A Toxoplasma gondii nucleic acid vaccine and its construction method

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a Toxoplasma gondii nucleic acid vaccine and a method for constructing the same. The present invention inserts two cDNA fragments SAG2 and SRS2 on the Toxoplasma RH strain into the eukaryotic vector pBudCE4.1 separately or together to obtain a recombinant plasmid as a Toxoplasma gondii nucleic acid vaccine. The present invention uses the Toxoplasma gondii nucleic acid vaccine to actively immunize 6-8 week old Kunming mice three times, each time at an interval of two weeks, and evaluates the immunogenicity of the vaccine by measuring the cellular and humoral immune indicators of the immunized mice, and evaluates the immune protection of the vaccine by an in vivo attack experiment. The present invention can effectively enhance the humoral immunity and cellular immune response of experimental animals, and effectively reduce the number of deaths of experimental animals. The Toxoplasma gondii nucleic acid vaccine of the present invention can be used as an effective candidate vaccine for preventing Toxoplasma infection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Toxoplasma gondii nucleic acid vaccine and a construction method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The traditional treatment for toxoplasmosis is a combination of sulfadiazine sodium and pyrimethamine. However, drug treatment has serious side effects, such as high toxicity. Because vaccines are non-toxic, easy to manufacture and apply on a large scale, they are considered to be an important means to replace drug treatment. Nucleic acid vaccines are to directly introduce exogenous genes (DNA or RNA) encoding a certain antigen protein into animal cells, and synthesize antigen proteins through the expression system of host cells, inducing the host to produce an immune response to the antigen protein to achieve the purpose of preventing and treating the disease. Compared with traditional inactivated vaccines, subunit vaccines and genetically engineered vaccines, nucleic acid vaccines have the following advantages: enhanced immune protection; simple preparation, time-saving and labor-saving; cross-protection between the same species and strains; safer application; long-lasting immune response; convenient storage and transportation. Although there are many types of toxoplasmosis vaccines, there is currently no commercial vaccine used in clinical practice. For this reason, the development of an efficient, cheap and safe vaccine is an urgent need for the prevention and control of toxoplasmosis. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a Toxoplasma gondii nucleic acid vaccine and a method for constructing the same. The present invention inserts two cDNA fragments SAG2 and SRS2 on the Toxoplasma RH strain into the eukaryotic vector pBudCE4.1 separately or together to obtain a recombinant plasmid as a Toxoplasma gondii nucleic acid vaccine. The present invention uses the Toxoplasma gondii nucleic acid vaccine to actively immunize 6-8 week old Kunming mice three times, each time at an interval of two weeks, and evaluates the immunogenicity of the vaccine by measuring the cellular and humoral immune indicators of the immunized mice, and evaluates the immune protection of the vaccine by an in vivo attack experiment. The present invention can effectively enhance the humoral immunity and cellular immune response of experimental animals, and effectively reduce the number of deaths of experimental animals. Therefore, the Toxoplasma gondii nucleic acid vaccine of the present invention can be used as an effective candidate vaccine for preventing Toxoplasma infection.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0006] In the first aspect, the present invention provides a Toxoplasma gondii nucleic acid vaccine, comprising a recombinant plasmid consisting of a gene fragment obtained by polymerase chain reaction amplification using the cDNA of Toxoplasma RH as a template and a vector; the gene fragment is one or more of SAG2 and SRS2, the nucleotide sequence of SAG2 is shown in GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown in GenBank: AF012276.1.

[0007] In a second aspect, the present invention provides a method for constructing a Toxoplasma gondii nucleic acid vaccine, comprising the following steps:

[0008] Infect HFF cells, collect Toxoplasma RH after the cells are completely ruptured, extract parasite RNA using Trizol method, and use Toxoplasma RH RNA as template to obtain cDNA using reverse transcription kit;

[0009] The SAG2 fragment was amplified using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SRS2 fragment was amplified using primers SRS2-F-SalI and SRS2--R-XbaI. The SAG2 fragment and the SRS2 fragment were inserted into a eukaryotic vector separately or together to obtain a recombinant plasmid. The plasmid was extracted and purified to obtain a Toxoplasma gondii nucleic acid vaccine.

[0010] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0011] (1) After the recombinant plasmid constructed by the present invention is transfected into cells in vitro, the protein is successfully expressed.

[0012] (2) The present invention uses the Toxoplasma gondii nucleic acid vaccine to actively immunize Kunming mice, and the immunized mice serum has a higher antibody titer.

[0013] (3) The survival rate of mice immunized with Toxoplasma gondii nucleic acid vaccine reached 30%. The immune protection effect of the vaccine is basically equivalent to that of sulfonamide drugs, and it is a potential alternative to drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] Figure 1 A is a schematic diagram of the construction of the pSAG2 plasmid in Example 1, B is a schematic diagram of the construction of the pSRS2 plasmid in Example 2, C is a schematic diagram of the construction of the pSAG2+SRS2 plasmid in Example 3, and D is the in vitro protein detection result after eukaryotic plasmid transfection;

[0016] Figure 2A is the construction of SAG2 prokaryotic expression vector, protein expression and purification, B is the construction of SRS2 prokaryotic expression vector, protein expression and purification, C is the immunoblotting verification of purified proteins, and D is the localization analysis of SAG2 and SRS2;

[0017] Figure 3 This is the result of the spleen lymphocyte proliferation experiment of immunized mice;

[0018] Figure 4 To measure the cytokine levels of spleen lymphocytes of immune mice after in vitro stimulation;

[0019] Figure 5 The results of antibody detection in immunized mice;

[0020] Figure 6 This is the survival curve of mice in the in vivo parasite attack experiment in immunized mice. DETAILED DESCRIPTION

[0021] The first typical embodiment of the present invention is a Toxoplasma gondii nucleic acid vaccine, comprising a gene fragment obtained by polymerase chain reaction amplification using Toxoplasma RH cDNA as a template and a recombinant plasmid consisting of a vector;

[0022] The gene fragment is one or more of SAG2 and SRS2. The nucleotide sequence of SAG2 is shown in GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown in GenBank: AF012276.1.

[0023] In one or more examples of this embodiment, the eukaryotic vector is pBudCE4.1.

[0024] A second typical embodiment of the present invention is a method for constructing a Toxoplasma gondii nucleic acid vaccine, comprising the following steps:

[0025] Infect HFF cells, collect Toxoplasma RH after the cells are completely ruptured, extract parasite RNA using Trizol method, and use Toxoplasma RH RNA as template to obtain cDNA using reverse transcription kit;

[0026] The SAG2 fragment was amplified using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SRS2 fragment was amplified using primers SRS2-F-SalI and SRS2--R-XbaI. The SAG2 fragment and the SRS2 fragment were inserted into a eukaryotic vector separately or together to obtain a recombinant plasmid. The plasmid was extracted and purified to obtain a Toxoplasma gondii nucleic acid vaccine.

[0027] In one or more examples of this embodiment, HFF cells are infected at an MOI of 5.

[0028] In one or more examples of this embodiment, the eukaryotic vector is pBudCE4.1.

[0029] In one or more examples of this embodiment, the nucleotide sequence of FLAG-SAG2-F-NotI is shown in Seq ID No.1, and the nucleotide sequence of SAG2-R-kPNI is shown in Seq ID No.2.

[0030] In one or more examples of this embodiment, the nucleotide sequence of SRS2-F-SalI is shown in Seq ID No.3, and the nucleotide sequence of SRS2--R-XbaI is shown in Seq ID No.4.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0032] The primers used in the present invention are shown in Table 1.

[0033] Table 1 Primers used in the present invention and their sequences

[0034]

[0035]

[0036] Example 1

[0037] HFF cells were infected with MOI=5, and Toxoplasma RH was collected after the cells were completely ruptured. The parasite RNA was extracted using the Trizol method, and cDNA was obtained using a reverse transcription kit using the RNA of Toxoplasma RH as a template;

[0038] The SAG2 fragment was amplified using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SAG2 fragment was inserted into the eukaryotic vector pBudCE4.1 to obtain the recombinant plasmid pSAG2. The plasmid was extracted and purified to obtain the Toxoplasma gondii nucleic acid vaccine. The schematic diagram of the pSAG2 plasmid construction is shown in the figure. Figure 1 As shown in A.

[0039] Example 2

[0040] HFF cells were infected with MOI=5, and Toxoplasma RH was collected after the cells were completely ruptured. The parasite RNA was extracted using the Trizol method, and cDNA was obtained using a reverse transcription kit using the RNA of Toxoplasma RH as a template;

[0041] The SRS2 fragment was amplified using primers SRS2-F-SalI and SRS2--R-XbaI, and the SRS2 fragment was inserted into the eukaryotic vector pBudCE4.1 to obtain the recombinant plasmid pSRS2. The plasmid was extracted and purified to obtain the Toxoplasma gondii nucleic acid vaccine. The schematic diagram of the pSRS2 plasmid construction is shown in the figure. Figure 1 As shown in B.

[0042] Example 3

[0043] HFF cells were infected with MOI=5, and Toxoplasma RH was collected after the cells were completely ruptured. The parasite RNA was extracted using the Trizol method, and cDNA was obtained using a reverse transcription kit using the RNA of Toxoplasma RH as a template;

[0044] The SAG2 fragment was amplified with primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SRS2 fragment was amplified with primers SRS2-F-SalI and SRS2--R-XbaI. The SAG2 fragment and the SRS2 fragment were co-inserted into the eukaryotic vector pBudCE4.1 to obtain the recombinant plasmid pSAG2+SRS2. The plasmid was extracted and purified to obtain the Toxoplasma gondii nucleic acid vaccine. The schematic diagram of the construction of the pSAG2+SRS2 plasmid is shown in Figure 1 As shown in C.

[0045] Experimental example

[0046] The recombinant plasmids of Examples 1-3 were transfected into 293T cells using liposome Lipofectamine 2000. Figure 1 As shown in D, protein electrophoresis confirmed that the expressed protein was consistent with the expected value.

[0047] like Figure 2 As shown in A, the SAG2 fragment was amplified using primers HA-SAG2-F and SAG2-R, the Pet32a backbone was amplified using primers Pet32a-F and Pet32a-R, and the Pet32a-SAG2 prokaryotic expression vector was constructed using homologous recombination. The nucleotide sequence of HA-SAG2-F is shown in Seq ID No.5, and the nucleotide sequence of SAG2-R is shown in Seq ID No.6. The nucleotide sequence of Pet32a-F is shown in Seq ID No.7, and the nucleotide sequence of Pet32a-R is shown in Seq ID No.8. Figure 2As shown in Figure 2, primers SRS2-F and SRS2-R were used to amplify the SRS2 fragment, and primers Pet32a-F and Pet32a-R were used to amplify the Pet32a backbone. The Pet32a-SRS2 prokaryotic expression vector was constructed by homologous recombination. The nucleotide sequence of SRS2-F is shown in Seq ID No.9, and the nucleotide sequence of SRS2-R is shown in Seq ID No.10. Escherichia coli was induced to express and purified, and the purified protein was verified by Western blot, as shown in Figure 2. Figure 2 As shown in C, the size of the expressed protein was in line with expectations. The purified protein was used to immunize mice, and mouse serum was collected after four immunizations. The indirect immunofluorescence method was used to analyze the localization of SAG2 and SRS2 proteins, as shown in Figure 2 As shown in D, both proteins were found to be located on the surface of the insect body.

[0048] Kunming mice were randomly divided into 4 groups, each with 16 mice. They were respectively Example 1 (pSAG2) immunization group, Example 2 (pSRS2) immunization group, Example 3 (pSAG2+SRS2) immunization group, and control (pBudCE4.1) group. Each mouse was injected with 100 μg of recombinant plasmid each time, and immunized three times in total. After the initial immunization, booster immunization was performed 2 weeks later and 4 weeks later. Blood was collected for antibody detection two weeks after the last immunization.

[0049] Two weeks after the last immunization, the spleen of the immunized mouse was aseptically removed and ground with 200-mesh gauze to prepare a spleen single-cell suspension. After removing the red blood cells from the spleen cells using red blood cell lysis buffer, the cells were resuspended in 1640 medium containing 10% FCS, 100 U / ml penicillin and 100 U / ml streptomycin, and the concentration of the cell suspension was adjusted to 2×10 5 After 72 hours of stimulation with PBS and recombinant protein (CA), lymphocyte proliferation assay was performed using CCK8 kit. Figure 3 As shown, the spleen lymphocytes in Example 2 and Example 3 groups increased significantly.

[0050] Two weeks after the last immunization, the spleen of the immunized mouse was aseptically removed and ground with 200-mesh gauze to prepare a spleen single-cell suspension. After removing the red blood cells from the spleen cells using red blood cell lysis buffer, the cells were resuspended in 1640 medium containing 10% FCS, 100 U / ml penicillin and 100 U / ml streptomycin, and the concentration of the cell suspension was adjusted to 2×10 5 Cells / well were stimulated with PBS and recombinant protein (CA), respectively. After 24 hours of stimulation, the cell supernatant was collected and the concentration of interleukin-4 (IL-4) was measured. After 96 hours of stimulation, the supernatant was collected and the concentration of interferon-γ (IFN-γ) was measured. Figure 4As shown, the IFN-γ level of the Example 3 group increased, and the IL-4 level of the Example 2 group increased.

[0051] On the 14th day after the third immunization, blood was collected from the mice by the inner canthus method. The blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 3000 rpm for 10 minutes to collect serum. The total anti-Toxoplasma immunoglobulin (IgG) content in the serum was determined by enzyme-linked immunosorbent assay. Figure 5 After three immunizations, the serum antibody titers of mice in Example 1-3 groups increased significantly compared with the control group (P<0.0001).

[0052] Two weeks after the last immunization, the immunized mice were subjected to an insect attack experiment. The mice were infected with 100 RH worms through the abdominal cavity. The mortality of the mice was recorded. Figure 6 As shown, the attack experiment of immunized mice showed that the survival time of mice in Example 1-3 groups was significantly prolonged compared with the control group. In particular, the survival rate of mice in Example 3 group reached 30%.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Toxoplasma gondii nucleic acid vaccine, characterized in that: It includes a recombinant plasmid consisting of a gene fragment obtained by polymerase chain reaction amplification using the cDNA of Toxoplasma RH as a template and a eukaryotic vector; The gene fragments are SAG2 and SRS2, the nucleotide sequence of SAG2 is shown in GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown in GenBank: AF012276.1; The eukaryotic vector is pBudCE4.1; The method for constructing the Toxoplasma gondii nucleic acid vaccine comprises the following steps: HFF cells were infected with MOI=5, and Toxoplasma RH was collected after the cells were completely ruptured. The parasite RNA was extracted using the Trizol method, and cDNA was obtained using a reverse transcription kit using the RNA of Toxoplasma RH as a template; The SAG2 fragment was amplified using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SRS2 fragment was amplified using primers SRS2-F-SalI and SRS2--R-XbaI. The SAG2 fragment and the SRS2 fragment were inserted into a eukaryotic vector separately or together to obtain a recombinant plasmid. The plasmid was extracted and purified to obtain a Toxoplasma gondii nucleic acid vaccine.

2. A method for constructing a Toxoplasma gondii nucleic acid vaccine, characterized in that: The following steps are involved: Infect HFF cells, collect Toxoplasma RH after the cells are completely ruptured, extract parasite RNA using Trizol method, and use Toxoplasma RH RNA as template to obtain cDNA using reverse transcription kit; The SAG2 fragment is amplified using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, the SRS2 fragment is amplified using primers SRS2-F-SalI and SRS2--R-XbaI, the SAG2 fragment and the SRS2 fragment are inserted into a eukaryotic vector separately or together to obtain a recombinant plasmid, the plasmid is extracted and purified, and a Toxoplasma gondii nucleic acid vaccine is obtained; The nucleotide sequence of SAG2 is shown in GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown in GenBank: AF012276.

1.

3. The method for constructing a Toxoplasma gondii nucleic acid vaccine according to claim 2, characterized in that: The nucleotide sequence of FLAG-SAG2-F-NotI is shown in Seq ID No.1, and the nucleotide sequence of SAG2-R-kPNI is shown in Seq ID No.

2.

4. The method for constructing a Toxoplasma gondii nucleic acid vaccine according to claim 2, characterized in that: The nucleotide sequence of SRS2-F-SalI is shown in Seq ID No.3, and the nucleotide sequence of SRS2-R-XbaI is shown in Seq ID No.4.

Images