Protective agent for toxoplasma attenuated vaccine, its preparation method and application

Abstract

The application discloses a protective agent for a Toxoplasma gondii attenuated vaccine, a preparation method and application thereof, and relates to the technical field of biotechnology.The protective agent comprises a basic buffer and a protective component; the protective component comprises fetal bovine serum, trypsin, sucrose and dimethyl sulfoxide.The protective agent for the Toxoplasma gondii attenuated vaccine provided by the application has excellent activity protection effect on Toxoplasma gondii, and can stably store Toxoplasma gondii tachyzoites for 18 days under the condition of 4 DEG C, which is much better than a gelatin protective preparation and a simple PBS system.Piglet immunization tests prove that after immunization of piglets with the Toxoplasma gondii attenuated strain stored for 18 days under the condition of 4 DEG C by the protective agent, the piglets can normally induce the body to produce mild immune stress such as fever; sustained antibody detection for 63 days shows that the IgG antibody level produced after addition of the protective agent continuously rises within four weeks, and the antibody is positive in the fourth week.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a protective agent for a Toxoplasma gondii attenuated vaccine, its preparation method, and its application. Background Technology

[0002] Toxoplasmosis is a disease caused by Toxoplasma gondii (… Toxoplasma gondii This zoonotic parasitic disease is caused by Toxoplasma gondii, an pathogen that can infect almost all warm-blooded animals, including humans. It has a global distribution and poses a persistent threat to public health and livestock production. The life cycle of Toxoplasma gondii is complex, involving both sexual reproduction in felines as the definitive host and asexual reproduction in intermediate hosts. It forms three main infectious morphologies: tachyzoites, bradyzoites within tissue cysts, and highly resistant sporulated oocysts in the environment. Human infection routes primarily include oral ingestion of water or food contaminated with oocysts, consumption of undercooked meat containing tissue cysts, and vertical transmission via the placenta.

[0003] Currently, there is no ideal method for the prevention and control of toxoplasmosis. Vaccination is the ideal technical approach for controlling toxoplasmosis. Research on toxoplasmosis vaccines has progressed through various explorations, including inactivated vaccines, live attenuated vaccines, subunit vaccines, and nucleic acid vaccines. However, each approach has its own problems, such as weak immunogenicity and poor safety. Therefore, in addition to optimizing the antigen, the development of toxoplasmosis vaccines requires the use of adjuvants to enhance vaccine immunogenicity and induce the production of immune-active molecules, regulate the strength and type of the immune response, and generate more effective and durable protective immunity.

[0004] Live attenuated vaccines can undergo limited proliferation within the host and mimic the natural infection process, simultaneously activating both cellular and humoral immune responses, resulting in comprehensive protection. Toxovax, a live attenuated vaccine, has been approved for use in sheep in some countries. ® It shows a certain preventive effect against congenital toxoplasmosis. Although attenuated vaccines prepared from the tachyzoite stage of Toxoplasma gondii can effectively control the spread and prevalence of toxoplasmosis, the short shelf life of attenuated vaccines at 4°C seriously affects their transportation, use, application, and promotion. Therefore, it is necessary to develop a protectant for attenuated Toxoplasma gondii vaccines to extend their shelf life. Summary of the Invention

[0005] The purpose of this invention is to provide a protectant for a Toxoplasma gondii attenuated live vaccine, its preparation method, and its application, in order to solve the problems existing in the prior art. This protectant can extend the shelf life of the Toxoplasma gondii attenuated live vaccine, thereby significantly reducing transportation and storage costs while preserving the vaccine's complete immunogenicity.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a protectant for a Toxoplasma gondii attenuated vaccine, the protectant comprising a basal buffer and protective components; the protective components comprising fetal bovine serum, trypsin, sucrose and dimethyl sulfoxide.

[0007] Furthermore, in the protective agent, the contents of the fetal bovine serum, the trypsin, the sucrose, and the dimethyl sulfoxide are 15-25 mL / L, 0.5-1.5 g / L, 5-15 g / L, and 5-15 mL / L, respectively.

[0008] Preferably, in the protective agent, the contents of the fetal bovine serum, the trypsin, the sucrose, and the dimethyl sulfoxide are 20 mL / L, 1 g / L, 10 g / L, and 10 mL / L, respectively.

[0009] Furthermore, the base buffer is PBS buffer.

[0010] The present invention also provides a method for preparing the above-mentioned protective agent, comprising the step of adding the fetal bovine serum, the trypsin, the sucrose and the dimethyl sulfoxide to the basal buffer and mixing them evenly to obtain the protective agent.

[0011] The present invention also provides the use of the above-mentioned protective agent in the preparation of a live attenuated Toxoplasma gondii vaccine.

[0012] Furthermore, the Toxoplasma gondii attenuated vaccine uses a Toxoplasma gondii RHΔ strain. gra47 Insect strain or Toxoplasma gondii PRUΔ gra72 Insect strains.

[0013] The present invention also provides a Toxoplasma gondii attenuated vaccine, comprising an attenuated Toxoplasma gondii strain and the aforementioned protective agent.

[0014] Furthermore, the attenuated strain of *Toxoplasma gondii* is *Toxoplasma gondii* RHΔ gra47 Insect strains.

[0015] Furthermore, the attenuated strain of *Toxoplasma gondii* is *PRUΔ*. gra72 Insect strains.

[0016] The present invention discloses the following technical effects: The Toxoplasma gondii attenuated vaccine protectant provided by this invention exhibits excellent protective effect against Toxoplasma gondii activity. It can stably preserve Toxoplasma gondii tachyzoites for up to 18 days at 4°C, far superior to gelatin-based protectants and the PBS system alone. Immunization experiments in piglets confirmed that the attenuated Toxoplasma gondii strain preserved at 4°C for 18 days using the protectant of this invention could induce mild immune stress such as fever in piglets after immunization. Antibody detection over 63 days showed that the IgG antibody level produced after adding the protectant continuously increased within four weeks, and the antibody was positive in the fourth week. The protectant provided by this invention is a compound of multiple components, which synergistically maintain the cell membrane structure of the parasite and reduce protein denaturation. It allows for long-term storage and transportation without a complex cold chain, significantly reducing transportation and storage costs while preserving the complete immunogenicity of the vaccine, and has good prospects for industrial application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Survival curves of mice infected with Toxoplasma gondii RH strain tachyzoites from different groups after 18 days of preservation; Figure 2 For RHΔ gra47 group RHΔ gra47+ Figure 1 shows the body temperature monitoring results of pigs in the protective agent group and the PBS group; Figure 3 For PRUΔ gra72 group 、PRUΔ gra72+ Figure 1 shows the body temperature monitoring results of pigs in the protective agent group and the PBS group; Figure 4 For RHΔ gra47 group RHΔ gra47+ Figure 1 shows the detection results of Toxoplasma gondii-specific antibody IgG levels in porcine serum from the protectant group and the PBS group. Figure 5 For PRUΔ gra72 group 、PRUΔ gra72+ The results of detecting Toxoplasma gondii-specific antibody IgG levels in porcine serum in the protectant group and the PBS group are shown in the figure. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The Toxoplasma gondii experimental materials involved in the following examples are as follows: Wild-type Toxoplasma gondii RH strain tachyzoites were provided by the Parasitic Diseases Laboratory of the College of Veterinary Medicine, Shanxi Agricultural University; Toxoplasma gondii strain RHΔ gra47, The invention has been published under the publication number "CN117731764A" and is entitled "Toxoplasma gondii attenuated vaccine strain RHΔ". gra47 The patent disclosure text for "and its applications" is disclosed; Toxoplasma gondii strain PRUΔ gra72, The invention has been published under the publication number "CN118846019A" and is entitled "Toxoplasma gondii PRUΔ". gra72 The patent publication text of "Attenuated live vaccine strain, its construction method and application" is disclosed.

[0025] The methods for culturing and counting Toxoplasma gondii tachyzoites are as follows: Tachyzoites were passaged in monolayers of cells. To maintain cell growth, the culture medium was replaced with DMEM infection medium containing 1% (v / v) fetal bovine serum before inoculation with Toxoplasma gondii tachyzoites. Tachyzoites proliferated continuously in the cells until they burst the host cells, releasing a large number of tachyzoites. Cells were scraped from the culture flask with a cell scraper, and the cells were repeatedly aspirated with a 27 G needle to fully release the tachyzoites. After centrifugation at 200×g for 5 min to remove host cell debris, the supernatant was filtered through a 5 μm pore membrane, centrifuged at 1500×g for 10 min, the supernatant was discarded, and the cells were resuspended in an appropriate infection medium and counted using a cell counting chamber.

[0026] Example 1 A method for preparing a protective agent for a Toxoplasma gondii attenuated vaccine: Fetal bovine serum, trypsin, sucrose, and dimethyl sulfoxide (DMSO) were added to PBS buffer and mixed thoroughly to obtain a final concentration of 20 mL / L for fetal bovine serum, 1 g / L for trypsin, 10 g / L for sucrose, and 10 mL / L for DMSO, thus preparing a protective agent for the attenuated Toxoplasma gondii vaccine.

[0027] Example 2 A method for preparing a protective agent for a Toxoplasma gondii attenuated vaccine: Fetal bovine serum, trypsin, sucrose, and DMSO were added to PBS buffer and mixed thoroughly to obtain a final concentration of 25 mL / L for fetal bovine serum, 0.5 g / L for trypsin, 15 g / L for sucrose, and 5 mL / L for DMSO, thus preparing a protective agent for the attenuated Toxoplasma gondii vaccine.

[0028] Example 3 A method for preparing a protective agent for a Toxoplasma gondii attenuated vaccine: Fetal bovine serum, trypsin, sucrose, and DMSO were added to PBS buffer and mixed thoroughly to obtain a final concentration of 15 mL / L for fetal bovine serum, 1.5 g / L for trypsin, 5 g / L for sucrose, and 15 mL / L for DMSO, thus preparing a protective agent for the attenuated Toxoplasma gondii vaccine.

[0029] Comparative Example 1 A method for preparing a gelatin formulation: Gelatin, lactalbumin hydrolysate, and sucrose are added to water to obtain a gelatin preparation with a final concentration of 15 g / L for gelatin, 1 g / L for lactalbumin hydrolysate, and 10 g / L for sucrose.

[0030] Comparative Example 2 Same as Example 1, except that trypsin is not added.

[0031] Example 1 of effect verification 1. Experimental Methods 1.1 Preservation of tachyzoites from Toxoplasma gondii RH strains In the first group, the tachyzoites of Toxoplasma gondii RH strain were suspended and added to the protective agent prepared in Example 1 to make the concentration of tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. The solution was then dispensed into 2 mL EP tubes and stored in a refrigerator at 4°C.

[0032] In the second group, the tachyzoites of Toxoplasma gondii RH strain were suspended and added to the protective agent prepared in Example 2 to make the concentration of tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. The solution was then dispensed into 2 mL EP tubes and stored in a refrigerator at 4°C.

[0033] In the third group, the tachyzoites of Toxoplasma gondii RH strain were suspended and added to the protective agent prepared in Example 3 to make the concentration of tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. The solution was then dispensed into 2 mL EP tubes and stored in a refrigerator at 4°C.

[0034] In the fourth group, the tachyzoites of Toxoplasma gondii RH strain were suspended and added to the gelatin preparation prepared in Comparative Example 1 to make the concentration of tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. The solution was then dispensed into 2 mL EP tubes and stored at 4°C.

[0035] In the fifth group, the tachyzoites of Toxoplasma gondii RH strain were suspended and added to the protective agent prepared in Comparative Example 2 to make the concentration of tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. The solution was then dispensed into 2 mL EP tubes and stored at 4°C.

[0036] The sixth group served as the control group. The tachyzoites of Toxoplasma gondii RH strain were suspended and added to PBS solution to make the concentration of Toxoplasma gondii RH strain tachyzoites 100 per 200 μL, thus obtaining the Toxoplasma gondii RH strain tachyzoite preservation solution. It was then dispensed into 2 mL EP tubes and stored at 4°C.

[0037] 1.2 Screening of Protective Agents From day 2 to day 21, six groups of Toxoplasma gondii RH strain tachyzoite preservation solutions, stored at 4°C, were inoculated into Kunming mice (200 μL intraperitoneally). The mice were observed twice a day for 30 consecutive days, and the mortality rate was recorded. The optimal vaccine protectant and effective storage time of the protectant were selected based on the specific mortality rate.

[0038] 2. Experimental Results After 18 days of preservation, groups 1, 2, and 3 were intraperitoneally injected with 6 Kunming mice, and all mice still died. Figure 1 Group 4: After 2 days of storage, 6 Kunming mice were intraperitoneally injected with the solution; all mice died. After 3 days or more of storage, mice were inoculated and none died. Group 5: After 8 days of storage, 6 Kunming mice were intraperitoneally injected with the solution; all mice died. After 8 days or more of storage, mice were inoculated and none died. Group 6: After 1 day of storage, 6 mice were inoculated; all mice died. After 2 days or more of storage, mice were inoculated and none died.

[0039] like Figure 1 As shown, after 18 days of storage, the mice in groups 1, 2, 3, 4, 5 and 6 were injected intraperitoneally into 6 Kunming mice. On the 9th day after inoculation, all 6 mice in groups 1, 2 and 3 died, while all mice in groups 4, 5 and 6 survived until the end of the experiment 30 days later.

[0040] Based on the above results, it is concluded that the first, second, and third groups of vaccine protectants have the best protective effect.

[0041] Example 2 of effect verification 1. Experimental Methods 1.1 Immunization of piglets with attenuated Toxoplasma gondii strains combined with protective agents Healthy piglets aged 50 days were randomly divided into 5 groups of 4 piglets each. The specific experimental groups and treatments for each group are shown in Table 1.

[0042] Table 1 Experimental Groups and Treatments 1.2 Post-immunization clinical body temperature monitoring After vaccination, the body temperature of pigs in each experimental group was measured and recorded at a fixed time every day for 14 consecutive days using rectal thermometry.

[0043] 1.3 Detection of anti-Toxoplasma gondii antibody levels in porcine serum Serum samples were collected from pigs at 7, 14, 21, 28, 35, 42, 49, 56, and 63 days after immunization as test samples. The levels of anti-Toxoplasma gondii specific antibodies were detected using an ELISA kit.

[0044] 2. Experimental Results 2.1 Results of monitoring clinical symptoms in pigs Compared with the PBS group, RHΔ gra47 + Protective agent group and PRUΔ gra72 Pigs in the +protectant group all exhibited decreased feed intake and lethargy, lasting for 1-2 days. Body temperature monitoring results for each group within 2 weeks post-immunization are shown below. Figures 2-3 .

[0045] 2.2 Results of detection of anti-Toxoplasma gondii antibody levels in porcine serum like Figure 4 As shown, RHΔ gra47 Group and RHΔ gra47 In the +protectant group, Toxoplasma gondii-specific antibody IgG in pig serum showed a continuous increase over four weeks, with antibody levels converging between the two groups, and the antibody becoming positive in the fourth week. In the PBS group, Toxoplasma gondii-specific antibody IgG remained at extremely low levels throughout the experiment.

[0046] like Figure 5 As shown, PRUΔ gra72 Group and PRUΔ gra72 In the protective agent group, the Toxoplasma gondii-specific antibody IgG in the serum of pigs continued to rise over four weeks, and the antibody levels in the two groups tended to be consistent, with the antibody being positive in the fourth week.

[0047] Based on the above experimental results, it can be seen that the protective agent of the present invention is effective against RHΔ gra47 and PRUΔ gra72 Preservation can enhance the immunization effect compared to RHΔ gra47 and PRUΔ gra72 The initial levels before saving were comparable.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A protective agent for a Toxoplasma gondii attenuated live vaccine, characterized in that, The protective agent comprises a base buffer and a protective component; the protective component comprises fetal bovine serum, trypsin, sucrose, and dimethyl sulfoxide.

2. The protective agent according to claim 1, characterized in that, In the protective agent, the contents of the fetal bovine serum, the trypsin, the sucrose, and the dimethyl sulfoxide are 15-25 mL / L, 0.5-1.5 g / L, 5-15 g / L, and 5-15 mL / L, respectively.

3. The protective agent according to claim 2, characterized in that, In the protective agent, the contents of the fetal bovine serum, the trypsin, the sucrose, and the dimethyl sulfoxide are 20 mL / L, 1 g / L, 10 g / L, and 10 mL / L, respectively.

4. The protective agent according to claim 1, characterized in that, The base buffer is PBS buffer.

5. A method for preparing a protective agent as described in any one of claims 1-4, characterized in that, The process includes the step of adding the fetal bovine serum, the trypsin, the sucrose, and the dimethyl sulfoxide to the basal buffer and mixing them thoroughly to obtain the protective agent.

6. The use of a protective agent as described in any one of claims 1-4 in the preparation of a Toxoplasma gondii attenuated vaccine.

7. The application according to claim 6, characterized in that, The Toxoplasma gondii attenuated vaccine used a Toxoplasma gondii RHΔ strain. gra47 Insect strain or Toxoplasma gondii PRUΔ gra72 Insect strains.

8. A live attenuated Toxoplasma gondii vaccine, characterized in that, Includes attenuated strains of Toxoplasma gondii and the protective agent as described in any one of claims 1-4.

9. The Toxoplasma gondii attenuated vaccine according to claim 8, characterized in that, The attenuated strain of Toxoplasma gondii was a Toxoplasma gondii RHΔ strain. gra47 Insect strains.

10. The Toxoplasma gondii attenuated vaccine according to claim 8, characterized in that, The attenuated strain of *Toxoplasma gondii* was *PRUΔ*. gra72 Insect strains.

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