition is completed. Stir for 15-20min, transfer the above-mentioned liquid to a centrifuge tube, and perform ultrasonication at 4°C. The power of the ultrasonic crusher is set to 40-50W, the ultrasonication is 4s-8s, the interval is 4s-5s, and the total ultrasonic time is 3-5min. Transfer the liquid in the centrifuge tube to an ultracentrifuge tube, centrifuge at 40,000 r/min for 50 min at 4°C, the liquid is divided into supernatant and jelly-like precipitate, separate the precipitate, and resuspend the precipitate with deionized water. The liquid is transferred to a vial, and freeze-dried to obtain the Toxoplasma gondii nanomaterial subunit vaccine freeze-dried powder.

In step (2), the rate of addition is more preferably 1 drop in 2s.

In step (2), the power of each ultrasonic break is 50W, the ultrasonic is 5s, and the interval is 5s.

The application of the Toxoplasma gondii nanomaterial-coated subunit vaccine of the present invention in the preparation of a medicament for preventing or treating toxoplasmosis infection in mice.

The present invention has the following advantages and effects:

(1) At present, there are few studies and reports on the preparation of Toxoplasma gondii subunit vaccines by using chitosan nanomaterials, and most of the vaccines currently have poor immunity to Toxoplasma gondii. The present invention fills in the chitosan Nanomaterials for Toxoplasma gondii subunit vaccine research. (2) Proteasome subunit 1, as a key enzyme in the protein metabolism of Toxoplasma gondii, plays an important role in the growth and development of Toxoplasma gondii. The protein is excreted from the body. After the PSMB1 recombinant protein and chitosan were coated with immunized mice, it was found that it could significantly reduce the occurrence of local inflammation and alleviate the suffering of animals. Compared with the control group, the survival time of the mice was prolonged (the total death time was extended from 11 days to 16 days), indicating that its immune protection effect against Toxoplasma gondii was enhanced. This indicates that the vaccine prepared by recombinant protein TgPSMB1 and chitosan nanomaterials can be used to prevent Toxoplasma gondii. (3) The present invention improves the embedding process of the reported nanomaterials, the embedding rate of the nanovaccine is significantly improved, and the particle size of the vaccine is 80-150 nm. (4) The Toxoplasma gondii nanomaterial subunit vaccine of the present invention can be used as a carrier for preparing a drug for preventing mammalian infection.

Description of drawings

Figure 1 SDS-PAGE analysis of purified rTgPSMB1 recombinant protein

M: Protein Mark (kDa); 1: Purified rTgPSMB1 recombinant protein

Figure 2 Western blot analysis of native PSMB1 protein and rTgPSMB1 protein

M: Protein Mark (kDa); 1: rTgPSMB1 protein recognized by artificially infected rat serum of Toxoplasma gondii; 2: rTgPSMB1 protein recognized by normal rat serum; 3: Toxoplasma gondii tachyzoite holozoite recognized by rTgPSMB1 immunized rat serum Soluble protein; 4: Normal rat serum recognizes the soluble protein of Toxoplasma gondii tachyzoites

Fig. 3 SEM results of nanomaterial subunit vaccine PSMB1-CS

Figure 4 Changes of antibody levels before and after immunization of mice with rTgPSMB1 and chitosan-coated rTgPSMB1

Figure 5 Changes of cytokines IFN-γ, IL-4, IL-10 and IL-17 before and after immunization of mice with rTgPSMB1 and chitosan-coated rTgPSMB1

Figure 6. Survival curve of mice immunized with rTgPSMB1 and chitosan-coated with rTgPSMB1 after intraperitoneal infection with 200 T. gondii tachyzoites

Figure 7 Changes of antibody levels before and after immunization of mice with nanomaterial vaccine

Figure 8 Changes of cytokines IFN-γ, IL-4, IL-10 and IL-17 before and after immunization of mice with nanomaterial vaccine

Figure 9 Survival curve of mice after intraperitoneal infection of 200 Toxoplasma gondii tachyzoites

Detailed ways

Basic Materials:

1. Toxoplasma gondii RH strain: The Toxoplasma gondii RH strain in our laboratory was stored in liquid nitrogen, and ascites was collected for rejuvenation after intraperitoneal infection of mice every 3 months.

2. Experimental animals: 18-22g SPF grade BABL/c mice and 200-220g SPF grade SD rats were purchased from the Experimental Animal Center of Yangzhou University, and were kept in the isolation barrier system from birth to the end of the experiment, and fed ad libitum and drinking water.

3. Plasmid vector: The plasmid containing pET-32a is stored in a -80℃ refrigerator in our laboratory.

4. Tool enzymes and reagents: EcoR I restriction endonuclease, Hind III restriction endonuclease, Ex Taq enzyme, and reverse transcription kit were purchased from TaKaRa Company; DAB chromogenic solution and protein molecular weight marker were purchased from Thermo Fisher Scientific Company ; Goat Anti-Mouse IgG(H+L)-HRP was purchased from abcam company; polyacrylamide, N,N'-methylenebisacryloyl and Coomassie brilliant blue were purchased from Shanghai Chemical Reagent Packing Factory; chitosan ( Molecular weight of 50,000-190,000 Da) and Freund's adjuvant were purchased from Sigma-Aldrich Company; sodium tripolyphosphate was purchased from Aladdin Company; plasmid mini kit, gel recovery kit and RNA extraction kit were purchased from Omega Company; DH5α competent cells, BL21(DE3) competent cells, and one-step homologous recombinase were purchased from Nanjing Novizan Company; PEG20,000 was purchased from Biyuntian Biotechnology Company; His-tagged protein purification column was purchased from from GE Company; other reagents are of domestic analytical grade.

5. Main equipment: gel imaging system (ChemiDocXRS+, Bio-Rad company); semi-dry transfer system (MiniTrans-Blot, American Bio-Rad company); protein electrophoresis system (Mini-PROTEAN, Bio-Rad company); air bath Shaker (THZ, Jiangsu Taicang Experimental Equipment Factory); Electrophoresis Apparatus (DYY-11B, Beijing Liuyi Instrument Factory); Refrigerated Ultracentrifuge (Beckman Coulter, USA); Vacuum Freeze Dryer (LABCONCO, USA); Scanning Electron microscope (SU8010, Hitachi).

Example 1 Preparation of Toxoplasma gondii nanomaterial subunit vaccine rTgPSMB1-CS

1. Preparation of genetically engineered bacteria

The total RNA of Toxoplasma gondii RH strain was extracted according to the instructions of the RNA extraction kit of Omega Company, and the extracted RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit of TaKaRa Company. .3 primers for PCR amplification. The PCR system is as follows:

The PCR procedure is as follows:

After the PCR product was electrophoresed on agarose gel at 110V for 30min, the amplified 768bp band was cut into the gel with reference to the instructions of the gel recovery kit of OMEGA company, and was used with reference to the EcoR I and Hind III restriction enzymes of TaKaRa company. According to the instructions, the empty vector pET-32a is double-enzyme digested, and the recovered PCR band is connected to the empty vector pET-32a after double-enzyme digestion according to the one-step homologous recombinase instruction of Novozem to form pET-32a. -PSMB1 recombinant plasmid. The recombinant plasmid pET-32a-PSMB1 was transformed into E.coli BL21(DE3) competent cells according to the instructions of the competent cells of Novozymes to obtain E. coli containing the recombinant expression plasmid of pET-32a-PSMB1.

2. Expression and purification of TgPSMB1 recombinant protein from Toxoplasma gondii and Western blot analysis

Expression and purification of Toxoplasma gondii recombinant protein TgPSMB1: Inoculate bacteria containing pET-32a-PSMB1 recombinant expression plasmid BL21 (DE3) into LB liquid medium at a volume ratio of 1:100, and place it on a shaker at 37°C and 200r/min , when the OD ₆₀₀ was 0.6, IPTG with a final concentration of 1 mmol/L was added to induce expression, and the culture was continued for 4 h in the above environment; the induced bacterial liquid was recovered, the supernatant was discarded by centrifugation, and the precipitate was dissolved in the supernatant Binding In the buffer, ultrasonically disrupted for 40min (ultrasonic power 35W, ultrasonic 3s, interval 2s); Purify PSMB1 recombinant protein according to the instructions of GE protein purification column; SDS-PAGE electrophoresis analysis of the collected protein samples was carried out. The results showed that the recombinant protein was purified by SDS-PAGE. The protein purification effect is good (Fig. 1); put the purified recombinant protein into a dialysis bag, and use PEG20,000 to concentrate and purify the obtained PSMB1 recombinant protein to saturation; then filter and sterilize with a 0.22 μm filter; refer to BCA of ThermoFisher Company The protein concentration was detected according to the instructions of the protein quantification kit; finally, the recombinant protein TgPSMB1 was diluted to 1 mg/mL with sterile PBS.

Western blot analysis: ① Preparation of serum from rTgPSMB1-immunized rats: 200 μg of the purified recombinant protein was mixed with an equal volume of Freund's adjuvant and emulsified, and the rats were subcutaneously injected at multiple points. Free and five free, each immunization is 2 weeks apart. One week after the five immunizations, blood was collected by orbital blood collection and serum was separated, and the antibody titer was detected by ELISA. ②Preparation of serum from rats artificially infected with Toxoplasma gondii: Take 200 tachyzoites of Toxoplasma gondii RH strain per normal rat by intraperitoneal immunization, and collect blood by orbital blood collection at the 4th week and separate serum. The parasite protein and recombinant protein were subjected to SDS-PAGE electrophoresis according to the method of Western blot. After the end, the gel was placed on the NC membrane for semi-dry transfer. After the transfer was completed, primary and secondary antibodies were added and DAB was added. Color solution and take pictures (Fig. 2). A specific band can be seen from Figure 2, but the control group does not appear, indicating that the recombinant protein has good antigenicity, and the rTgPSMB1 immunized rat serum can recognize the natural parasite protein.

3. Preparation of Nanomaterial Subunit Vaccine PSMB1-CS

Preparation of nanomaterial subunit vaccine PSMB1-CS: First, prepare 2 mg/mL chitosan (pH 5.0) as follows: take a small beaker and place it on a magnetic stirrer, put in a magnetic rotor and start the magnetic stirrer (can not be heated) , Measure 50 mL of 1% glacial acetic acid and add it to the beaker. A small amount of 0.1 g of chitosan was weighed into the beaker several times. This process should be slow, otherwise it is easy to agglomerate. After all the chitosan is dissolved, place the pH meter in the solution, and adjust the pH of the solution to 5.0 with 2mol/L NaOH; prepare 2mg/mL sodium tripolyphosphate as follows, take a small beaker and place it under magnetic stirring On the device, add about 8 mL of deionized water and stir, and add 0.02 g of sodium tripolyphosphate. After the sodium tripolyphosphate is dissolved, add deionized water to make up to 10 mL. Take 20 mL of 2 mg/mL chitosan (pH 5.0) in a small beaker, place the small beaker on a magnetic stirrer, turn on the magnetic stirrer and adjust the speed to 500 r/min. Use a pipette to add 2 mg/mL sodium tripolyphosphate dropwise to a total of 4 mL, and add 1 drop at a rate of 2 s. After the dropwise addition was completed, stirring was continued for 20 min. 4 mg of the target protein to be coated was added dropwise to a beaker placed on a magnetic stirrer. The drop rate of the target protein to be coated is 1 drop in 2 s. After the dropwise addition was completed, stirring was continued for 20 min. The liquid in the small beaker was transferred to a 50 mL centrifuge tube, and sonicated at 4° C. The power of the sonicator was set to 50 W, the sonication was performed for 4 s, the interval was 4 s, and the total sonication time was 3 min. Transfer the liquid in the 50mL centrifuge tube to the ultracentrifuge tube, and centrifuge at 40,000r/min for 50min at 4°C according to the instructions of the ultracentrifuge. The liquid is divided into supernatant and jelly-like precipitate. Care should be taken not to touch the precipitate during separation. The supernatant was stored at 4°C to determine the concentration of the target protein, and the coating rate was calculated according to the following formula: coating rate = (total amount of protein added - protein content in supernatant)/total amount of protein added × 100%. Resuspend the pellet with deionized water, transfer the liquid to a vial after resuspending, seal it with a sealing tape, and store it at -80°C for at least 2 hours. After completion, the frozen samples were placed in a freeze dryer for lyophilization (in a vacuum environment) for 24 hours, and the samples were taken out and flicked to form a freeze-dried powder. Encapsulated vaccines can be stored at 4°C.

A small amount of chitosan nanomaterial subunit vaccine freeze-dried powder was taken out for scanning electron microscope observation. The results showed that the encapsulation rate of the chitosan-coated recombinant protein subunit vaccine was 67.39%, and the particle size of the chitosan nano-subunit vaccine was about 80-150 nm (Fig. 3).

Example 2 Immunoprotective study of Toxoplasma gondii nanomaterial subunit vaccine 1

1. Experimental Design

All experimental protocols in the experimental research of this project comply with the relevant regulations on animal ethics and welfare of Nanjing Agricultural University, as well as the animal welfare protection regulations of the Jiangsu Provincial Department of Science and Technology.

SPF grade male BABL/c mice weighing 18-22 g were kept in an isolation barrier system from birth to the end of the experiment, free of Toxoplasma gondii, with free access to food and water. Mice were randomly divided into groups of 15, and the mice were immunized with PBS (CONTROL group), rTgPSMB1 (PSMB1 group) and chitosan-coated rTgPSMB1 (PSMB1-CS group) by subcutaneous injection at multiple points. Mouse 100 μL (Table 1). Orbital blood was collected from mice on days 0, 7 and 14 to determine immune-related cytokines IFN-γ, IL-4, IL-17, IL- ₁₀ and antibody titers IgGi, _IgG2a . In the 4th week, 10 mice in each group were selected, and 200 fresh tachyzoites of Toxoplasma gondii RH strain were injected intraperitoneally to draw the survival curve.

Table 1 Trial grouping and immunization schedule

2. Observation of immune protection effect

2.1 Antibody titer

On the 0th, 7th and 14th day of the experiment, the mouse serum was collected by orbital blood collection, and the serum levels of IgG ₁ and IgG _2a were determined. As shown in Figure 4, on day 7, the PSMB1 group (P<0.01) and PSMB1-CS group (P<0.001) produced significantly higher levels of IgG1 _than the CONTROL group, while on day 14, the PSMB1-CS group produced significantly higher levels of IgG1 than the CONTROL group. produced a higher level of IgG ₁ (P<0.05); on the 7th and 14th days, the PSMB1 group and PSMB1-CS group secreted and produced IgG _2a significantly higher than that of the CONTROL group (P<0.001), and at the 7th and 14th day On day 14, the PSMB1-CS group produced significantly higher IgG _2a than the PSMB1 group (P<0.01).

2.2 Levels of immune-related cytokines

On the 0th day, 7th day and 14th day of the experiment, mouse serum was collected by orbital blood collection, and the serum levels of IFN-γ, IL-4, IL-17 and IL-10 were determined. As shown in Figure 5, both the PSMB1 group and the PSMB1-CS group produced higher levels of IFN-γ (P<0.001), and on the 14th day, the PSMB1-CS group was higher than the PSMB1 group (P<0.001); Compared with the CONTROL group, the PSMB1 group (P<0.01) and the PSMB1-CS group (P<0.001) both produced higher levels of IL-4; while on day 14, the PSMB1-CS group produced IL-10 Significantly lower than that of PSMB1 group (P<0.05); and on the 14th day, compared with CONTROL group, PSMB1 group (P<0.05) and PSMB1-CS group (P<0.01) both produced higher levels of IL-17.

2.3 Insect attack test results

After intraperitoneal infection of 200 T. gondii tachyzoites, the CONTROL, PSMB1 and PSMB1-CS groups all died within 15 days, while the CONTROL group died within 11 days (Fig. 6). Although all the mice in the PSMB1 group and PSMB1-CS group died, compared with the CONTROL group, the mice in the PSMB1 group started to die for 1 day longer, the mice in the PSMB1-CS group started to die for 4 days, and all the mice in the PSMB1 group died. The time was prolonged by 3 days, and the death time of all mice in the PSMB1-CS group was prolonged by 4 days.

Example 3 Immunoprotective study of Toxoplasma gondii nanomaterial subunit vaccine 2

3. Experimental Design

All experimental protocols in the experimental research of this project comply with the relevant regulations on animal ethics and welfare of Nanjing Agricultural University, as well as the animal welfare protection regulations of the Jiangsu Provincial Department of Science and Technology.

SPF grade male BABL/c mice weighing 18-22 g were kept in an isolation barrier system from birth to the end of the experiment, free of Toxoplasma gondii, with free access to food and water. Mice were randomly divided into groups of 15, and pET-32a-tagged protein ( pET32a-CS group), chitosan-coated recombinant protein TgPSMB1 (PSMB1-CS group) and Freund's adjuvant-coated recombinant protein TgPSMB1 (PSMB1-CF group) were subcutaneously injected into the mice at multiple points, the dose of each small 100 μL of mice; booster immunization in the second week after the first immunization, the immunization method and dose are the same as the first immunization; blood was collected from the mouse orbit in the first week, the second week and the fourth week to measure the immune-related cytokines IFN-γ, IL -4, IL-17, IL-10 and antibody titers IgG ₁ , IgG _2a , 5 mice in each group; 10 mice in each group (non-blood-collecting mice) in the 4th week, intraperitoneal injection of fresh Toxoplasma gondii RH strain tachyzoite 200 / only to draw its survival curve. The grouping and immunization schedule are shown in Table 2.

Table 2 Trial grouping and immunization schedule

4. Observation of immune protection effect

2.1 Antibody titer

On the 7th day and the 14th day of the experiment, mouse serum was collected by orbital blood collection, and used to determine the content of IgG ₁ and IgG _2a in the serum. On day 7, the level of IgG ₁ in PSMB1-CF group was significantly higher than that in Control group and PBS-CS group (P<0.05); on day 14, the level of IgG ₁ in PSMB1-CS group was significantly higher than that in Control group (P<0.05). P<0.01) and PBS-CS group (P<0.05) (Fig. 7). However, higher levels of IgG _2a were detected in PSMB1-CS group and PSMB1-CF group, which were significantly higher than the other four groups (P<0.001), and the OD value of IgG _2a increased with time.

2.2 Levels of immune-related cytokines

On the 7th day and the 14th day of the experiment, mouse serum was collected by orbital blood collection, and the serum levels of IFN-γ, IL-4, IL-17 and IL-10 were determined. As shown in Figure 8, on the 7th and 14th days, the levels of IFN-γ produced by the PSMB1-CS group and PSMB1-CF group were significantly higher than those of the other four groups (P<0.001); On the 14th day and the 14th day, the level of IL-4 produced by the PSMB1-CS group was significantly higher than that of the Control group (P<0.01); while the IL-4 produced by the PSMB1-CF group was significantly higher than that of the Control group on the 14th day (P<0.01). P<0.001); on the 7th day, the PSMB1-CF group produced significantly higher IL-17 than the PSMB1-CS group (P<0.01), and on the 14th day it was significantly higher than the Control group (P<0.01). However, the IL-10 and IL-17 produced by mice in PSMB1-CS group had no significant changes before and after immunization compared with the control group.

2.3 Insect attack test results

After intraperitoneal infection of 200 T. gondii tachyzoites, the Control group, pET-32a group, PBS-CS group and pET32a-CS group all died within 12 days (Fig. 9). All the mice in the PSMB1-CF group died on the 15th day, and the onset of death was 2 days longer than that in the Control group; although all the mice in the PSMB1-CS group died, compared with the Control group, the onset of death was 3 days longer. The time of death was extended by 6 days.

3. Analysis of immune protection effect

Humoral immunity plays an important role in the infection of Toxoplasma gondii. Specific antibodies can inhibit the binding of the parasite to the host cell surface and promote macrophages to kill the intracellular Toxoplasma gondii. In this study, mice in PSMB1-CS group and PSMB1-CF group could produce higher levels of IgG _2a , and the level of antibodies produced in mice increased with time. IgG ₁ was mediated by Th2 cells Humoral immunity was generated. Compared with the PBS-CS group, a higher level of IgG ₁ was detected in the PSMB1-CS group, indicating that the chitosan-coated recombinant protein PSMB1 could induce the mice to produce persistent Th1-predominant Humoral immunity also provokes a Th2-type cellular immune response. Cytokines play an important role in the activation of helper T cells. As an effector molecule of Th1-type cellular immunity, IFN-γ can activate macrophages to produce NO to kill parasites. Therefore, IFN-γ plays an important role in the process of Toxoplasma gondii infection, and IL-4 is produced by Th2 cells. It can also enhance the antigen-presenting ability of B cells while promoting the activation of Th2 cells. IL-10 is mainly produced by iTreg cells and has the functions of inhibiting cellular immunity and mediating humoral immunity. IL-17 is produced by Th17 cells and plays an important role in host defense against intracellular infection and is also involved in resistance to Toxoplasma gondii infection. Compared to the four control groups, PSMB1-CS and PSMB1-CF mice produced IFN-γ and IL-4, but PSMB1-CS produced less IL-17 compared to PSMB1-CF. , which indicates that nanomaterials and Freund's adjuvant-coated recombinant protein TgPSMB1 can activate helper T cells and enhance immunity. Currently, Freund's adjuvant, as a traditional type of adjuvant, has not been widely used because of its side effects such as redness, swelling and induration at the injection site, and because of its high viscosity and inconvenient use. It has been reported that long-term use of Freund's adjuvant increases the incidence of encephalopathy, so it is particularly important to find alternatives to Freund's adjuvant.

To evaluate the immunoprotective efficacy of the vaccine, mice were acutely intraperitoneally infected with 200 T. gondii tachyzoites. Compared with other control group mice, the survival time of PSMB1-CS group and PSMB1-CF group was significantly prolonged, which indicated that the vaccine could provide partial immune protection; compared with PSMB1-CF group, PSMB1-CS group mice Start to die later and survive longer. This indicates that the recombinant protein TgPSMB1 vaccine coated with chitosan nanomaterials has fewer side effects than traditional Freund's adjuvant, and can induce strong humoral and cellular immune responses in the body, and can prolong acute infection of Toxoplasma gondii. survival time of mice.

sequence listing

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<212> DNA

<213> Artificial Sequence

<400> 3

ctcgagtgcg gccgcaagct tttagtcagc gcggaggtcc a 41

Claims (9)

1. Application of a recombinant protein TgPSMB1 shown in SEQ ID NO.1 in preparation of a vaccine for preventing Toxoplasma gondii infection.
2. 2. A Toxoplasma gondii nanomaterial subunit vaccine, characterized in that: the Toxoplasma gondii nano-material subunit vaccine is prepared from chitosan-coated recombinant protein TgPSMB1, the recombinant protein is derived from Toxoplasma gondii proteasome subunit 1, and the amino acid sequence of the recombinant protein is shown in SEQ ID No. 1.
3. 3. The toxoplasma gondii nanomaterial subunit vaccine of claim 2, wherein: the preparation method mainly comprises the following steps:

   (1) preparing recombinant protein rTgPSMB 1;

   (2) coating the recombinant protein TgPSMB1 by using chitosan: dropwise adding 2mg/mL of sodium tripolyphosphate into 2mg/mL of chitosan under a stirring state, continuously stirring for 15-20min after dropwise adding is finished, keeping the stirring state, dropwise adding the recombinant protein TgPSMB1 to be coated, continuously stirring for 15-20min after dropwise adding is finished, transferring the liquid into a centrifuge tube, carrying out ultrasonic crushing at 4 ℃, setting the power of an ultrasonic crusher to be 40-50W, carrying out ultrasonic treatment for 4-8 s at an interval of 4-5 s, wherein the total ultrasonic treatment time is 3-5 min, transferring the liquid in the centrifuge tube into an ultracentrifuge tube, centrifuging for 50min at 4 ℃ at 40,000r/min, separating the liquid into supernatant and jelly-like precipitates, separating out the precipitates, carrying out heavy suspension precipitation by using deionized water, transferring the liquid into a penicillin bottle after heavy suspension is finished, and carrying out freeze drying to obtain the Toxoplasma gondii nano-material subunit vaccine freeze-dried powder.
4. 4. The method for preparing a nanomaterial subunit vaccine against Toxoplasma gondii as claimed in claim 2, comprising the steps of:

   (1) preparing a recombinant protein TgPSMB 1;

   (2) coating the recombinant protein TgPSMB1 by using chitosan: dropwise adding 2mg/mL of sodium tripolyphosphate into 2mg/mL of chitosan under a stirring state, continuously stirring for 15-20min after dropwise adding, keeping the stirring state, dropwise adding the recombinant protein TgPSMB1 to be coated, continuously stirring for 15-20min after dropwise adding, transferring the liquid into a centrifuge tube, carrying out ultrasonic crushing at 4 ℃, setting the power of an ultrasonic crusher to be 40-50W, carrying out ultrasonic treatment for 4-8 s, carrying out interval of 4-5 s, carrying out total ultrasonic treatment for 3-5 min, transferring the liquid in the centrifuge tube into an ultracentrifuge tube, centrifuging for 50min at 4 ℃ at 30,000-40,000 r/min, separating the liquid into a supernatant and a jelly-like precipitate, separating out the precipitate, carrying out heavy suspension precipitation by using deionized water, transferring the liquid into a penicillin bottle after heavy suspension is completed, and carrying out freeze drying to obtain the toxoplasma gondii nanomaterial freeze-dried powder.
5. 5. The method according to claim 4, wherein the method of step (1) is: extracting total RNA of Toxoplasma gondii and carrying out reverse transcription to obtain cDNA, carrying out amplification by using PCR primers shown in SEQ ID NO.2 and SEQ ID NO.3, recovering an amplification product, connecting the amplification product with an empty vector pET-32a to form a recombinant expression plasmid pET-32a-PSMB1, transferring the prepared plasmid into escherichia coli BL21(DE3) for expression, and purifying the expressed recombinant protein TgPSMB1 by a His protein purification column to obtain the recombinant protein TgPSMB 1.
6. 6. The preparation method according to claim 4, wherein the nanomaterial subunit vaccine PSMB1-CS prepared in the step (2) is prepared by the following specific method: dropwise adding 2mg/mL of sodium tripolyphosphate into 2mg/mL of chitosan solution under the stirring state, wherein the stirring speed is 450-500 r/min, and the volume ratio of the chitosan solution to the sodium tripolyphosphate is 4-6: 1; and after the dripping is finished, continuously stirring for 15-20min, dripping 4-5mg of target protein to be coated into the mixture, after the dripping is finished, continuously stirring for 15-20min, transferring the liquid into a centrifuge tube, carrying out ultrasonic crushing at 4 ℃, setting the power of an ultrasonic crusher to be 40-50W, carrying out ultrasonic treatment for 4-8 s at intervals of 4-5 s, keeping the total ultrasonic treatment time to be 3-5 min, transferring the liquid in the centrifuge tube into an ultracentrifuge tube, centrifuging for 50min at 4 ℃ at 40,000r/min, separating the liquid into supernatant and jelly-like precipitate, separating out the precipitate, carrying out heavy suspension precipitation by using deionized water, transferring the liquid into a penicillin bottle after the heavy suspension is finished, and carrying out freeze drying to obtain the toxoplasma gondii nanomaterial lyophilized powder.
7. 7. The production method according to claim 2, wherein the dropping speed in the step (2) is 1 drop for 2 seconds.
8. 8. The method according to claim 2, wherein the power per sonication is 50W, 5s sonication, and 5s separation.
9. 9. The toxoplasma gondii nanomaterial-coated subunit vaccine of claim 2, for use in the preparation of a medicament for preventing or treating toxoplasmosis infection in animals.

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