CN109554390B

CN109554390B - Toxoplasma gondii SAG2 gene and MIC3 gene recombinant adenovirus construction method, recombinant adenovirus and application

Info

Publication number: CN109554390B
Application number: CN201811322996.2A
Authority: CN
Inventors: 陈启军; 张东超; 姜宁
Original assignee: Shenyang Agricultural University
Current assignee: Shenyang Agricultural University
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2022-05-10
Anticipated expiration: 2038-11-08
Also published as: CN109554390A

Abstract

The invention discloses a method for constructing a recombinant adenovirus of a toxoplasma gondii SAG2 (surface antigen 2) gene and an MIC3 (microlin 3) gene, the recombinant adenovirus and application. FromToxoplasma gondiiRespectively amplifying SAG2 gene and MIC3 gene in the RH strain, cloning the obtained target gene into a shuttle vector pHBAd-EF1a-MCS-3flag-CMV-EGFP, co-transfecting HEK293A cells with a shuttle plasmid pHBAd-SAG2-MIC3-EGFP and an adenovirus large skeleton pBHGlox (delta) E1 and 3Cre by a liposome transfection method, recombining and packaging to obtain the recombinant adenovirus Ad-SAG2-MIC3-EGFP containing SAG2 gene and MIC3 gene; after the recombinant adenovirus is used for immunizing Balb/c mice, the antibody level of the mice is obviously improved, the proportion of activated TCL and Th lymphocytes is obviously improved, cytokines such as IF-gamma, IL-6, TN-alpha and the like are obviously improved, and a certain protection effect is generated on the Balb/c mice.

Description

Toxoplasma gondii SAG2 gene and MIC3 gene recombinant adenovirus construction method, recombinant adenovirus and application

Technical Field

The invention belongs to the field of genetic biological engineering, relates to a method for constructing recombinant adenovirus, and particularly relates to a method for constructing recombinant adenovirus by utilizing Toxoplasma gondii SAG2 gene and MIC3 gene, the recombinant adenovirus and application.

Background

Toxoplasma gondii (Toxoplasma gondii) is obligate intracellular top-poda protozoa parasitized by karyocyte, is a serious-harm food-borne zoonosis parasite, and can infect over 200 animals including livestock, poultry and human. The human infection rate is about 25% -50%, and about 5-10 hundred million people infect toxoplasmosis globally; the death of livestock and poultry caused by the lethal outbreak of the toxoplasma can be achieved, and the death rate reaches 60 percent when the toxoplasmosis of pigs occurs. Toxoplasma not only directly endangers the development of animal husbandry and the safety of product quality, but also can cause public health and biological panic accidents due to the safety of meat and food, and influence the social stability.

Currently, the general vaccine categories for toxoplasma include inactivated vaccines, attenuated live vaccines, somatic specific component vaccines and recombinant protein vaccines. However, no toxoplasma vaccine is available for human, and the toxoplasma vaccine for animal use used in some countries is only one: s48 low strain vaccine. Therefore, the development of new vaccines is an urgent need to control toxoplasma infection and spread. The genetic engineering vaccine has the advantages of both attenuated vaccine and inactivated vaccine, and especially multivalent genetic engineering vaccine has more advantages in controlling the spread of epidemic diseases.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for constructing a recombinant adenovirus of an SAG2 gene and an MIC3 gene, the recombinant adenovirus and application thereof, wherein the genes SAG2 and MIC3 of T.gondii invasion-related protein are combined with the characteristic that an adenovirus vector system efficiently and stably expresses exogenous genes to construct the recombinant adenovirus capable of expressing the protein, indexes such as the level, lymphocyte proportion, cytokine change and the like of an anti-T.gondii antibody generated in an organism after an immunized mouse are respectively detected, the immune protection effect of the recombinant adenovirus is detected through an immune attack protection test, and the developed bivalent genetic engineering live vector vaccine for preventing toxoplasmosis is evaluated.

In order to solve the technical problems, the invention adopts the technical scheme that: a method for constructing a recombinant adenovirus by using SAG2 gene and MIC3 gene comprises the following steps:

1) SAG2 gene sequences GI were amplified separately from t.gondii RH strain: m33572.1 and MIC3 gene sequences GI: AJ 132530.1;

the amplification of the SAG2 gene and the MIC3 gene comprises the following steps: using SAG2-F SEQ ID NO.1 and SAG2-R SEQ ID NO.2 as primers and RNA of an RH strain as a template, and carrying out RT-PCR amplification to obtain an SAG2 gene; using MIC3-F SEQ ID NO.3 and MIC3-R SEQ ID NO.4 as primers and using RNA of an RH strain as a template, and carrying out RT-PCR amplification to obtain an MIC3 gene;

2) recombining a target gene to an adenovirus shuttle plasmid, co-transfecting HEK293A cells by using an obtained recombinant plasmid pHBAd-SAG2-MIC3-EGFP and an adenovirus large-skeleton plasmid pBHGlox E1 and 3Cre, recombining and packaging to obtain a recombinant adenovirus Ad-SAG2-MIC 3-EGFP.

The recombinant adenovirus prepared by the construction method of the T.gondii SAG2 gene and MIC3 gene recombinant adenovirus.

The recombinant adenovirus prepared by the construction method of the T.gondii SAG2 gene and MIC3 gene recombinant adenovirus is applied to preventing toxoplasma gondii.

The invention has the beneficial effects that: combining genes SAG2 and MIC3 related to the invasion of T.gondii with the characteristic that an adenovirus vector system stably expresses exogenous genes with high efficiency to construct a recombinant adenovirus Ad-SAG2-MIC3-EGFP capable of expressing the genes, thereby developing a bivalent gene engineering live vector vaccine for preventing toxoplasmosis. The immune effect of the recombinant adenovirus is evaluated by detecting indexes such as the anti-toxoplasma gondii antibody level, lymphocyte proportion, cytokine change and the like of an immunized Balb/c mouse and carrying out an immune attack test. Research results show that the antibody in the Balb/c mouse immunized with the recombinant adenovirus Ad-SAG2-MIC3-EGFP can reach the effective antibody level after immunizing for 28 days, the cellular immunity of the mouse is activated, and effective immune protection effect can be generated. The invention provides a bivalent gene engineering live vector vaccine for preventing toxoplasmosis.

Drawings

FIG. 1 shows the result of the SAG2 gene RT-PCR amplification (M: Trans 2K DNA molecular mass standard; 1: SAG2 gene RT-PCR product);

FIG. 2 shows the result of RT-PCR amplification of MIC3 gene (M: Trans 2K DNA molecular mass standard; 1: MIC3 gene RT-PCR product);

FIG. 3 is a diagram of HEK293A cells infected with recombinant adenovirus;

FIG. 4 is a Western blot for detecting the expression of SAG2 protein and MIC3 protein after HEK293A cells are infected by recombinant adenovirus;

FIG. 5 shows the PCR amplification result of SAG2 gene of recombinant adenovirus Ad-SAG2-MIC3-EGFP (M: Trans 2K DNA molecular quality Standard; 1: SAG2 gene PCR product);

FIG. 6 shows the PCR amplification results of MIC3 gene of recombinant adenovirus Ad-SAG2-MIC3-EGFP (M: Trans 2K DNA molecular mass standard; 1: MIC3 gene PCR product);

figure 7 is the level of mouse anti-t.gondii antibodies following ELISA detection of immune recombinant adenovirus;

FIG. 8 is a graph showing the ratio of mouse spleen T lymphocytes to non-T lymphocytes after flow detection of the immune recombinant adenovirus;

FIG. 9 is a graph showing the ratio of mouse spleen B lymphocytes to activated B lymphocytes after flow assay of the immune recombinant adenovirus;

FIG. 10 is a graph showing the ratio of mouse spleen killer T lymphocytes to activated killer T lymphocytes after flow detection of the immune recombinant adenovirus;

FIG. 11 shows the ratio of mouse spleen Th lymphocytes to activated Th lymphocytes after flow assay of the immune recombinant adenovirus;

FIG. 12 shows the Th1 cytokine profile in sera of mice immunized with recombinant adenovirus;

FIG. 13 shows other cytokine changes in the serum of mice with immune recombinant adenovirus;

figure 14 is survival and survival time of 100 t.gondii tachyzoites from mice immunized with recombinant adenovirus;

figure 15 is the survival rate and survival time of the 1000 t.gondii tachyzoites of the immune recombinant adenovirus mouse.

Detailed Description

The invention is described in further detail below with reference to the following figures and detailed description:

compared with other vectors, the adenovirus vector has obvious advantages, and a plurality of reports of preparing vaccines by taking adenovirus as the vector exist at home and abroad, and the adenovirus vector system is quite mature at present. The research on T.gondii is quite clear, and the genetic engineering vaccine of SAG2 or MIC3 is reported to be developed by using other vectors, and an immune attack test proves that the vaccine has a certain protective effect on mice, so that the recombinant adenovirus Ad-SAG2-MIC3-EGFP is constructed by using T.gondii SAG2 and MIC3 genes, and the bivalent genetic engineering live vector vaccine for preventing toxoplasmosis is developed.

The method for constructing the recombinant adenovirus Ad-SAG2-MIC3-EGFP by the T.gondii SAG2 gene and the MIC3 gene comprises the following steps:

1) amplification of t.gondii SAG2 gene and MIC3 gene fragments;

2) the virus Ad-SAG2-MIC3-EGFP was recombined and packaged in HEK293A cells.

The method specifically comprises the following steps:

2) recombining a target gene to an adenovirus shuttle plasmid, co-transfecting HEK293A cells by the obtained recombinant plasmid pHBAd-SAG2-MIC3-EGFP and an adenovirus large-skeleton plasmid pBHGlox E1 and 3Cre, recombining and packaging to obtain the recombinant adenovirus Ad-SAG2-MIC 3-EGFP.

That is to say: amplifying target genes (SAG2 and MIC3) from a T.gondii RH strain; cloning a target gene onto a pMD19-T vector through T-A; after enzyme digestion and sequencing identification, connecting the product with a shuttle vector pHBAd-EF1a-MCS-3 flag-CMV-EGFP; transfecting HEK293A cells with a shuttle plasmid pHBAd-SAG2-MIC3-EGFP and an adenovirus large skeleton pBHGlox (delta) E1 and 3Cre by a liposome transfection method to obtain a recombinant adenovirus Ad-ROP9-MIC3-EGFP, and identifying the recombinant adenovirus by PCR and Western blot; after purification of recombinant adenovirus, TCID was added₅₀The method determines the titer of the recombinant adenovirus; immunizing Balb/c mice with the recombinant adenovirus vaccine, and detecting an immune mouse antibody by ELISA; detecting indexes such as lymphocyte proportion and cytokine change of the immune mice; the immune attack detects the morbidity and mortality of the immunized mice, and evaluates the effect of developing bivalent vaccine immunized mice.

The present invention is further illustrated in detail below with reference to specific examples:

1 materials and methods

1.1 amplification of SAG2 Gene and MIC3 Gene

Inoculating a T.gondii RH strain to Balb/c mice in an abdominal cavity, collecting abdominal cavity liquid of the mice, purifying tachyzoites and extracting polypide RNA by using a Trizol method. Primers were designed based on the SAG2 gene (M33572.1) and the MIC3 gene sequence (AJ132530.1) of the T.gondii RH strain in GenBank, and RT-PCR was performed to amplify the gene fragment of interest. SAG2 gene and MIC3 gene primers are shown in Table 1 (the underlined part is an enzyme cutting site, and the bold part is a Kozak sequence), and the PCR reaction systems of the SAG2 gene and the MIC3 gene are shown in Table 2.

Table 1 primer sequences for amplifying SAG2 and MIC3 gene fragments

TABLE 2 RT-PCR reaction System for SAG2 Gene and MIC3 Gene

Note: the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; {95 ℃ denaturation for 30 s; annealing at 62 ℃ for 45 s; 72 ℃ extension for 60s } for 30 cycles, 72 ℃ extension for 10 min.

1.2 construction of recombinant shuttle adenovirus vectors

Carrying out double enzyme digestion on the obtained SAG2 gene and MIC3 gene and shuttle vector pHBAd-EF1a-MCS-3flag-CMV-EGFP by using corresponding enzymes (the enzyme digestion reaction system is shown in tables 3 and 4), and then using T to carry out double enzyme digestion on the obtained SAG2 gene and MIC3 gene₄Ligase 16 ℃ for 12h ligation (see Table 5 for reaction system). And screening out correct recombinant shuttle plasmid pHBAd-SAG2-MIC3-EGFP through bacterial liquid PCR, enzyme digestion and sequencing identification.

TABLE 3 digestion system

TABLE 4 digestion system

TABLE 5T₄Ligase ligation systems

1.3 recombination and packaging of adenoviruses

Taking 15.0 mu L of liposome, mixing the liposome with 3 mu g of adenovirus shuttle plasmid pHBAd-SAG2-MIC3-EGFP and 6 mu g of adenovirus large-skeleton plasmid pBHGlox (delta) E1 and 3Cre uniformly, co-transfecting HEK293A cells, recombining the cells in HEK293A cells, packing for 14d, collecting CPE cells, repeatedly freezing and thawing, collecting viruses, and freezing and storing at-80 ℃.

The collected recombinant adenovirus is inoculated to normal HEK293A cells, and the cytopathic condition and the fluorescence condition of the cells are observed by a fluorescence microscope. Extracting DNA of the recombinant adenovirus by using a virus genome extraction kit, and detecting a target gene by PCR; HEK293A cells infected by the recombinant adenovirus for 48h are collected, and then the expression conditions of SAG2 protein and MIC3 protein are detected by Western blot.

1.4 determination of the titer of recombinant adenovirus

Purifying the recombinant adenovirus by using adenovirus purification kit, and then purifying the recombinant adenovirus according to TCID₅₀The method detects the titer of the recombinant adenovirus.

1.5ELISA for detecting antibody level of immune Balb/c

Selecting 50 SPF-grade Balb/c mice of 20-22 g, and randomly grouping the mice into 5 groups and 10 mice per group; the Ad-SAG2-MIC3-EGFP group, the Ad-SAG2-EGFP group, the Ad-MIC3-EGFP group, the Ad-EGFP (control) group, and the PBS (blank) group were set, respectively. Two immunizations, separated by 14 days, were administered by intramuscular injection 10⁹CPU recombinant adenovirus particles. During this period, blood was collected from the tail vein and serum was isolated every 7 days, and the antibody levels of the immunized mice were measured at different times using an ELISA kit. 28 days after the first immunization, blood is collected by eyeballs, serum is separated, and cytokine changes are detected; spleen lymphocyte changes were detected by flow cytometry.

1.6 immune protection assay

Selecting 100 SPF-grade Balb/c mice of 20-22 g, and randomly grouping 10 groups of the mice, wherein each group comprises 10 mice; respectively setting Ad-SAG2-MIC3-EGFP group (a), Ad-SAG2-EGFP (a) group, Ad-MIC3-EGFP (a)) Group, Ad-EGFP (a; control) group, PBS (a; blank group, Ad-SAG2-MIC3-EGFP group (b), Ad-SAG2-EGFP (b) group, Ad-MIC3-EGFP (b) group, Ad-EGFP (b; control), PBS (b; blank) groups, and immunization was performed separately. Group a was inoculated with 1X 10 vaccine 28 days after the first immunization²Gondii RH strain tachyzoite; group b seed 1X 10³Gondii RH strain tachyzoite; the status of the mice was observed daily and the morbidity and mortality of the mice were counted.

2 results and analysis

2.1 RT-PCR amplification results of SAG2 Gene and MIC3 Gene

Extracting RNA of T.gondiiRH by using SAG2 gene primer pair designed according to SAG2 gene sequence in GenBank to perform RT-PCR amplification, and obtaining an expected fragment with the size of about 561bp through sequencing; the T-A clone obtains PMD-SAG2 plasmid, PCR amplification is carried out by using designed SAG2 gene primer, and the mutexpected target fragment with the size of about 561bp is obtained by sequencing (figure 1).

Carrying out RT-PCR amplification on RNA extracted from T.gondiiRH by using an MIC3 gene primer pair designed according to an MIC3 gene sequence in GenBank, and sequencing to obtain an expected fragment with the size of about 1080 bp; the T-A clone obtains PMD-MIC3 plasmid, PCR amplification is carried out by using designed MIC3 gene primer, and the mutexpected target fragment with the size of about 1080bp is obtained by sequencing (figure 2).

2.2 sequencing results analysis of amplified SAG2 Gene and MIC3 Gene

The nucleotide sequences of the sequenced SAG2 gene and MIC3 gene are subjected to Blast comparative analysis with the nucleotide sequence (M33572.1) of the SAG2 gene and the nucleotide sequence (AJ132530.1) of the MIC3 gene in GenBank respectively, the homology of the SAG2 gene sequence reaches 100 percent, the homology of the MIC3 gene sequence reaches 100 percent, and no alkali deletion or insertion exists.

2.3 obtaining recombinant adenovirus

By T₄The target fragments SAG2 and MIC3 are respectively connected to the adenovirus shuttle plasmid by ligase to obtain recombinant plasmids pBHAd-SAG2-EGFP, pBHAd-MIC3-EGFP and pBHAd-SAG2-MIC 3-EGFP. The adenovirus large skeleton and the recombinant shuttle plasmid are respectively cotransfected with HEK293A cells by using liposome, and after about 14 days of culture, the antigen is harvestedGeneration recombinant adenovirus Ad-SAG2-EGFP, Ad-MIC3-EGFP and Ad-SAG2-MIC 3-EGFP. Screening monoclonal viruses through plaques, infecting normal HEK293A cells with recombinant adenoviruses respectively, and exciting the cells by blue-violet light under a fluorescence microscope to see that the cells show green fluorescence; the uninfected cells were normal and no green fluorescence was observed under a fluorescence microscope (FIG. 3).

2.4Western blot results of detecting SAG2 protein and MIC3 protein expression

Western blot results show that: the recombinant adenoviruses Ad-SAG2-MIC3-EGFP and Ad-SAG2-EGFP show specific bands at about 22kDa, which are equivalent to the expected SAG2 protein molecular mass; specific bands appeared at about 38kDa for Ad-SAG2-MIC3-EGFP and Ad-MIC3-EGFP, comparable to the expected molecular mass of the MIC3 protein; the specific bands of Ad-SAG2-MIC3-EGFP, Ad-SAG2-EGFP, Ad-MIC3-EGFP and Ad-EGFP appear at about 42kDa, which is equivalent to the expected mass of the internal reference beta-actin protein (FIG. 4).

2.5 detection of target Gene of recombinant Virus

Extracting DNA from the packaged recombinant adenovirus, performing PCR amplification by respectively using designed SAG2 gene and MIC3 gene primers, and performing sequencing analysis on a target gene sequence, wherein the result shows that: bands consistent with the expected fragment size appeared around 561bp and around 1080bp, respectively (FIGS. 5 and 6).

2.6 titer determination of recombinant viruses

The purified and concentrated recombinant adenovirus is diluted in a multiple ratio and then infected with HEK293A cells, after 7d of infection, the positive hole ratio is calculated according to the KaBer method, and the titer of Ad-SAG2-MIC3-EGFP, Ad-SAG2-EGFP, Ad-MIC3-EGFP and Ad-EGFP is determined to be 1.58 multiplied by 10¹¹CPU、1.58×10¹¹CPU、1.58×10¹¹CPU and 1.58 × 10¹¹CPU。

2.7ELISA test results

The antibody OD was detected using the separated serum according to the ELISA kit instructions₄₀₅。

From day 14 and day 28 post-immunization, the levels of mouse antibodies from the Ad-SAG2-MIC3-EGFP immunized differed very significantly from the levels of mouse antibodies from the Ad-EGFP and PBS injected (P < 0.001); there was no significant difference in mouse antibody levels between the Ad-EGFP injected group and the PBS group (P > 0.05; fig. 7).

2.8 lymphocyte changes

On the 28 th day after the mice are immunized for the first time, spleens of the mice are picked up, and the proportion of lymphocytes in the spleens is detected. The non-T lymphocytes of the mice of the Ad-SAG2-MIC3-EGFP group were significantly lower than those of the PBS group (P < 0.001; FIG. 8), and the T lymphocytes were significantly higher than those of the PBS group (P <0.001) (FIG. 8); the activated lymphoid B cells of mice in the Ad-SAG2-MIC3-EGFP group were significantly higher than those of mice in the PBS group (P <0.001) (FIG. 9); the activated CTL of mice in Ad-SAG2-MIC3-EGFP group was significantly higher than that of mice in PBS group (P <0.001), significantly higher than that of mice in Ad-EGFP group (P < 0.05; FIG. 10); the Th lymphocyte level of mice in the Ad-SAG2-MIC3-EGFP group was significantly higher than that of mice in the PBS group (P < 0.001; FIG. 10), and the activated Th lymphocyte level of mice in the Ad-SAG2-MIC3-EGFP group was significantly higher than that of mice in the PBS (P <0.001) group and significantly higher than that of mice in the Ad-EGFP (P <0.05) (FIG. 11).

2.9 cytokine changes

On day 28 after the first immunization of the mice, blood was collected from the eyeballs and serum was separated, and serum cytokine levels among different groups were detected by flow cytometry. The cytokines such as IL-6, TN-alpha, IL-10, IL-22 and IL-17A in the serum of the mice of the Ad-SAG2-MIC3-EGFP group are all significantly higher than those of the mice of the Ad-EGFP and PBS groups (P <0.001 or P < 0.01; FIG. 12 and FIG. 13), and IF-gamma and IL-17A are significantly higher than those of the PBS group (P < 0.05; FIG. 12 and FIG. 13); there was no significant difference in cytokines between the Ad-EGFP group and the PBS group injected mice (P > 0.05; fig. 12 and 13).

2.10 immunoprotection results

The test result of the attack of the insects after immunization shows that: the insect-attacking dose is 1 × 10²Gondii RH tachyzoite, protection of 60% for Ad-SAG2-MIC3-EGFP immunized mice, 20% for Ad-SAG2-EGFP immunized mice, 50% for Ad-MIC3-EGFP immunized mice, and no protection for all deaths on day 11 after injection of Ad-EGFP and PBS mice (FIG. 14).

The insect-attacking dose is 1 × 10³Protection rate of Ad-SAG2-MIC3-EGFP immune mice at T.gondii RH tachyzoite of 30%(ii) a All mice in the Ad-SAG2-EGFP immune group die at day 13, all mice in the Ad-MIC3-EGFP immune group die at day 17, and the survival time of the mice is prolonged to a certain extent; mice injected with Ad-EGFP and PBS all died at day 8 post-challenge, and no protection was produced (FIG. 15).

3 conclusion

3.1 amplifying SAG2 gene and MIC3 gene from T.gondii RH strain respectively;

3.2 successfully packaging the recombinant adenovirus Ad-SAG2-MIC 3-EGFP; the recombinant adenovirus can well express SAG2 protein and MIC3 protein;

3.3 the titer of the harvested recombinant adenovirus Ad-SAG2-MIC3-EGFP is: 1.58X 10¹¹PFU, required titer requirements for subsequent animal trials;

3.4 the recombinant adenovirus immune mouse constructed by the method generates higher antibody level, lymphocytes are activated, and cytokines are activated;

3.5 the recombinant adenovirus has certain immune protection effect on mice.

The innovation of the invention is that the genes SAG2 and MIC3 of T.gondii invasion-related protein are combined with the characteristic that an adenovirus vector system efficiently and stably expresses an exogenous gene to construct a recombinant adenovirus capable of expressing the protein, the indexes such as the level of an anti-T.gondii antibody generated in an organism after an immunized mouse, lymphocyte proportion, cytokine change and the like are respectively detected, the immune protection effect is detected through an immune challenge protection test, and the bivalent gene engineering live vector vaccine for preventing toxoplasmosis is successfully developed.

The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to carry out the same, and the present invention shall not be limited to the embodiments, i.e. the equivalent changes or modifications made within the spirit of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

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Claims

1. A construction method of a recombinant adenovirus of a toxoplasma SAG2 gene and an MIC3 gene is characterized by comprising the following steps:

1) fromToxoplasma gondii SAG2 gene sequences GI are respectively amplified from the RH strain: m33572.1 and MIC3 gene sequence AJ 132530.1;

2. The recombinant adenovirus prepared by the method for constructing the SAG2 gene and MIC3 gene recombinant adenovirus according to claim 1.

3. The use of the recombinant adenovirus prepared by the method for constructing the recombinant adenovirus containing the SAG2 gene and the MIC3 gene according to claim 1 in preparing a medicament for preventing toxoplasmosis.

4. The method for constructing the recombinant adenovirus of the Toxoplasma gondii SAG2 gene and the MIC3 gene of claim 1, wherein in the step 1, the T. gondii RH strain is inoculated to Balb/c mice in the abdominal cavity, the collected abdominal cavity fluid of the mice is purified and tachyzoite is purified, and the RNA of the insect body is extracted by the Trizol method.

5. The method for constructing the recombinant adenovirus of the Toxoplasma gondii SAG2 gene and the MIC3 gene according to claim 1, wherein in the step 2, the obtained SAG2 gene and the MIC3 gene are subjected to double enzyme digestion with corresponding enzymes together with a shuttle vector pHBAd-EF1a-MCS-3flag-CMV-EGFP, then are subjected to 16 ℃ ligation with T4 ligase for 12 hours, and a correct recombinant shuttle plasmid pHBAd-SAG2-MIC3-EGFP is screened out through bacterial liquid PCR, enzyme digestion and sequencing identification.

6. The method for constructing the recombinant adenovirus of the Toxoplasma gondii SAG2 gene and the MIC3 gene of claim 1, wherein in the step 2, 15.0 μ L of liposome is taken, mixed with 3 μ g of adenovirus shuttle plasmid pHBAd-SAG2-MIC3-EGFP and 6 μ g of adenovirus large-framework plasmid pBHGloxE1,3Cre, co-transfected with HEK293A cells, recombined and packaged in HEK293A cells for 14d, and the CPE cells are collected, repeatedly frozen and thawed and the virus is collected, and frozen and stored at-80 ℃.