CN115998852A - Toxoplasma gondii nucleic acid vaccine and construction method thereof - Google Patents
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biology, and particularly relates to a toxoplasma gondii nucleic acid vaccine and a construction method thereof. According to the invention, two cDNA fragments SAG2 and SRS2 on the toxoplasma RH strain are respectively or jointly inserted into a eukaryotic vector pBud CE4.1 to obtain a recombinant plasmid as a toxoplasma nucleic acid vaccine. According to the invention, toxoplasma nucleic acid vaccine is used for 3 times of active immunization of a Kunming mouse with 6-8 weeks, each time is divided into two weeks, the immunogenicity of the vaccine is evaluated by measuring cell and humoral immunity indexes of the immunized mouse, and the immunoprotection of the vaccine is evaluated by an in vivo attack experiment. The invention can effectively enhance humoral immunity and cellular immune response of experimental animals and effectively reduce death quantity of the experimental animals. The toxoplasma nucleic acid vaccine provided by the invention can be used as an effective candidate vaccine for preventing toxoplasma infection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a toxoplasma gondii nucleic acid vaccine and a construction method thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
The traditional toxoplasmosis treatment scheme is that sulfadiazine sodium and pyrimethamine are used for compatibility treatment. However, drug therapy has serious side effects such as high toxicity. Vaccines are considered to be an important means for substituting for drug therapy because of their non-toxicity, ease of manufacture, and ease of large-scale use. The nucleic acid vaccine is to introduce the foreign gene encoding some antigen protein into animal cell directly, synthesize antigen protein via the expression system of host cell and induce the host to produce immune response to the antigen protein for the purpose of preventing and treating diseases. Compared with the traditional inactivated vaccine, subunit vaccine and genetic engineering vaccine, the nucleic acid vaccine has the following advantages: enhancing immunity; the preparation is simple, and the time and the labor are saved; cross protection of the same species of different strains; the application is safer; generating a persistent immune response; the storage and the transportation are convenient. Despite the wide variety of toxoplasma vaccines, no commercially available vaccine is currently available for clinical use. Therefore, developing a vaccine with high efficiency, low cost and safety is an urgent need for controlling toxoplasmosis.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a toxoplasma nucleic acid vaccine and a construction method thereof. According to the invention, two cDNA fragments SAG2 and SRS2 on the toxoplasma RH strain are respectively or jointly inserted into a eukaryotic vector pBud CE4.1 to obtain a recombinant plasmid as a toxoplasma nucleic acid vaccine. According to the invention, toxoplasma nucleic acid vaccine is used for 3 times of active immunization of a Kunming mouse with 6-8 weeks, each time is divided into two weeks, the immunogenicity of the vaccine is evaluated by measuring cell and humoral immunity indexes of the immunized mouse, and the immunoprotection of the vaccine is evaluated by an in vivo attack experiment. The invention can effectively enhance humoral immunity and cellular immune response of experimental animals and effectively reduce death quantity of the experimental animals. Therefore, the toxoplasma nucleic acid vaccine provided by the invention can be used as an effective candidate vaccine for preventing toxoplasma infection.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the invention provides a toxoplasma nucleic acid vaccine, comprising a recombinant plasmid composed of a gene fragment obtained by amplification of a cDNA of toxoplasma RH as a template through polymerase chain reaction and a vector; the gene fragment is one or more of SAG2 and SRS2, the nucleotide sequence of SAG2 is shown as GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown as GenBank: AF 012276.1.
In a second aspect, the invention provides a method for constructing toxoplasma nucleic acid vaccine, comprising the following steps:
infecting HFF cells, collecting toxoplasma RH after the cells are completely ruptured, extracting the insect RNA by using a Trizol method, and obtaining cDNA by using a reverse transcription kit by taking the toxoplasma RH RNA as a template;
the SAG2 fragment is amplified by using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, the SRS2 fragment is amplified by using primers SRS2-F-SalI and SRS 2-R-XbaI, the SAG2 fragment and the SRS2 fragment are respectively or jointly inserted into eukaryotic vectors to obtain recombinant plasmids, and the recombinant plasmids are extracted and purified to obtain toxoplasma gondii nucleic acid vaccine.
The beneficial effects obtained by one or more of the technical schemes of the invention are as follows:
(1) After the recombinant plasmid constructed by the invention is transfected into cells in vitro, the protein is successfully expressed.
(2) According to the invention, toxoplasma nucleic acid vaccine is used for actively immunizing a Kunming mouse, and the immune mouse serum has higher antibody titer.
(3) The survival rate of immunized mice using toxoplasma nucleic acid vaccine reaches 30%, the immune protection effect of the vaccine is basically equivalent to that of sulfanilamide drug effect, and the vaccine is a potential scheme for substituting drug treatment.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1A is a schematic diagram of construction of the pSAG2 plasmid in example 1, B is a schematic diagram of construction of the pSRS2 plasmid in example 2, C is a schematic diagram of construction of the pSAG2+SRS2 plasmid in example 3, and D is an in vitro protein detection result after eukaryotic plasmid transfection;
in fig. 2, a is SAG2 prokaryotic expression vector construction and protein expression and purification, B is SRS2 prokaryotic expression vector construction and protein expression and purification, C is purified protein immunoblotting verification, and D is SAG2 and SRS2 localization analysis;
FIG. 3 is a graph showing the results of an experiment for proliferation of spleen lymphocytes in immunized mice;
FIG. 4 is a cytokine level assay following in vitro stimulation of immune murine spleen lymphocytes;
FIG. 5 shows the results of detection of immunized murine antibodies;
FIG. 6 is a graph showing survival of mice in vivo challenge experiments in immunized mice.
Detailed Description
In a first exemplary embodiment of the present invention, a toxoplasma nucleic acid vaccine comprises a recombinant plasmid composed of a gene fragment obtained by amplification of a cDNA of toxoplasma RH as a template through polymerase chain reaction and a vector;
the gene fragment is one or more of SAG2 and SRS2, the nucleotide sequence of SAG2 is shown as GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown as GenBank: AF 012276.1.
In one or more embodiments of this embodiment, the eukaryotic vector is pbudce4.1.
In a second exemplary embodiment of the present invention, a method for constructing a toxoplasma nucleic acid vaccine, comprises the steps of:
infecting HFF cells, collecting toxoplasma RH after the cells are completely ruptured, extracting the insect RNA by using a Trizol method, and obtaining cDNA by using a reverse transcription kit by taking the toxoplasma RH RNA as a template;
the SAG2 fragment is amplified by using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, the SRS2 fragment is amplified by using primers SRS2-F-SalI and SRS 2-R-XbaI, the SAG2 fragment and the SRS2 fragment are respectively or jointly inserted into eukaryotic vectors to obtain recombinant plasmids, and the recombinant plasmids are extracted and purified to obtain toxoplasma gondii nucleic acid vaccine.
In one or more examples of this embodiment, HFF cells are infected with a ratio of moi=5.
In one or more embodiments of this embodiment, the eukaryotic vector is pbudce4.1.
In one or more examples of this embodiment, the nucleotide sequence of FLAG-SAG2-F-NotI is shown as Seq ID No.1 and the nucleotide sequence of SAG2-R-kPNI is shown as Seq ID No. 2.
In one or more examples of this embodiment, the nucleotide sequence of SRS2-F-SalI is shown as Seq ID No.3, and the nucleotide sequence of SRS2- -R-XbaI is shown as Seq ID No. 4.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
The primers used in the present invention are shown in Table 1.
TABLE 1 primers used in the present invention and sequences thereof
Example 1
Infecting HFF cells in a MOI=5 ratio, collecting toxoplasma RH after the cells are completely ruptured, extracting insect RNA by using a Trizol method, taking the toxoplasma RH RNA as a template, and obtaining cDNA by using a reverse transcription kit;
the SAG2 fragment was amplified with primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, and the SAG2 fragment was inserted into eukaryotic vector pBudCE4.1 to obtain recombinant plasmid pSAG2, which was extracted and purified to obtain toxoplasma gondii nucleic acid vaccine. The construction scheme of the pSAG2 plasmid is shown in FIG. 1A.
Example 2
Infecting HFF cells in a MOI=5 ratio, collecting toxoplasma RH after the cells are completely ruptured, extracting insect RNA by using a Trizol method, taking the toxoplasma RH RNA as a template, and obtaining cDNA by using a reverse transcription kit;
the primers SRS2-F-SalI and SRS 2-R-XbaI are used for amplifying SRS2 fragments, the SRS2 fragments are inserted into a eukaryotic vector pBudCE4.1 to obtain a recombinant plasmid pSRS2, and the recombinant plasmid pSRS2 is extracted and purified to obtain the toxoplasma gondii nucleic acid vaccine. The construction scheme of the pSRS2 plasmid is shown in FIG. 1B.
Example 3
Infecting HFF cells in a MOI=5 ratio, collecting toxoplasma RH after the cells are completely ruptured, extracting insect RNA by using a Trizol method, taking the toxoplasma RH RNA as a template, and obtaining cDNA by using a reverse transcription kit;
the SAG2 fragment is amplified by using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, the SRS2 fragment is amplified by using primers SRS2-F-SalI and SRS 2-R-XbaI, the SAG2 fragment and the SRS2 fragment are jointly inserted into a eukaryotic vector pBudCE4.1 to obtain a recombinant plasmid pSAG 2+SRSr2, and the plasmid is extracted and purified to obtain the toxoplasma gondii nucleic acid vaccine. The construction scheme of the pSAG2+SRS2 plasmid is shown in FIG. 1C.
Experimental example
The recombinant plasmids of examples 1-3 were used to transfect 293T cells using liposome Lipofectamine 2000, as shown in FIG. 1D, and protein electrophoresis was performed to confirm that the expressed proteins were consistent with expectations.
As shown in FIG. 2A, the SAG2 fragment was amplified by using primers HA-SAG2-F and SAG2-R, the Pet32A skeleton was amplified by using primers Pet32A-F and Pet32A-R, and a Pet32A-SAG2 prokaryotic expression vector was constructed by using a homologous recombination method. The nucleotide sequence of HA-SAG2-F is shown as Seq ID No.5, and the nucleotide sequence of SAG2-R is shown as Seq ID No. 6. The nucleotide sequence of the Pet32a-F is shown as Seq ID No.7, and the nucleotide sequence of the Pet32a-R is shown as Seq ID No. 8. As shown in FIG. 2B, the SRS2 fragment was amplified by using primers SRS2-F and SRS2-R, the Pet32a skeleton was amplified by using primers Pet32a-F and Pet32a-R, and a Pet32a-SRS2 prokaryotic expression vector was constructed by using a homologous recombination method. The nucleotide sequence of SRS2-F is shown as Seq ID No.9, and the nucleotide sequence of SRS2-R is shown as Seq ID No. 10. And E.coli induced expression and purification are carried out, western blot verification is carried out on the purified protein, and as shown in figure 2C, the size of the expressed protein accords with the expectations. Mice were immunized with purified protein and mouse serum was collected after four-immunization. The SAG2 and SRS2 proteins were subjected to localization analysis by indirect immunofluorescence, as shown in FIG. 2D, and both proteins were found to be on the surface of the insect.
The Kunming mice were randomly divided into 4 groups of 16 mice each. Examples were respectively the example 1 (pSAG 2) immunized group, the example 2 (pSRS 2) immunized group, the example 3 (pSAG 2+SRS2) immunized group, and the control (pBudCE4.1) group. Each mouse was injected with 100. Mu.g of recombinant plasmid three times, and after primary immunization, each immunization was boosted 2 weeks later and 4 weeks later. Antibody detection was performed by taking blood two weeks after the last immunization.
At 2 weeks after the last immunization, spleens of immunized mice were aseptically taken and ground through 200 mesh gauze to prepare spleen single cell suspensions. After removing erythrocytes from spleen cells using an erythrocyte lysis buffer, the cells were resuspended in 1640 medium containing 10% FCS,100U/ml penicillin and 100U/ml streptomycin, and the concentration of the cell suspension was adjusted to 2X 10 5 Lymphocyte proliferation experiments were performed using CCK8 kit after 72 hours of stimulation of individual cells/well with PBS and recombinant protein (CA), respectively. As shown in fig. 3, the spleen lymphocytes of the example 2 and example 3 groups were significantly increased.
At 2 weeks after the last immunization, spleens of immunized mice were aseptically taken and ground through 200 mesh gauze to prepare spleen single cell suspensions. After removing erythrocytes from spleen cells using an erythrocyte lysis buffer, the cells were resuspended in 1640 medium containing 10% FCS,100U/ml penicillin and 100U/ml streptomycin, and the concentration of the cell suspension was adjusted to 2X 10 5 Cells/well were stimulated with PBS and recombinant protein (CA), respectively, cell supernatants were collected 24 hours after stimulation and assayed for interleukin-4 (IL-4) concentration, and supernatants were collected 96 hours after stimulation for interferon-gamma (IFN-gamma) concentration. As shown in FIG. 4, IFN-gamma levels were increased in the group of example 3 and IL-4 levels were increased in the group of example 2.
Mice were bled at the inner canthus on day 14 after the third immunization, and the blood samples were allowed to stand at room temperature for 2h, then centrifuged at 3000rpm for 10min, and serum was collected. The content of total anti-toxoplasma immunoglobulin (IgG) in serum was determined using an enzyme-linked immunosorbent assay. As shown in fig. 5. After 3 immunizations, the serum antibody titers were significantly increased in the mice of examples 1-3 (P < 0.0001) compared to the control group.
The immunized mice were subjected to an insect challenge experiment 2 weeks after the last immunization. Mice were intraperitoneally infected with 100 RH worms. Mice were recorded for mortality. As shown in fig. 6, the challenge experiments of immunized mice showed that the mice of examples 1-3 survived for a significantly longer period of time than the control group. In particular, the survival rate of the mice in the group of the example 3 reaches 30 percent.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A toxoplasma nucleic acid vaccine is characterized by comprising a recombinant plasmid formed by a gene fragment obtained by amplification of a polymerase chain reaction by taking cDNA of toxoplasma RH as a template and a eukaryotic vector;
the gene fragment is one or more of SAG2 and SRS2, the nucleotide sequence of SAG2 is shown as GenBank: JX045478.1, and the nucleotide sequence of SRS2 is shown as GenBank: AF 012276.1.
2. The toxoplasma nucleic acid vaccine of claim 1, wherein the eukaryotic vector is pbudce4.1.
3. The construction method of the toxoplasma nucleic acid vaccine is characterized by comprising the following steps:
infecting HFF cells, collecting toxoplasma RH after the cells are completely ruptured, extracting the insect RNA by using a Trizol method, and obtaining cDNA by using a reverse transcription kit by taking the toxoplasma RH RNA as a template;
the SAG2 fragment is amplified by using primers FLAG-SAG2-F-NotI and SAG2-R-kPNI, the SRS2 fragment is amplified by using primers SRS2-F-SalI and SRS 2-R-XbaI, the SAG2 fragment and the SRS2 fragment are respectively or jointly inserted into eukaryotic vectors to obtain recombinant plasmids, and the recombinant plasmids are extracted and purified to obtain toxoplasma gondii nucleic acid vaccine.
4. The method of constructing a toxoplasma nucleic acid vaccine according to claim 1, wherein HFF cells are infected with a ratio of MOI = 5.
5. The method for constructing toxoplasma nucleic acid vaccine according to claim 1, wherein the eukaryotic vector is pbudce4.1.
6. The method of constructing a toxoplasma gondii nucleic acid vaccine according to claim 1, wherein the nucleotide sequence of FLAG-SAG2-F-NotI is represented by Seq ID No.1, and the nucleotide sequence of SAG2-R-kPNI is represented by Seq ID No. 2.
7. The method for constructing a toxoplasma gondii nucleic acid vaccine according to claim 1, wherein the nucleotide sequence of SRS2-F-SalI is represented by Seq ID No.3, and the nucleotide sequence of SRS 2-R-XbaI is represented by Seq ID No. 4.
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| US20070286871A1 (en) * | 2004-10-05 | 2007-12-13 | Diversa Corporation | Vaccines |
| US20130273094A1 (en) * | 2010-11-04 | 2013-10-17 | The University Of Chicago | Vaccines against toxoplasma gondii |
| CN103251960A (en) * | 2013-05-27 | 2013-08-21 | 山东大学 | DNA (deoxyribonucleic acid) vaccine for preventing chronic toxoplasmosis of humans or animals |
| CN105561343A (en) * | 2016-02-04 | 2016-05-11 | 山东大学 | DNA vaccine for preventing toxoplasmosis of humans or animals |
| CN109554391A (en) * | 2018-11-08 | 2019-04-02 | 沈阳农业大学 | The method and recombined adhenovirus and application of toxoplasma SAG2 gene and the building of ROP9 gene recombinant adenovirus |
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