CN116814437A - Toxoplasma gondii live vaccine for expressing S1 protein of cat infectious peritonitis virus, construction method and application thereof - Google Patents

Abstract

The invention relates to the technical field of toxoplasma vaccine, in particular to a toxoplasma live vaccine for expressing S1 protein of cat infectious peritonitis virus, and a construction method and application thereof. The invention provides a toxoplasma gondii strain for expressing S1 protein, wherein the amino acid sequence of the S1 protein is shown as SEQ ID NO. 1. Compared with the original insect strain, the insect strain expressing the S1 protein has no obvious change in toxicity, has higher pathogenicity in mice, almost has no pathogenicity in cats, and has higher safety to cats. The insect strain expressing the S1 protein can realize reinfection on toxoplasma gondii strain and provide effective protection against FIPV virus infection, and has the potential of preventing toxoplasma gondii oocysts from being discharged and preventing cat infectious peritonitis virus.

Description

Toxoplasma gondii live vaccine for expressing S1 protein of cat infectious peritonitis virus, construction method and application thereof

Technical Field

The invention relates to the technical field of toxoplasma vaccine, in particular to a toxoplasma live vaccine for expressing S1 protein of cat infectious peritonitis virus, and a construction method and application thereof.

Background

Feline Infectious Peritonitis Virus (FIPV) is a type of feline coronavirus that can cause high mortality disease resulting from abnormal immune responses in felines, and although multiple drugs have been developed clinically, the effect is not obvious. Meanwhile, FIPV vaccines developed by conventional technology cannot generate effective protection effect on hosts, and can also trigger antibody dependence enhancement effect of FIPV infection.

Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic protozoa capable of infecting most homeothermic animals, including humans. Toxoplasmosis is a zoonosis caused by toxoplasmosis, and for people with normal immunity, most of toxoplasmosis is recessive infection after toxoplasmosis infection, and clinical manifestations are not generally caused. However, toxoplasmosis can be caused by the vertical transmission of toxoplasmosis through the placenta, and thus the primary infection of toxoplasmosis by pregnant women can have serious consequences. In the case of toxoplasma, it has been demonstrated that some proteins of plasmodium and leishmania are transfected into toxoplasma and expressed successfully, and that toxoplasma is used as a carrier to stimulate the body to produce a stronger Th 1-dominated immune process. Toxoplasma should be considered as a new candidate for vaccine vectors, since it has an unusually strong adjuvant effect and is easy to genetically manipulate.

The market scale of pets is continuously expanding, the pet economy is continuously heating, and the pet vaccine is or becomes a new growth point of animal vaccine industry. Based on the important role of cats in the transmission of the infectious peritonitis virus and toxoplasma in cats, and the fact that toxoplasma can guide the organism to generate nonspecific antibodies to other pathogens, whether toxoplasma can be used as a live vaccine vector or not is considered to present immunodominant antigens expressing the infectious peritonitis virus of cats, so that the immunodominant antigens can be used for constructing immune responses against toxoplasma in cats and simultaneously triggering specific immune responses against the infectious peritonitis of cats.

Disclosure of Invention

The invention provides a construction method and application of toxoplasma live vaccine for expressing S1 gene of cat infectious peritonitis virus.

The invention aims to achieve the aim, and firstly provides a construction method of transgenic toxoplasma gondii expressing S1 protein. The invention constructs an S1 expression vector, applies CRISPR/Cas9 mediated homologous recombination technology to transfer an S1 gene into toxoplasma gondii, and obtains a transgenic toxoplasma gondii strain capable of expressing an S1 protein through screening and identification.

In a first aspect, the present invention provides a transgenic toxoplasma gondii strain expressing the S1 protein of the feline peritonitis virus, wherein the toxoplasma gondii UPRT gene is replaced with the S1 gene, and FIPV S1 protein is expressed, thereby obtaining the transgenic toxoplasma gondii strain; the nucleotide sequence of the S1 gene is shown as SEQ ID NO. 2.

The S protein consists of two subunits, S1 (receptor binding domain-RBD) and S2 (fusion domain). The S1 subunit is divided into two functional domains, the N-terminal domain (NTD) and the C-terminal domain (RBD), and binding to the receptor of cells is considered to be a determinant of viral tropism. The S1 protein has strong specificity, can avoid antigen cross reaction, and is an important protein for determining virus antigenicity and inducing neutralizing antibodies.

In a second aspect, the invention provides the use of a transgenic toxoplasma strain as described above for the prevention of infectious peritonitis in cats, said transgenic toxoplasma strain providing a specific immune response against cat abdominal viruses after immunization as a vaccine.

In the application provided by the invention, the initial toxoplasma strain for preparing the transgenic toxoplasma strain is a toxoplasma RH toxoplasma strain or other toxoplasma strains with delta Ku80 gene knocked out.

In a third aspect, the present invention provides a method for constructing the transgenic toxoplasma strain, comprising:

(1) Constructing a CRISPR/Cas9 plasmid pSAG1-CAS9-TgU6-sgUPRT plasmid;

(2) Constructing an expression vector pUPRT-Tub-Gra8ss-S1-3HA-UPRT: the vector is formed by sequentially connecting a 5 'homologous arm of a UPRT gene, a Tubulin gene, a gra gene, an S1 gene, an HA gene and a 3' homologous arm of the UPRT gene;

(3) The pSAG1-CAS9-TgU6-sgUPRT plasmid and an expression vector pUPRT-Tub-Gra8ss-S1-3HA-UPRT are co-transfected into toxoplasma, and a monoclonal insect strain expressing the S1 gene is obtained through screening and identification, namely the transgenic toxoplasma strain.

In the construction method of the transgenic toxoplasma gondii strain, the primer sequence for amplifying the S1 gene shown as SEQ ID NO.2 is shown as SEQ ID NO. 3-4.

In a fourth aspect, the invention provides a feline infectious peritonitis virus S1 protein expressed from the transgenic toxoplasma strain described above; the amino acid sequence of the S1 protein of the feline infectious peritonitis virus is shown as SEQ ID NO. 1.

The invention also provides application of the transgenic toxoplasma strain or the feline infectious peritonitis virus S1 protein in preparing medicines for preventing or treating feline peritonitis virus infection.

The application of the transgenic toxoplasma strain or the cat infectious peritonitis virus S1 protein in preparing cat peritonitis virus vaccine.

In a fifth aspect, the present invention provides a live vaccine, which is obtained by preparing a suspension from tachyzoites or cysts of the transgenic toxoplasma strain and a vaccine protecting agent. The live vaccine provided by the invention is a novel vaccine capable of preventing toxoplasma oocysts from being discharged and preventing cat coronaviruses.

The invention has the beneficial effects that: the invention provides a method for constructing a transgenic toxoplasma gondii strain by using a CRISPR/Cas9 technology, and the toxoplasma gondii strain is used as a live vector to express S1 protein of infectious peritonitis cat, so that the effect of simultaneously preventing toxoplasma felis and infectious peritonitis cat is achieved. Compared with the starting insect strain, the transgenic insect strain expressing the S1 gene has obviously reduced proliferation speed in vitro, and can induce stronger humoral immunity and certain cellular immunity after the mice are immunized by the transgenic insect strain expressing the S1 gene. Therefore, the transgenic insect strain expressing the S1 gene has the potential of preparing attenuated genetic engineering live vaccine for preventing toxoplasmosis and infectious peritonitis of cats.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing the insertion and identification of the S1 gene in example 1 of the present invention.

FIG. 2 is a graph showing the PCR identification of RH ΔKu80-S1 insect strains in example 1 of the present invention, wherein Clone5, clone9, clone11 and Clone16 represent different clones of transgenic insect strains RH ΔKu80-S1; in the figure, RH represents the wild strain. PCR (1) for identifying 5' homologous recombination (1515 bp); PCR (2) for identifying 3' homologous recombination (1010 bp); PCR (3) for identifying the gene coding region (187 bp) of UPRT; PCR (4) for identifying the S1 gene fragment (2241 bp).

FIG. 3 shows the results of intracellular proliferation rates of RH Δku80-S1 strain in example 2 of the present invention, and the ratio of the sodium worm vacuoles of RH Δku80 strain and RH Δku80-S1 strain at different proliferation stages was counted (100 sodium worm vacuoles were counted randomly for each strain).

FIG. 4 shows the plaque count and area size of RH Δku80 insect strain and RH Δku80-S1 insect strain in example 2 of the present invention.

FIG. 5 shows the toxicity test result of Toxoplasma gondii strain RH delta ku80-S1 in example 2 of the present invention.

Fig. 6 is a schematic diagram of the experimental procedure of the mouse immunoprotection experiment and the schematic diagram of the weight change of mice in the experimental group and the control group in example 2 of the present invention.

FIG. 7 shows ELISA detection of specific antibody levels against Toxoplasma in mouse serum in example 3 of the present invention.

FIG. 8 shows the detection of specific antibody levels against S1 in mouse serum by ELISA in example 3 of the present invention.

FIG. 9 shows the results of the detection of the change in the levels of antibodies and cytokines in serum at different times when mice were immunized in example 3 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The toxoplasma RH ΔKu80 strain used in the present invention is disclosed in (Huynh, M.H.and Carruther, V.B. (2009). Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80.Eukaryot Cell 8, 530-539.).

EXAMPLE 1 construction of Toxoplasma gondii S1 Gene-expressing insect Strain

The S1 gene expression insect strain is constructed by taking RH delta Ku80 as a basic insect strain.

RH ΔKu80 is a toxoplasma species type I insect strain that loses the ability to form cysts in animals. The S1 protein has strong specificity, can avoid antigen cross reaction, is an important protein for determining virus antigenicity and inducing neutralizing antibodies, and has the amino acid sequence shown in SEQ ID NO.1 and the nucleotide sequence shown in SEQ ID NO. 2.

1. Amplification of FIPV S1 Gene of interest

(1) Preparation of DNA templates

Collecting ascites from abdominal cavity of suspected case, adding 250 μl of ascites into 1.5ml centrifuge tube, adding 750 μl of Trizol, mixing, standing at room temperature for 10min, adding 0.2ml of chloroform, oscillating for 15s, and standing for 2min. Centrifuging at 4deg.C, 12000g×15min, and collecting supernatant. 0.5ml of isopropyl alcohol was added, and the liquid in the tube was gently mixed and allowed to stand at room temperature for 10min. Centrifuge at 4℃12000 g.times.10 min, discard supernatant. 1ml of 75% ethanol was added and the precipitate was gently washed. The supernatant was discarded at 4℃and 7500 g.times.5 min. Air drying, adding proper amount of DEPC H ₂ O-dissolution (dissolution promotion at 65 ℃ C. For 10-15 min). UsingNorth ViewIII 1st Strand cDNA Synthesis Kit (+gDNA wind) reverse transcription kit.

(2) PCR amplification

S1 primers were designed in NCBI (https:// www.ncbi.nlm.nih.gov /).

An upstream primer: S1F: TTGGCTGGCAATGAAAACCTTAT (SEQ ID NO. 3);

a downstream primer: S1R: GTAGTAATAAAAATTAGGTGTCGTTGTCCA (SEQ ID NO. 4).

Using the extracted genome DNA as a template, and adopting the primer to carry out PCR amplification, wherein a PCR reaction system and a PCR reaction program are as follows:

PCR reaction system: 1.5. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the DNA template, 1. Mu.L of dNTP solution, 10. Mu.L of 5 Xreaction buffer solution, 0.5. Mu.L of Q5 enzyme, and 50. Mu.L of the solution were made up with deionized water.

PCR reaction procedure: pre-denaturation at 98 ℃ for 30s; denaturation at 98℃for 15s; annealing at 68 ℃ for 2min; the extension temperature is 72 ℃ for 30s; cycling for 35 times; finally, the extension is carried out for 10min at 72 ℃.

2. Amplification of pUPRT-Tub-Gra8ss-S1-3HA-UPRT vector

The original pUPRT-Tub-Gra8ss-RBD-3HA-UPRT plasmid of a laboratory is taken as a template, and the primer sequence is as follows:

an upstream primer: backbone-F TTGCCAGCCAACAAAGGACCGTTCATGGC (SEQ ID NO. 5); a downstream primer: backbone-R TACTACGGTGGAGGTGGAAGTTACCCG (SEQ ID NO. 6);

fragment two:

an upstream primer: backbone-2Fw: AATCAGGTTCTCGTTGCCTGCCAACAAAGGACCG TTCATGGCGC (SEQ ID NO. 7); a downstream primer: backbone-2Rv: TACCCCTAACTTTTAC TACTACGGTGGAGGTGGAAGTTACCCG (SEQ ID NO. 8).

All the above DNA fragments were treated with the NEB Q5 high-fidelity DNA polymerase @ CoHigh-Fidelity DNA Polymerase) and PCR reactionThe reaction system is shown in Table 1.

TABLE 1PCR reaction System

The PCR reaction conditions are shown in Table 2.

TABLE 2PCR reaction conditions

Note that: q5 high-fidelity DNA polymerase extends at least 2kb per minute, so the extension time is determined by the specific amplified fragment length.

After the PCR reaction is finished, the target fragment is recovered by running gel, and gel is purified by using a gel recovery kit of TransGen Biotech companyQuick Gel Extraction Kit) purifying and recovering each DNA fragment.

(1) Fragment ligation

The purified DNA product was ligated using a multi-fragment seamless cloning kit from TransGen Biotech companyBasic Seamless Cloning and Assembly Kit). The reaction system is shown in Table 3.

TABLE 3 reaction conditions for the multi-fragment ligation

The reaction system was gently mixed and reacted at 50℃for 15min, after the reaction was completed, the mixture was cooled on ice. The product was transferred to 50. Mu.L of Trans1-T1 competent cells (TransGen Biotech, trans1-T1 Phage Resista nt Chemically Competent Cell), gently mixed, placed on ice for 30min, heat-shocked on a metal bath at 42℃for 1min, immediately thereafter onto ice for 2min. mu.L of LB medium was added and incubated for 1h at 250rpm on a shaker at 37 ℃. mu.L of the solution was uniformly spread on an ampicillin-resistant plate. After 24 hours, several monoclonal samples were picked for sequencing, and the sequencing primers were used to measure one reaction each using the universal primers M13F and M13R. Sequencing results showed no errors at the plasmid junction and plasmid construction was successful.

(2) The plasmid pUPRT-Tub-Gra8ss-S1-3HA-UPRT was extracted for use using the Beijing Aidelai Biotechnology Co.Ltd PL 04-endotoxin-free high-purity plasmid small-volume rapid extraction kit (centrifugal column type).

(3) The laboratory original plasmid pSAG1-CAS9-TgU6-sgUPRT was extracted for use using the PL 14-Large Scale plasmid extraction kit (Aidlab biotechnologies CO.Ltd).

3. Linearization of pUPRT-Tub-Gra8ss-S1-3HA-UPRT plasmid

(1) The 5 'and 3' homology arms of the UPRT gene are amplified by using the Q5 high-fidelity DNA polymerase and the pUPRT-Tub-Gra8ss-S1-3HA-UPRT plasmid with correct sequence as a template, and the amplification primer sequences are as follows:

an upstream primer: TCTGGCCCCCCTGTTTCGGTTG (SEQ ID NO. 9);

a downstream primer: GAAGAGAGAAGTTGTGTGCT (SEQ ID NO. 10).

(2) 50 mu L of reaction system is amplified into 4 tubes, 3 mu L of PCR products are taken for gel running identification, and if target bands exist, DNA purification is carried out on the rest PCR products, and the operation steps are as follows:

a: adding 10 times volume of absolute ethyl alcohol into the PCR product, and standing at-20 ℃ for more than 1 h;

b: centrifuging at 10000rpm for 10min at 4deg.C, and discarding supernatant;

c: adding 500 μL of 75% ethanol, centrifuging at 10000rpm at 4deg.C for 5min, discarding supernatant, volatilizing ethanol, adding 50 μL of sterile water to dissolve precipitate, and preserving at-20deg.C.

4. Construction of Toxoplasma gondii expressing exogenous protein S1

(1) Collect 5X 10 ⁶ The freshly released Δku80 RH tachyzoites were filtered off with a 5 μm filter to remove cell debris and centrifuged at 2000rpm for 9min, and the supernatant was discarded.

(2) Add 2ml cytomix buffer (120mM KCl,0.15mM CaCl) ₂ ，10mM K ₂ HPO ₄ /KH ₂ PO ₄ ，25mM HEPES，2mM EGTA，5mM MgCl ₂ Ph=7.6) the pellet was resuspended, centrifuged at 2000rpm for 5min and the supernatant discarded.

(3) mu.L of cytomix buffer was taken, and pSAG1-CAS9-TgU6-sgUPRT plasmid (50. Mu.g) and UPRT homologous recombination template (10. Mu.g) were added in a volume of about 100. Mu.L, mixed well, and added to an electric beaker having a diameter of 4 mm.

(4) Setting an electrotometer program: voltage 2000V, capacitance 25F, resistance infinity. Toxoplasma transfection, inoculation into Vero cells, and 24h later, replacement into culture medium containing 3 mu M5-fluorodeoxyuridine.

(5) Screening of monoclonal: after three generations of drug screening, monoclonal screening was performed. A bottle of confluent HFF cells (T25) was digested, added to a 96-well plate (100. Mu.L per well volume), worms were collected, counted with a cell counting plate, diluted to 300 tachozoites/ml, 100. Mu.L worms were added to each end of the 96-well plate, and serial multiple dilutions (30, 15, 7.5, 3.75, 1.875, 0.9375) were performed, and 100. Mu.L of medium was supplemented per well after the dilution was completed.

(6) Culturing in a cell culture incubator (37 ℃,5% CO) ₂ ) After 7 days, wells with only one plaque were selected under a microscope and transferred to a 12-well plate confluent with Vero cells for further 3 days.

(7) The monoclonal insect strain in a part of 12-well plates is taken for identification (Tiangen biochemical technology (Beijing) limited company, blood/cell/tissue genome DNA extraction kit), and the DNA extraction method refers to the cell DNA extraction method of the kit. The identification primers were as follows:

5’-UPRT-Fw：TCCCTTCCAACTCTCCGCTT(SEQ ID NO.11)；

5’-UPRT-Rv：ATGGCGCGAGCTACACCAAA(SEQ ID NO.12)(PCR1)；

3’-UPRT-Fw：TTTCAGATTTGCAAAAGTCC(SEQ ID NO.13)；

3’-UPRT-Rv：TAGTTCAAATAACTCGATAAATTA(SEQ ID NO.14)(PCR2)；

UPRT-CDS-Fw：TTCCCAATGTGGTGCTCATGAAG(SEQ ID NO.15)；

UPRT-CDS-Rv：ATCGATTCGACGCGGCTTCCT(SEQ ID NO.16)(PCR3)

S1-Fw：TTGGCAGGCAACGAGAACCTGATT(SEQ ID NO.17)；

S1-Rv：GTAGTAGTAAAAGTTAGGGGTA(SEQ ID NO.18)(PCR4)。

the S1 gene insertion and identification strategy is shown in figure 1, the PCR identification result is shown in figure 2, wherein the transgenic insect strain PCR1/PCR2/PCR4 has target bands, and the PCR3 has no target band; the wild insect strain PCR1/PCR2/PCR4 has no target band, and the PCR3 has target band, so the transgenic insect strain is a positive monoclonal insect strain. The identified S1 gene insert strain was designated RH Δku80-S1 strain.

Example 2 detection of proliferation Rate, virulence and immunoprotection of RH Δku80-S1 insect strains

1. In vitro proliferation assay of RH delta ku80-S1 insect strain

The intracellular proliferation rate of the toxoplasma strain RH delta ku80-S1 expressing the S1 protein constructed in example 1 is detected, and the specific method is as follows:

collecting the tachyzoites of the freshly released RH delta Ku80 and RH delta Ku80-S1 insect strains, and inoculating 10 respectively ⁵ Tachyzoites were plated into 12-well plates (sterile cell slide prior to plating) filled with HFF cells (human foreskin fibroblasts, purchased from ATCC). After 1h inoculation, uninjured worms were washed off and the culture was continued in an incubator. After 24 hours or 48 hours of cultivation, IFA test was performed as follows:

(1) toxoplasma-infected cells were fixed at 37℃for 30min in 4% paraformaldehyde (1 mL per well).

(2) Permeabilization was carried out in 0.25% Triton X-100 (500 μl per well) for 30min at 37deg.C.

(3) Blocking was performed in 3% BSA (500 μl per well) at 37deg.C for 30min.

(4) A primary antibody against the rabbit-derived Toxoplasma gondii GAP45 protein (Plattner, F., yarovinsky, F., romero, S., didry, D., carlier, M.F., sher, A.and Soldati-Favre, D. (2008) Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12response.CELL HOST MICROBE 3,77-87.) was added and incubated at 37℃for 1h and washed 3 times with PBS.

(5) Secondary anti-FITC/Cy 3-labeled goat anti-mouse IgG (h+l) and nuclear dye Hoechst33258 (both purchased from beijing michaeli technologies limited) were added and incubated at 37 ℃ for 1H in the dark. The PBS was washed 3 times.

(6) 10 mu L of anti-fluorescence quenching agent sealing sheet is added on the flying sheet, and the number of tachyzoites in the artemia cavity membrane is counted under a fluorescence microscope.

As a result, as shown in FIG. 3, after 24 hours of growth in HFF cells, it was found that RH ΔKu80-S1 was able to undergo normal binary division and form a nano-insect vacuole, but the proliferation rate was significantly slower and the proliferation rate was reduced by 34.25% as compared with the starting strain. RH ΔKu80-S1 can form plaques, but the area and number of plaques formed are significantly different from those of the starting strain, see FIG. 4 (p < 0.05).

2. Mouse virulence experiment of RH delta ku80-S1 insect strain

The virulence of the toxoplasma strain RH delta ku80-S1 expressing the S1 gene constructed in example 1 is detected by the following specific method: intracellular RH Deltaku 80-S1 strain was collected, and BALB/C mice (female, 5 weeks) were inoculated intraperitoneally at a dose of 100 tachyzoites, while 100 RH DeltaKu 80 tachyzoites and the same volume of PBS were inoculated simultaneously as a control group, 5 mice per group. The 3 groups of mice were individually kept in the same environment, and survival of the mice was recorded daily for 10 days.

The results are shown in FIG. 5. The results showed that mice vaccinated with the Δku80 RH strain died all over day 8, while mice vaccinated with the RH Δku80-S1 strain died all over day 9. There was no significant difference in toxicity of RH Δku80-S1 from RH ΔKu80.

3. Mouse immunoprotection experiments with RH Δku80-S1 insect strains

The RH delta ku80-S1 strain is adopted to immunize mice, the immune protection efficacy, immunization, toxicity attack, detection and other experimental procedures are shown in the figure 6, and the specific method is as follows:

(1) 6 weeks of ageBALB/C mice were immunized 10 each ³ RH Δku80-S1 tachyzoites (immunized group), in non-immunized mice (non-immunized group), immune protein group (positive control group) and immunization 10 ³ RH Δku80 tachyzoites served as controls (control group), 15 mice per group, each group being kept under the same conditions.

The secondary immunization was performed 14 days later, blood was collected weekly from the beginning of the primary immunization, serum was isolated, and stored at-80℃for use (for humoral and cellular immune monitoring of example 3).

(2) After 21 days of second immunization, three mice were taken from each group to examine toxoplasma in their brain, lung, spleen, liver, ascites and to laparoscopically attack the remaining immunized and non-immunized mice, 10 ⁴ /only. The results show that RH Δku80-S1 immunized mice and 10 ³ When the RH delta ku80 control group is infected with different toxoplasma strains, no death is caused, and the non-immune group and the protein immune group all die in the acute phase.

EXAMPLE 3 monitoring of humoral and cellular immune response of RH Δku80-S1 insect strain immunized hosts

The humoral immunity and the cellular immune response of mice immunized with the RH delta ku80-S1 strain in example 2 were examined by the following methods:

detection of toxoplasma related IgG antibodies in mouse serum:

(1) Antigen coating: toxoplasma whole insect antigen was prepared by adding 100. Mu.L (5. Mu.g/ml of antigen coating solution) to 96-well plates overnight at 4 ℃.

(2) Closing: 300 μl of PBST was added to each well, the shaking table was spun dry at 200r/min for 5min, the washing was repeated 3 times, and the mixture was blocked at 5% BSA for 1 hour at room temperature.

(3) Primary antibody (mouse serum) was added: the mixture was washed 5 times in the same manner as in the step (2), and 100. Mu.L (1:100 dilution) of mouse serum was added thereto and incubated at 37℃for 1 hour.

(4) Adding a secondary antibody: washing 5 times in the same step (2), adding HRP-marked goat anti-mouse IgG secondary antibody and incubating for 1 hour at 37 ℃.

(5) Color development: washing 5 times in the same step (2), adding TMB, and developing at 37 ℃ for 15min.

(6) And (3) terminating: the reaction was terminated by adding 2mol/L sulfuric acid solution, and the value of 450nm was immediately read on a microplate reader. The level of specific antibodies against toxoplasma in the serum of mice is shown in figure 7.

The detection of novel coronavirus S1 protein-related IgG antibodies in mouse serum was also performed by ELISA, and the S1 protein used for coating was prepared by prokaryotic expression, the coating concentration was 2. Mu.g/ml, and the specific detection method was referred to the ELISA detection method above. The level of specific antibodies against S1 in mouse serum is shown in figure 8.

Concentration of cytokines (IFN-. Gamma., TNF-. Alpha.) in serum: the detection was also performed by ELISA method, and specific detection method was referred to above ELISA detection method, and cytokine kit was purchased from the biotechnology company, inc (Dakewe Biotech co., ltd.).

The results are shown in FIG. 9, and the RH delta ku80-S1 strain immunized mice were significantly higher in serum than the non-immunized group when the mice were immunized for 14 days for both first and second immunization; the concentration of IFN-gamma, TNF-alpha cytokines in serum increased significantly after 14 days of immunization. The result shows that the RH delta ku80-S1 insect strain can provide better humoral immunity and cellular immune response for the host after being immunized.

In summary, the invention provides a toxoplasma live vaccine expressing S1 protein, and determines safe immune dosage and immune program. Through mouse experiments, the toxoplasma strain expressing the S1 protein can generate stronger humoral immune response and cellular immune response level after being immunized. The infection of the toxoplasma strain and the cat abdominal transmission virus can provide effective protection, and the secondary immunity can provide long-term protection effect. Therefore, the S1 gene insert strain has the potential of preventing the discharge of toxoplasma oocysts and preventing new coronaviruses.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (9)

1. The transgenic toxoplasma strain for expressing the S1 protein of the feline peritonitis virus is characterized in that a toxoplasma UPRT gene is replaced by an S1 gene, and FIPV S1 protein is expressed to obtain the transgenic toxoplasma strain; the nucleotide sequence of the S1 gene is shown as SEQ ID NO. 2.

2. Use of a transgenic toxoplasma strain according to claim 1 for the prevention of infectious peritonitis in cats, wherein said transgenic toxoplasma strain provides a specific immune response against cat abdominal viruses after immunization as a vaccine.

3. The use according to claim 2, wherein the starting strain from which the transgenic toxoplasma gondii strain is prepared is a toxoplasma gondii RH strain or other toxoplasma gondii strain from which the Δku80 gene is knocked out.

4. The method for constructing a transgenic toxoplasma strain according to claim 1, comprising:

(1) Constructing a CRISPR/Cas9 plasmid pSAG1-CAS9-TgU6-sgUPRT plasmid;

(2) Constructing an expression vector pUPRT-Tub-Gra8ss-S1-3HA-UPRT: the vector is formed by sequentially connecting a UPRT gene 5 'homology arm, a Tubulin gene promoter, a gra gene, an S1 gene, an HA gene and a UPRT gene 3' homology arm;

(3) The pSAG1-CAS9-TgU6-sgUPRT plasmid and an expression vector pUPRT-Tub-Gra8ss-S1-3HA-UPRT are co-transfected into toxoplasma, and a monoclonal insect strain expressing the S1 gene is obtained through screening and identification, namely the transgenic toxoplasma strain.

5. The construction method according to claim 4, wherein the primer sequence for amplifying the S1 gene shown in SEQ ID NO.2 is shown in SEQ ID NO. 3-4.

6. The S1 protein of the cat infectious peritonitis virus, which is characterized by being expressed by the transgenic toxoplasma strain in claim 1; the amino acid sequence of the S1 protein of the feline infectious peritonitis virus is shown as SEQ ID NO. 1.

7. Use of a transgenic toxoplasma strain according to claim 1 or a feline infectious peritonitis virus S1 protein according to claim 6 for the manufacture of a medicament for preventing or treating feline peritonitis virus infection.

8. Use of the transgenic toxoplasma strain of claim 1 or the S1 protein of feline infectious peritonitis virus of claim 6 for the preparation of a feline peritonitis virus vaccine.

9. A live vaccine, characterized in that the tachyzoite or the capsule of the transgenic toxoplasma strain of claim 1 and a vaccine protecting agent are prepared into a suspension.