Trichinella spiralis vaccine kit based on prime-boost sequential immunization program and use thereof

Abstract

Provided are a Trichinella spiralis vaccine kit based on a prime-boost sequential immunization program and use thereof, improving the protective effect against trichinosis. The kit is divided into two parts; the first part is a Trichinella spiralis recombinant protein vaccine having an amino acid sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 2, and the second part is a recombinant adenovirus expressing an amino acid sequence set forth in SEQ ID NO. 1. The prime-boost immunization strategy not only effectively avoids the deficiency associated with a single-type Trichinella spiralis vaccine, but also significantly improves the efficacy and durability of the immune response.

Description

A Trichinella spiralis vaccine kit based on a Prime-boost sequential immunization program and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Trichinella spiralis vaccine kit based on the Prime-boost sequential immunization program and its application. Background Technology

[0002] Trichinosis is a zoonotic parasitic disease caused by the nematode *Trichinella spiralis*. It poses a significant threat to livestock farming, leading to stunted animal growth, decreased meat quality, and even death, resulting in direct economic losses. Furthermore, humans who consume meat infected with *Trichinella spiralis* may also contract the disease, experiencing serious health problems and further impacting food safety and public health. Therefore, trichinosis not only affects the sustainable development of livestock farming but also poses a potential risk to human health, necessitating effective prevention and control measures to ensure the safety of livestock production and human health.

[0003] Despite the severe impact of trichinosis on animal economy and livestock farming, there is currently no effective vaccine for trichinosis. Traditional vaccine development methods, such as live attenuated vaccines and natural antigen vaccines, are insufficient because the antigenic composition of Trichinella spiralis is complex, and Trichinella spiralis cannot complete its entire life cycle through in vitro culture. Furthermore, it is difficult to prepare such antigens through standardized mass production methods using artificially infected animals, thus failing to meet the needs for antigens used in immunodiagnosis and immunoprophylaxis. With the rapid development of genetic engineering technology, novel vaccines such as recombinant protein vaccines, peptide vaccines, and DNA vaccines have been continuously explored and applied in the field of trichinosis control. While they possess some protective efficacy, none have achieved complete protection against Trichinella spiralis infection. Summary of the Invention

[0004] The purpose of this invention is to improve the protective effect against trichinosis.

[0005] The present invention provides a Trichinella spiralis vaccine kit, which is divided into two parts: the first part is a Trichinella spiralis recombinant protein vaccine with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the second part is a recombinant adenovirus expressing an amino acid sequence as shown in SEQ ID NO.1.

[0006] Further specifying, the content of the Trichinella spiralis recombinant protein is 60 μg; the content of the recombinant adenovirus expressing the amino acid sequence as shown in SEQ ID NO.1 is 10 μg. 8 PFU; the recombinant trichinella protein vaccine is administered via intramuscular injection; the recombinant adenovirus expressing the amino acid sequence shown in SEQ ID NO.1 is administered via nasal drop injection.

[0007] Further specifying, the Trichinella recombinant protein vaccine is a Trichinella recombinant protein with amino acids as shown in SEQ ID NO.2, and the ratio of the CPG1018 adjuvant is 1:1 / v:v.

[0008] This invention provides the application of the above-mentioned kit in the preparation of drugs for the prevention or treatment of trichinosis.

[0009] The present invention provides a recombinant protein of Trichinella spiralis, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] The present invention provides the nucleic acid molecule of the above-mentioned vaccine.

[0011] This invention provides a recombinant vector for the above-mentioned nucleic acid molecules.

[0012] The present invention provides recombinant microbial cells containing the above-mentioned nucleic acid molecules.

[0013] The present invention provides the use of the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or the above-mentioned recombinant microbial cell in the preparation of vaccines or drugs for the prevention or treatment of trichinosis.

[0014] Further specifying, vaccines are available in the form of nasal sprays, oral preparations, suppositories, or parenteral preparations.

[0015] Beneficial Effects: By using a recombinant adenovirus vaccine for primary immunization and a recombinant protein vaccine for booster immunization, the immunogenicity of the vaccines can be effectively improved, thereby stimulating the body to produce a stronger immune response. This strategy combines the highly effective immunogenicity of recombinant adenovirus vaccines with the high safety of recombinant protein vaccines, synergistically enhancing the immune response and providing the body with robust immune protection. Furthermore, the prime-boost immunization regimen can also stimulate the body to form long-term immune memory, meaning that once the body is reinfected with the parasite, it can quickly initiate a strong immune response to rapidly clear the pathogen, thereby effectively reducing the risk of disease recurrence.

[0016] The Prime-Boost immunization strategy has demonstrated unique advantages in the development of Trichinella spiralis vaccines, aiming to overcome the limitations of single-type vaccines. This strategy first utilizes a recombinant adenovirus vaccine for primary immunization, which efficiently and briefly activates humoral and cellular immunity. This includes stimulating B lymphocytes to produce antibodies such as IgG secreted by plasma cells to neutralize the virus, and activating cytotoxic T lymphocytes to recognize and kill virus-infected target cells, thereby clearing the virus. However, homologous immunization with Trichinella spiralis recombinant adenovirus vaccines may cause pre-existing immune problems, reduced immunogenicity, and adverse reactions such as fever. Trichinella spiralis recombinant protein vaccines, as booster shots, can compensate for the shortcomings of recombinant adenovirus vaccines. They have strong immunogenicity, significantly increase neutralizing antibody levels, and are highly safe with few adverse reactions, good stability, and less demanding storage and transportation requirements. Therefore, the Prime-Boost immunization strategy not only effectively avoids the shortcomings of single-type Trichinella spiralis vaccines but also significantly improves the efficacy and durability of the immune response, providing the body with more efficient and comprehensive immune protection.

[0017] Adenovirus vaccines have the issue of pre-existing immunity; if both doses are administered with adenovirus vaccine, the immunization effect may be affected. Recombinant protein vaccines require adjuvants and 2-3 doses. Considering that trichinosis vaccines are mainly used in pigs (pigs are the primary source of infection), and pigs have a limited time to market (approximately six months), adenovirus vaccines do not require adjuvants and are highly effective with a single dose. Therefore, using adenovirus vaccines for initial immunization activates the immune system, and booster immunization requires only one dose of recombinant protein vaccine, avoiding the problem of a long immunization cycle. Attached Figure Description

[0018] Figure 1 shows the recombinant adenovirus plasmid rAd5TsCLP and its identification results. M is the DL15000 DNA Marker; lane 1 shows the recombinant adenovirus plasmid rAd5TsCLP; lane 2 shows the PacI restriction enzyme linearization identification results.

[0019] Figure 2 shows the results of IFA analysis of Ts-CLP protein expression in HEK293A cells infected with recombinant adenovirus rAd5TsCLP.

[0020] Figure 3 shows the prediction results of the signal peptide of Trichinella spiralis protein;

[0021] Figure 4 is a flowchart of prokaryotic expression and immunization of recombinant protein;

[0022] Figure 5 shows the results of SDS-PAGE and WB verification of Ts-CLP protein expression in the Trichinella spiralis recombinant protein vaccine.

[0023] Figure 6 shows the Prime-boost animal immunization program and specific immunization dosage results; A is the animal immunization program; B is the immunization dosage results.

[0024] Figure 7 shows the protective analysis results of rAd5TsCLP-immunized mice after being challenged with 250 Trichinella spiralis muscle larvae. A represents the average number of larvae per gram of muscle (LPG), and B represents the average number of AD3 adult worms per gram of muscle. The data are mean ± SEM.

[0025] Figure 8 shows the results of HE staining of diaphragmatic tissue;

[0026] Figure 9 shows the results of detecting the levels of specific antibodies against IgG, IgG1, IgG2a, IgA, IgM, and NAb in mouse serum (*, P<0.05; **, P<0.01; ***, P<0.001); A: Detection results of IgG specific antibody levels in mouse serum; B: Detection results of IgG1 specific antibody levels in mouse serum; C: Detection results of IgG2a specific antibody levels in mouse serum; D: Detection results of IgA specific antibody levels in mouse serum; E: Detection results of IgM specific antibody levels in mouse serum; F: Detection results of neutralizing antibody NAb levels in mouse serum.

[0027] Figure 10 shows the cytokine levels in mouse serum (*, P < 0.05; **, P < 0.01; ***, P < 0.001); A shows the IFN-γ level in mouse serum; B shows the TNF-α level in mouse serum; C shows the IL-4 level in mouse serum; D shows the IL-10 level in mouse serum.

[0028] Figure 11 shows the number of CD3+CD4+, CD3+CD8+, and CD3+CD4+CD8+ T lymphocytes (*, P<0.05; **, P<0.01; ***, P<0.001). Detailed Implementation

[0029] Example 1: Preparation of Recombinant Adenovirus Vaccine

[0030] 1. Construction of rAd5TsCLP recombinant adenovirus

[0031] The Ts-CLP target gene was obtained from PubMed, and its CDS sequence (SEQ ID NO.3) was designed to optimize its expression in mammalian cells. The full-length sequence is 1218 bp. The Ts-CLP fragment was amplified and identified. A recombinant adenovirus expressing the Ts-CLP gene was constructed using the E1 and E3-deleted type 5 adenovirus AdEasy system. The pShuttle-CMV-Ts-CLP shuttle plasmid was constructed, transformed into DH5α competent cells for amplification, linearized by PmeI digestion, and then transformed into BJ5183 bacteria already carrying the pAdEasy-1 plasmid for homologous recombination. Finally, selection was performed using kanamycin.

[0032] SEQ ID NO.3:

[0033] A small number of amplified bacteria were used to extract plasmids, which were then identified by PacI digestion. PacI digestion of the recombinant adenovirus plasmid yielded a large fragment of approximately 30 kb and a smaller fragment of 3 kb or 4.5 kb. The correctly identified recombinant adenovirus plasmid rAd5TsCLP was transformed into XL10-Gold supercompetent bacteria for amplification. The linearized recombinant adenovirus plasmid, after PacI digestion and DNA purification, was used for adenovirus packaging (Figure 1).

[0034] Packaging of 2.rAd5TsCLP recombinant adenovirus

[0035] 7-8×10 5 HEK293A cells / mL were transferred to 60-mm cell culture dishes and cultured for 24 hours. The recombinant adenovirus plasmid, digested with PacI, was transfected into the overnight-cultured HEK293A cells using Lipofectamine 3000. Six hours after transfection, the cells were replaced with fresh cell culture medium and cultured for another 7 to 10 days. The cells clearly exhibited a floating state, and the adenovirus was collected to obtain the recombinant adenovirus vaccine rAd5TsCLP.

[0036] 3. Collection and amplification of rAd5TsCLP recombinant adenovirus

[0037] Adenovirus packaging cell culture medium was aspirated, and cells were scraped off with 0.5 mL of PBS and transferred to centrifuge tubes. Cells were lysed by repeated freeze-thaw cycles at -80°C and 37°C four times. The supernatant was collected by centrifugation to obtain P0 generation adenovirus, which was stored for later use. HEK293A cells were infected with P0 generation virus for amplification. After small-scale amplification and repeated freeze-thaw cycles, P1 generation virus was obtained. This process was repeated for three generations up to P4 for large-scale amplification, purification, and concentration. After titer determination, recombinant adenovirus rAd5TsCLP was aliquoted and stored. The results showed that IFA confirmed that rAd5TsCLP expressed Ts-CLP protein in HEK293A cells (Figure 2).

[0038] SEQ ID NO.1:

[0039] Example 2: Preparation of Recombinant Protein Vaccine

[0040] 1. Preparation of expression bacteria

[0041] SignalP 6.0 software was used to predict the signal peptide in this sequence. The advantage of using a prokaryotic expression system to remove the signal peptide (amino acid sequence such as SEQ ID NO.2) is that it can avoid protein insolubility, inclusion body formation and potential processing problems caused by the signal peptide, thereby improving the efficiency of soluble protein expression and purification (Figure 3).

[0042] SEQ ID NO.2:

[0043] The Trichinella spiralis protein gene without the signal peptide was cloned into the pET-28a(+) vector and transformed into BL21 competent cells. After overnight culture, single clones were picked and cultured in LB medium to prepare the expression strain pET28a-TsCLP, which was then stored at -80℃.

[0044] 2. Recombinant protein-induced expression

[0045] The expression strain pET28a-TsCLP, stored at -80℃, was taken out and streaked onto an LB agar plate containing kanamycin using an inoculation loop. A single colony was picked from the LB agar plate and inoculated into 5 mL of LB liquid medium. The culture was then expanded using 1 L of liquid LB medium. When the OD600 of the bacterial culture was measured to be 0.4-0.6 using a microplate reader, 1 mL of 1 mol / L IPTG was added, and the culture was incubated on a horizontal shaker at 37℃ for 6 h. The induced bacterial culture was aliquoted into 500 mL centrifuge bottles, centrifuged at 5000 g for 30 min, the supernatant was discarded, and the bacterial pellet was resuspended in PBS. The resuspended bacterial pellet was added to 50 mL centrifuge tubes and subjected to three freeze-thaw cycles. The resuspended bacterial pellet was then subjected to ultrasonic disruption under the following conditions: ultrasonic power 200 W, duration 3 s, interval 3 s, total time 45 min, and temperature 4℃. Centrifuge 5000g of the ultrasonically disrupted bacterial solution for 30 minutes, discard the supernatant, and collect the bacterial precipitate.

[0046] 3. Gel purification

[0047] The purified recombinant protein sample was mixed thoroughly with protein loading buffer in a specific ratio, boiled, and then subjected to SDS-PAGE electrophoresis. After electrophoresis, the stacking gel was removed, and the separating gel was placed in 2.5% KCl solution and then placed in a 4°C refrigerator. After 5 minutes, a white band appeared on the stacking gel, which was cut off with a sterile knife. The white band was chopped and placed in a 2 mL centrifuge tube, and an appropriate amount of autoclaved PBS solution was added. The tube was then incubated overnight at 4°C with shaking. After centrifugation, the supernatant was collected by pipette, and the target protein was concentrated to the desired concentration by centrifugation using a sterile ultrafiltration tube. Endotoxins in the purified protein were removed using a large-volume endotoxin removal kit to obtain the recombinant protein rTsCLP. The Ts-CLP protein was then validated by SDS-PAGE and Western blot (Figure 5).

[0048] The recombinant protein vaccine rTsCLP was prepared by adjuvant and amino acid formulations as shown in SEQ ID NO.2, in a 1:1 / v:v ratio, followed by immunization.

[0049] Example 3: Prime-boost Immunization Vaccine Kit

[0050] 1. Kit composition: Part 1 is the recombinant protein obtained in Example 2; Part 2 is the recombinant adenovirus obtained in Example 1.

[0051] 2. Specific protocols for strategic animal experiments

[0052] Six- to eight-week-old female BALB / c mice were randomly divided into five groups of ten mice each. Ten mice were vaccinated intranasally at week 0. 8 In week 2, PFU rAd5TsCLP was administered via bilateral intramuscular injection in the thigh, with 60 μg of recombinant protein vaccine rTsCLP (rTsCLP:CPG1018 = 1:1 / v:v) for a total immunization dose of 100 μL (rAd5TsCLP:rTsCLP). Simultaneously, two immunizations with the recombinant protein vaccine (rTsCLP:rTsCLP) and a booster immunization with the recombinant protein vaccine (Ad5Neg:rTsCLP) from the initial immunization served as controls. At each immunization time point, an equal volume of PBS was administered as a blank control. 8Empty adenovirus Ad5Neg (PFU) without the target gene was used as an empty vector control. Two weeks after the last immunization, each mouse was orally infected with 250 Trichinella spiralis muscle larvae. Mice were euthanized on day 3 and at week 5 post-infection, and the number of adult intestinal worms and muscle larvae was measured. The number of muscle larvae per gram of muscle (LPG) was calculated based on the number of collected muscle larvae and muscle weight. The worm reduction rate was calculated by comparing the average LPG of each group with the PBS control group. Serum, spleen, and small intestine were collected at 1, 3, and 9 weeks to detect humoral immunity, cellular immunity, and mucosal immunity levels in each group. Simultaneously, the diaphragm of mice was stained with hematoxylin and eosin (HE) for parasite load assessment (Figure 6).

[0053] Example 4: Evaluation of the Immunization Effect of the Prime-boost Immunization Strategy

[0054] 10 were administered via both intramuscular injection and nasal drops. 8 PFU recombinant adenovirus rAd5TsCLP was injected into 6-8 week old female BALB / c mice (10 mice per group). A treatment with adenovirus Ad5Neg was set up as a vaccine control, and a treatment with PBS was set up as a blank control.

[0055] Detection of adult intestinal worms and muscle larvae in mice: Mice were euthanized on day 3 and week 5 after Trichinella infection. The number of adult intestinal worms and muscle larvae was measured, and the reduction rate was calculated. The number of muscle larvae in mice was determined using a pepsin digestion method. The specific method is as follows: Pepsin and hydrochloric acid were added to distilled water at 37°C to prepare a digestion solution. The final concentration of both pepsin and hydrochloric acid was 1%. Each mouse was digested with 500 mL of the digestion solution and placed in a 37°C incubator with a magnetic stirrer for 2 hours. After the entire digestion process, the residue in the digestion solution was filtered out through a 60-mesh sieve. The solution was allowed to stand at room temperature for 1 hour. The supernatant was gently aspirated with a sterile syringe, and 100 mL of the liquid was collected. 400 mL of distilled water was added, and the solution was washed repeatedly. Finally, the liquid was transferred to a 50 mL beaker, and the Trichinella muscle larvae were counted under a microscope.

[0056] 1. Calculation of the reduction rate of muscle larvae

[0057] 500 mL of digestive solution was prepared by adding 1% pepsin and hydrochloric acid to distilled water at 37°C for 2 hours of digestion in each mouse. After digestion, the residue was filtered through a 60-mesh sieve, allowed to stand for 1 hour, and the supernatant was collected, with 100 mL retained. After repeated washing, the residue was transferred to a 50 mL beaker. The number of Trichinella spiralis muscle larvae was counted under a microscope, and the number of muscle larvae per gram of muscle (LPG) in each mouse was calculated. The results were compared with the PBS control group to calculate the larval reduction rate of each experimental group, using the following formula:

[0058] The results showed that, 5 weeks after infection, compared with the PBS group, the myolarial load of Ad5Neg (adenovirus empty vector), Ad5Neg:rTs-CLP (adenovirus empty vector primary immunization, recombinant protein secondary immunization), rAd5TsCLP:rTs-CLP (adenovirus primary immunization, recombinant protein secondary immunization), and rTs-CLP:rTs-CLP (recombinant protein two immunizations) decreased by 9.88%, 21.29%, 58.22%, and 53.39%, respectively, and the adult load decreased by 9.28%, 34.36%, 61.17%, and 54.3%, respectively (Figure 7).

[0059] Combined immunization with rAd5TsCLP reduced rTsCLP myolarvae by 58.22%, while immunization with rAd5TsCLP alone reduced them by 45.61% via intramuscular injection and 47.91% via nasal drops.

[0060] Combined immunization with rAd5TsCLP reduced adult rTsCLP worms by 61.17%, while immunization with rAd5TsCLP alone reduced worms by 36.49% via intramuscular injection and 42.57% via nasal drops.

[0061] 2. HE staining of diaphragmatic tissue

[0062] After dehydration, clearing, paraffin embedding, and other steps, the diaphragm tissue was sectioned and then stained with hematoxylin and eosin (HE) to observe its tissue structure and pathological changes. The results showed that, compared with the PBS and Ad5Neg groups, the rAd5TsCLP:rTs-CLP cysts showed more severe inflammatory cell infiltration and entered the cyst to directly contact the parasite (Figure 8).

[0063] 3. Specific antibody levels

[0064] Specific antibody ELISA detection

[0065] Specific antibodies (IgG, IgG1, IgG2a, IgM, IgA) in the serum of mice in each group were detected by indirect enzyme-linked immunosorbent assay (ELISA). 5 μg / mL of purified recombinant protein rTsCLP was coated onto 96-well plates and incubated overnight at 4°C. Subsequently, the plates were blocked with 100 μL / well blocking buffer (5% skim milk) at 37°C for 1 hour. The collected serum was serially diluted 10-fold, incubated at 37°C for 1 hour, and then incubated with goat anti-mouse IgG-HRP. Finally, TMB substrate was added for color development, and the reaction was terminated with 2M H2SO4 sulfuric acid. The OD450 value was measured using a microplate reader.

[0066] 4. Neutralizing antibody (NAb) detection

[0067] The collected serum was inactivated at 56°C for 30 min, and then serially diluted 10-fold in 96-well plates (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 in 100 μL DMEM), and mixed with an equal volume of 100 TCID50. 50 Recombinant adenovirus rAd5TsCLP was mixed and incubated at 37°C for 1 hour. Then, 200 μL of the serum-virus mixture was added to a monolayer of HEK293A cells. Ad5Neg and HEK293A cells served as controls. The cells were incubated at 37°C and 5% CO2 for 72 hours, and the expression of viral fluorescent protein was observed. The neutralizing antibody titer was calculated. A positive result for neutralizing antibodies was defined as a reduction in the number of green fluorescent lesions in the diluted sample compared to the infected control group by more than 50%.

[0068] The results showed that rAd5TsCLP:rTs-CLP could stimulate higher levels of IgG, IgG1, IgG2a, IgA, IgM and neutralizing antibody NAb. After the challenge, the levels of these antibodies continued to rise throughout the entire life cycle of the parasite from invasion to completion of its life cycle. This may mean that the body's immune system continuously strengthens its defense against parasite invasion in an attempt to resist the potential harm caused by the parasite (Figure 9).

[0069] The average antibody titers of neutralizing antibodies (NAb) at week 9 (35 days post-challenge) were as follows: PBS group: 40; Ad5Neg group: 40; Ad5Neg:rTsCLP group: 320; rTsCLP:rTsCLP group: 640; rAd5TsCLP:rTsCLP group: 1024 (confirmed after serial dilution and retesting). At week 7 (35 days post-challenge) after a single intramuscular injection in the rAd5TsCLP group, the average antibody titer was 512 (confirmed after serial dilution and retesting). At week 7 (35 days post-challenge) after a single nasal drop injection in the rAd5TsCLP group, the average antibody titer was 320.

[0070] 5. Serum cytokine detection

[0071] The secretion levels of IFN-γ, TNF-α, IL-4, and IL-10 cytokines in mouse serum were detected using the Luminex detection platform.

[0072] The results showed that, compared with other immunization groups, the concentrations of IFN-γ, TNF-α, IL-4, and IL-10 cytokines in the serum of mice in the rAd5TsCLP:rTsCLP group were the highest. Although Th2 response and mucosal immunity play an important role in the clearance of Trichinella spiralis, Th1 response also plays an equally important role (Figure 10).

[0073] 6. T lymphocyte detection

[0074] After euthanasia of mice, the spleen was removed and isolated into a single-cell suspension, which was then passed through a 100-mesh cell sieve. The cells were lysed with erythrocyte lysis buffer and resuspended in PBS. In the dark, the cells were stained with anti-mouse CD3-APC, CD4-FITC, and CD8-PE surface-labeled antibodies at 4°C for 30 minutes. All samples were analyzed using a BD FACSCanton flow cytometer, and data were analyzed using FlowJo 10 software.

[0075] The results showed that, compared with the PBS group, the number of CD3+CD4+T, CD3+CD8+T, and CD3+CD4+CD8+T lymphocytes increased in other immunization groups. Among them, the rAd5TsCLP:rTs-CLP group showed the greatest increase in the number of secreted T lymphocytes. Therefore, the prime-boost immunization strategy can stimulate the proliferation of T lymphocytes, promote the secretion of cytokines, and thus regulate and enhance the strength and breadth of the immune response (Figure 11).

[0076] The above results indicate that: (1) This invention provides a method for preparing a recombinant adenovirus vaccine containing Trichinella protein; (2) This invention provides a method for preparing a recombinant protein vaccine containing Trichinella protein; (3) This invention combines the advantages of recombinant adenovirus vaccines and recombinant protein vaccines, and uses a prime-boost immunization strategy to immunize BALB / c mice; (4) The prime-boost immunization strategy used in this invention has many advantages, which can effectively enhance the body's systemic immune level, strongly stimulate the body to produce a strong mucosal immune response, promote the dynamic balance of Th1 / Th2, and enhance the proliferation capacity of T lymphocytes; (5) The prime-boost immunization strategy described in this application shows unique advantages in dealing with trichinosis. It can effectively save the immunization dose, avoid the inconvenience and potential risks that may be caused by large-scale vaccination, and significantly improve the immunization effect, enabling the body to more effectively resist the invasion of Trichinella, providing a new idea and reliable approach for the prevention and control of trichinosis.

Claims

1. A Trichinella spiralis vaccine kit, characterized in that, The kit consists of two parts: the first part is a Trichinella spiralis recombinant protein vaccine with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the second part is a recombinant adenovirus expressing an amino acid sequence as shown in SEQ ID NO.

1.

2. The reagent kit according to claim 1, characterized in that, The content of the recombinant Trichinella spiralis protein is 60 μg; the content of the recombinant adenovirus expressing the amino acid sequence as shown in SEQ ID NO.1 is 10 μg. 8 PFU; the recombinant trichinella protein vaccine is administered via intramuscular injection; the recombinant adenovirus expressing the amino acid sequence shown in SEQ ID NO.1 is administered via nasal drop injection.

3. The reagent kit according to claim 1, characterized in that, The Trichinella recombinant protein vaccine is a mixture of Trichinella recombinant protein (amino acids as shown in SEQ ID NO.2) and CPG1018 adjuvant in a volume ratio of 1:

1.

4. The use of the kit according to any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of trichinosis.

5. A recombinant Trichinella spiralis protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. Encoding the nucleic acid molecule of the vaccine according to claim 5.

7. A recombinant vector containing the nucleic acid molecule of claim 6.

8. A recombinant microbial cell containing the nucleic acid molecule of claim 6.

9. The use of the recombinant protein of claim 5, the nucleic acid molecule of claim 6, the recombinant vector of claim 7, or the recombinant microbial cell of claim 8 in the preparation of vaccines or drugs for the prevention or treatment of trichinosis.

10. The application according to claim 9, characterized in that, Vaccines are available in the form of nasal sprays, oral preparations, suppositories, or parenteral preparations.

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