CN103275182B - CD8+T cell dominant epitopes based on toxoplasmagondii bradyzoite antigens - Google Patents

Abstract

The present invention discloses CD8 ⁺ T cell dominant epitopes of two toxoplasma-specific surface antigens of bradyzoites: VPNSSLVEN and SQFLSLSLL, which are based on the protein sequences of bradyzoite-specific surface antigens SAG2C, -2D, -2X , 6 H-2-restricted, high-affinity CD8 ⁺ T cell epitopes were screened out. BALB/c mice were actively immunized with the screened epitope polypeptides. After immunization, the level of lymphocyte proliferation and cytokine content were measured, and the immune protection of epitope vaccine was evaluated. The results showed that the mice induced by the epitope vaccine could produce strong cellular immunity, and two of the antigenic peptides: VPNSSLVEN and SQFLSLSLL, proved to have a strong ability to stimulate T cell proliferation and induce mice to secrete protective cytokines And immunoprotection, it can be used as an effective epitope vaccine against Toxoplasma gondii infection, and can reduce the cyst formation rate in the host brain tissue.

Description

Predominant epitopes of CD8+ T cells based on bradyzoite antigen of Toxoplasma gondii

technical field

The present invention relates to CD8 ⁺ T cell dominant epitope based on toxoplasma gondii bradyzoite antigen.

Background technique

Toxoplasmagondii is a worldwide distributed parasitic protozoan, which widely parasitizes in the nucleated cells of human and animals, and can cause great clinical damage to human health and huge economic losses in animal husbandry. For the treatment of toxoplasmosis, no ideal control drug has been found so far. The epitope vaccine has become a research hotspot of Toxoplasma gondii vaccine in recent years because of its easy synthesis, simple development and safety. Antigenic epitopes are antigenic determinants on the surface of protein antigens, which are special chemical groups that determine antigen specificity. Antigens bind to corresponding antigen receptors on the surface of lymphocytes through antigenic epitopes, thereby activating lymphocytes and causing immune responses; antigens also use epitopes to specifically bind to corresponding antibodies or sensitized lymphocytes to exert immune effects. Toxoplasma gondii is an obligate intracellular parasite whose surface antigens are degraded into (8-12aa) long amino acid fragments that are presented to cells with MHC-class I molecules, MHC-class I molecules and peptides recognized by CD8 ⁺ T cells complex, which kills Toxoplasma-infected cells by stimulating CD8 ⁺ T cells to secrete IFN-γ. In this study, we screened 6 CD8 ⁺ T cell epitopes with high affinity in SAG2C, -2D, and SAG2X proteins, among which, two epitope peptides showed good immunogenicity , can stimulate splenocyte proliferation, induce mice to secrete protective cytokines, and produce effective immune protection.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention has screened out two dominant epitopes of CD8 ⁺ T cell antigens based on the Bradyzoite SAG2C, SAG2D and SAG2X antigens of Toxoplasma gondii: it has been proved to have a strong ability to stimulate T cell proliferation and immunity Protective and can be used as an epitope vaccine effective against Toxoplasma gondii infection.

The present invention is achieved through the following technical solutions:

A dominant epitope of the CD8 ⁺ T cell antigen based on the bradyzoite antigen of Toxoplasma gondii: it is an antigenic peptide whose amino acid sequence is VPNSSLVEN, as shown in SEQ ID NO.1.

Another dominant epitope of the CD8 ⁺ T cell antigen based on the bradyzoite antigen of Toxoplasma gondii is an antigenic peptide with the amino acid sequence of SQFLSLSLL, as shown in SEQ ID NO.4.

The inventors of the present application analyzed the amino acid sequence of the SAG2CDX protein of Toxoplasma gondii bradyzoite, and screened out 6 CD8 ⁺ T cell epitopes with H-2 restriction and high affinity (Table 1); After dissolving in PBS, mix and immunize BALB/c mice, and inject 100ul of the peptide mixture at the base of the tail of the mice, wherein the content of each peptide is 100ug. Immunization 2 times, 3 weeks apart. Four weeks after immunization, the spleen was taken under aseptic conditions, and the spleen cell proliferation level and cytokine content were measured, as well as the immunoprotective evaluation of the epitope vaccine. The results showed that the mice induced by the epitope vaccine could produce strong cellular immunity. Two of the antigenic peptides, VPNSSLVEN and SQFLSLSLL, showed good ability to stimulate T cell proliferation. These screened epitopes can be used as good candidate epitopes in the design of epitope vaccines to reduce the rate of cyst formation in the host brain tissue.

The CD8 ⁺ T cell antigen dominant epitope vaccine based on the toxoplasma gondii antigen of the present invention can be designed by adding conventional medical vaccine adjuvants during specific application.

Description of drawings

Figure 1: Cell proliferation assay induced by epitope peptide vaccine. Four weeks after immunization, splenocytes from the mice were harvested to assess the proliferative immune response of the splenocytes to the epitope vaccine. The results showed that the lymphocyte proliferative response was not significantly different in mice treated with PBS. Among the six epitope peptides, VPNSSLVEN and SQFLSLSLL showed high OD absorbance values, demonstrating a better ability to stimulate splenocytes to generate a strong lymphocyte response. the

Figure 2: The level of IFN-γ secreted by CD8 ⁺ T cells of immunized mice, wherein, A: PBS control group; B: epitope mixed immunization group. Staining of surface molecule CD8 and intracellular cytokine IFN-γ of splenocytes of immunized mice. B1 and B2 are the proportion of cells positive for IFN-γ staining. B2 and B4 are the proportion of cells positively stained for CD8 ⁺ . B2 is the ratio of IFN-γ and CD8 ⁺ double-stained positive cells.

Detailed ways

The present invention will be further described below in conjunction with embodiment. the

Example 1 Screening dominant epitopes, immunizing BALB/c mice

The SAG2C, SAG2D and SAG2X protein sequences were retrieved from the Toxoplasma gondii database (ToxoDB: http://toxodb.org/). Antigenic epitopes were screened using the (ANN, SMM) algorithm in the Immunological Epitope Database (IEDB) (http://www.iedb.org/). Six H-2 (Ld, Kd, Dd) high-affinity CD8 ⁺ T cell epitope peptides (Table 1) were screened out, and the specific sequences were shown in 1-6 in the sequence table, which were artificially synthesized. Dissolve the peptides in PBS and mix them to immunize BALB/c mice. Inject 100ul of the peptide mixture at the base of the tail of the mice, in which the content of each peptide is 100ug. Immunization 2 times, 3 weeks apart.

Example 2 Cell Proliferation Experiment of Immunized Mice

Four weeks after immunization, the spleen was taken from the immunized mice under aseptic conditions, the spleen cell suspension was prepared, stimulated by adding epitope peptides, and cultured at 37°C and 5% _CO2 for 48 hours, each 100 μl of cell culture medium was added with 10 μl of WST-8 dye, CCK-8 method for lymphocyte proliferation experiment, measure the absorbance value of each hole at 450nm. (See Figure 1 for the results).

The results showed that the two epitope peptides: VPNSSLVEN and SQFLSLSLL showed high absorbance values, which proved that they had a strong ability to stimulate the proliferation of splenocytes, and could be used as superior candidate epitopes for the design of epitope vaccines. the

Example 3 Determination of dominant epitope vaccines inducing mice to produce cytokines

After dissolving the polypeptide VPNSSLVEN and SQFLSLSLL in PBS to 10ug/ul, inject the immunized BALB/c mice into the tail of the mice, divide them into control group, single peptide immunization group and mixed immunization group, and the single peptide immunization group was injected with single peptide VPNSSLVEN100ug respectively Or SQFLSLSLL100ug, the mixed immunization group was injected with two kinds of single peptides at the same time, and the content of each peptide was 100ug. Immunization 2 times, 3 weeks apart. The preparation of the splenocyte suspension of immunized mice was as described in Example 2. After the splenocytes from mice were stimulated, the culture supernatants were collected at different times to measure the contents of IL-2 and IL-10 in cells. The results are shown in Table 2. A large amount of IL-2 was detected in the splenocyte culture supernatant of mice immunized with epitope vaccine. The IL-2 content in the culture supernatant of mouse splenocytes in the VPNSSLVEN immunized group was 141.2±10.2pg/ml; the IL-2 content in the culture supernatant of the mouse splenocytes in the SQFLSLSLL immunized group was 125.6±11.4pg/ml. It was higher than that of the control group (PBS: 57.4±3.5pg/ml) (P<0.05); while the content of IL-2 in the culture supernatant of mouse splenocytes in the epitope mixed immunization group was 221.1±12.7pg/ml, which was the same as significantly different from the control. However, the amount of IL-10 was not significantly different between the immunized group and the control group (P>0.05). It was revealed that the epitope vaccine mainly induced Th1 type cellular immune response in mice. the

Splenocyte suspension was added with FITC-labeled anti-CD8a monoclonal antibody and anti-IFN-γ-PE antibody, and the expressions of CD8 ⁺ and IFN-γ in spleen cells were analyzed by Beckman Coulter flow cytometer and CXP analysis software. The results of flow cytometry (Figure 2) showed that the splenocytes of mice in the epitope immunization group expressed IFN-γ in 28.2%, which was significantly higher than that in the PBS control group (17.4%), and the expression of IFN-γ increased significantly.

Example 4. Epitope vaccine immune protection study. Four weeks after the last immunization, the immunized mice were challenged. Mice were orally infected with 30 Toxoplasma gondii cysts suspended in 0.1 ml of PBS. Two months after the infection, all the mice were sacrificed, and the brain tissue of the mice was separated for physical grinding to prepare a homogenate of the brain tissue. The brain tissue homogenate was mixed evenly, and 10 microliters were taken and counted three times under a microscope to obtain the average value. The number of cysts in the mouse brain was the average value of 10 times. The challenge experiment of immunized mice showed that compared with the control group PBS group, the number of cysts detected in the brains of the mice immunized with the epitope vaccine was significantly reduced. (See Table 3 for the results). Compared with the PBS control group (1156±205), the number of cysts in the brain tissue of the mice in the VPNSSLVEN immunized group and SQFLSLSLL immunized group decreased to 492±45 and 510±29 respectively (P<0.05). It can significantly reduce the number of cysts formed in the mouse brain tissue to 380±34 (P<0.01). the

Schedule description

Table 1. Antigen epitopes predicted by bioinformatics methods. the

The SAG2C, SAG2D and SAG2X protein sequences were retrieved from the Toxoplasma gondii database (ToxoDB: http://toxodb.org/). Antigenic epitopes were screened using the (ANN, SMM) algorithm in the Immunological Epitope Database (IEDB) (http://www.iedb.org/). Six H-2 (Ld, Kd, Dd) high-affinity CD8 ⁺ T cell epitope peptides were screened out.

Table 1. Results of bioinformatics prediction of H-2-restricted CD8 ⁺ T cell epitopes

Table 2. Cytokines induced by epitope vaccines. Spleen cells were stimulated for 24 hours and 72 hours to collect cell supernatants to measure the concentration of IL-2 and IL-10, respectively. IL-2, IL-10 concentrations were determined using commercial ELISA kits according to the manufacturer's instructions. The IL-2 content in the culture supernatant of mouse splenocytes in the VPNSSLVEN immunized group was 141.2±10.2pg/ml; the IL-2 content in the culture supernatant of the mouse splenocytes in the SQFLSLSLL immunized group was 125.6±11.4pg/ml. It was higher than that of the control group (PBS: 57.4±3.5pg/ml) (P<0.05); while the content of IL-2 in the culture supernatant of mouse splenocytes in the epitope mixed immunization group was 221.1±12.7pg/ml, which was the same as significantly different from the control. However, the amount of IL-10 was not significantly different between the immunized group and the control group (P>0.05). It was revealed that the epitope vaccine mainly induced Th1 type cellular immune response in mice. the

Table 2. Determination of cytokines induced by epitope vaccines by ELISA

Table 3. Challenge experiment of immunized mice. Four weeks after the last immunization, the immunized mice were orally infected with 30 Toxoplasma gondii cysts of the PRU strain per mouse. After 8 weeks, the brain tissues of the mice were collected for cyst counting. Compared with the PBS control group (1156±205), the number of cysts in the brain tissue of the mice in the VPNSSLVEN immunized group and SQFLSLSLL immunized group decreased to 492±45 and 510±29 respectively (P<0.05). It can significantly reduce the number of cysts formed in the mouse brain tissue to 380±34 (P<0.01). the

table 3

*P<0.05, **P<0.01, compared with the control group. the

Claims (3)

1. The dominant epitope of the CD8 ⁺ T cell antigen based on the bradyzoite antigen of Toxoplasma gondii, characterized in that it is an antigenic peptide whose amino acid sequence is SQFLSLSLL. 2. The application of the dominant epitope of the CD8 ⁺ T cell antigen based on the toxoplasma bradyzoite antigen described in claim 1 in the preparation of an epitope vaccine against toxoplasma infection. 3. The application according to claim 2, characterized in that: during application, a medical vaccine adjuvant is added.